Journal of Developmental Biology doi: 10.3390/jdb14020024

Authors: Shir Maizus Dorit Kalo Tanya Kogan Ariel Michaelov Zvi Roth

The proportion of spermatozoa with progressive motility is widely used to evaluate the quality of a single ejaculate. However, the cellular and physiological mechanisms underlying this trait remain unclear. The present study examined the association between the progressive motility of bovine spermatozoa, their quality and their fertilization competence in vitro, and subsequently the association with the developmental morphokinetics of the formed embryos. Fresh ejaculates were classified and divided into groups with high (HPM), medium (MPM), or low (LPM) progressive motility. Then, spermatozoa were evaluated for their morphology, plasma membrane integrity, mitochondrial membrane potential, oxidative status, and acrosome integrity. The findings revealed that spermatozoa from HPM ejaculates enhanced motility in association with higher mitochondrial membrane potential relative to the LPM group, suggesting higher metabolic potential. No differences were recorded in fertilization competence among groups; however, the developmental kinetics of the formed embryos, determined by a time-lapse system, differed; embryos derived from HPM spermatozoa cleaved earlier to the two-, three-, and four-cell stages than embryos derived from MPM spermatozoa, suggesting that HPM-derived embryos are of good quality. Our findings suggest that progressive motility is not only a motility characteristic; it also reflects cellular quality of spermatozoa and the formed embryo.

Journal of Developmental Biology doi: 10.3390/jdb14020023

Authors: Ashina Nagra Ling-Yi Chen Soheil Saeidiborojeni Jessica M. Rosin Siddharth R. Vora

In osteopetrotic mice with homozygous inactivating mutations in the colony stimulating factor 1 (Csf1op/op) or its receptor (Csf1r−/−) gene, teeth fail to erupt due to severe reduction in osteoclastogenesis. Dental abnormalities have been described in the unerupted teeth of these models, but it remains unclear whether these defects arise from direct roles of CSF1R in odontogenesis or indirectly from impaired bone remodeling associated with failed eruption. Here, we examined the spatiotemporal expression of CSF1R during tooth development and inhibited CSF1R pharmacologically in utero using PLX5622 during early stages of tooth morphogenesis. Teeth and surrounding bone were analyzed at embryonic and postnatal stages using histology and high-resolution micro-computed tomography. Embryonic CSF1R inhibition resulted in reproducible abnormalities in incisor and molar morphology that were evident before and after birth and were associated with loss of normal bone remodeling at the tooth–bone interface. In contrast, postnatal CSF1R inhibition did not affect the structure or continuous growth of adult incisors. Together, these findings demonstrate a temporally restricted, indirect role for CSF1R in odontogenesis that is independent of tooth eruption and associated with remodeling of the bony crypts surrounding developing teeth by CSF1R-dependent cells.

Journal of Developmental Biology doi: 10.3390/jdb14020022

Authors: Le Sun Lu Ping Fuyu Zhang Ruzhen Gao Bo Zhang Xiaowei Chen

Microtia–atresia is a rare craniofacial malformation primarily affecting the first and second pharyngeal arches, leading to the deformity of the auricle and atresia of the external ear canal. Its etiology is heterogenous and largely unknown, including both genetic and environmental factors. The HOXA4 gene has been identified as potentially pathogenetic for microtia–atresia in three twin families. A hoxa4a mosaic knockdown zebrafish model was constructed using CRISPR/Cas9. hoxa4a was expressed in the mandible during early development in zebrafish, while the F0 mosaic knockdowns exhibited craniofacial malformations with abnormal chondrocyte morphologies. Specifically, hoxa4a knockdown reduced cranial neural crest cell proliferation while increasing apoptosis, markedly downregulating chondrogenic markers sox9a and col2a1a. Consequently, pharyngeal arch chondrocytes exhibited disorganized arrangement and morphological abnormalities, resulting in mandibular hypoplasia. Our findings provide important insights into the role of hoxa4a in zebrafish mandibular development and the pathology of microtia–atresia caused by HOXA4 gene mutations in humans.

Journal of Developmental Biology doi: 10.3390/jdb14020021

Authors: Saif Rehman William G. Willmore Kenneth B. Storey

The poly(A) tail has long been known to play a central role in mRNA stability, storage, and translational competence, making it a potential key regulator during hypometabolic states. During seasonal torpor, hibernating mammals must frequently enter these hypometabolic states to survive. In this study, we examined protein abundance changes in key enzymes involved in poly(A) tail synthesis, binding, and removal during torpor in the brown adipose tissue of the 13-lined ground squirrel, Ictidomys tridecemlineatus, using immunoblots. BAT during late torpor exhibited significantly reduced abundance of the catalytic cleavage enzyme CPSF73, but increased abundance of poly(A) polymerase PAPOLA. In contrast, poly(A)-binding proteins and major complex subunits of deadenylases, including CCR4-Not, exhibited no significant changes. Furthermore, despite unchanged levels of the translation initiation factor eIF4E, the phosphorylated variant of 4E-BP1, a potent inhibitor of the initiation factor when hypophosphorylated, was significantly reduced during late torpor. Overall, constrained mRNA maturation, preserved transcript stability, and reversible translational inhibition suggest that an important role exists for poly(A) tail regulatory machinery in hypometabolic survival throughout the torpid state.

Journal of Developmental Biology doi: 10.3390/jdb14020020

Authors: Martina Contestabile Jacqueline Fátima Martins de Almeida Chiara De Cesari Ilaria Tonazzini Paolo Giovanni Artini Simona Daniele

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A expression, leading to disrupted proteostasis and synaptic dysfunction. Adenosine is a ubiquitous neuromodulator whose G protein-coupled receptors (ARs) regulate neuronal differentiation and neurite outgrowth during development. Here, we investigated AR signaling and their influence on survival–autophagy balance and neuronal morphology in an AS cellular model. Using SH-SY5Y cells with silenced UBE3A, we found that UBE3A loss markedly decreased A1AR, A2BAR, and A3AR protein levels while significantly increasing A2AR expression. Ligand affinity was preserved across genotypes, but A1AR and A2AAR desensitization kinetics were significantly slower in UBE3A-deficient cells. These effects were associated with reduced recruitment of G protein-coupled receptor kinase 2 (GRK2) to the plasma membrane and decreased GRK2–AR association in UBE3A-deficient cells, suggesting a possible contribution of altered GRK2 dynamics to prolonged AR signaling. Functionally, A1AR and A2AR agonists preferentially promoted survival of UBE3A-deficient cells and modulated the MDM2–p53 axis and autophagy markers; A1R stimulation also increased neurite density in UBE3A-deficient cells. Together, these results identify AR-level alterations and defective desensitization machinery in AS neuronal cells and link receptor changes to downstream proteostasis and morphological phenotypes relevant to AS pathophysiology.

Journal of Developmental Biology doi: 10.3390/jdb14020019

Authors: Jorge A. De los Santos Funes

Feto-maternal microchimerism (Mc) refers to the exchange of cells between the fetus and mother, and fetal–fetal Mc to the exchange between fetuses during pregnancy. This phenomenon occurs across mammalian species, including humans, mice, and cattle. Key data on Mc cells and theoretical considerations regarding the presence of fetal-derived material, such as trophoblast cells, cell-free fetal DNA (cffDNA), and exosomes in maternal blood are summarized. This review aims to first, synthesize current knowledge on feto-maternal and fetal–fetal Mc across mammals, second, address three core questions: how and where Mc has been demonstrated in animals, what techniques have been used over time to detect fetal-derived material and Mc, and how placental structures influence the frequency of Mc. Finally, it aims to identify gaps in the literature for species such as horses, goats, and pigs. This article concludes that Mc is a widespread phenomenon among mammals, but detection methods and reported frequencies vary significantly by species and placental type. A biological model is presented in this article in which multinucleated trophoblast cells undergo apoptosis, releasing cffDNA that enters the maternal blood circulation after multinucleated trophoblast invasion. Advances in molecular biology technology have improved the ability to detect fetal-derived material, cells, DNA, and exosomes in maternal blood. However, notable research gaps remain for Mc in horses, goats, and pigs, highlighting the need for targeted studies to better understand species-specific patterns or a general biological model.

Journal of Developmental Biology doi: 10.3390/jdb14020018

Authors: Ann-Kathrin Schmitt Victoria Tjora Nela Kelam Marija Jurić Gunjača Petar Todorović Clelia Picard Manel Loche-Dalmon Katarina Vukojević Anita Racetin

Background/Objectives: Growing evidence indicates that melatonin contributes to kidney development and function, while disruptions of fetal circadian signaling have been linked to congenital anomalies of the kidney and urinary tract (CAKUT). This study aimed to characterize the developmental and spatial expression patterns of melatonin receptors MTNR1A and MTNR1B in normal human fetal kidneys and in CAKUT phenotypes. Methods: This study analyzed 40 human fetal kidney specimens, including healthy controls and CAKUT cases (horseshoe kidneys, duplex kidneys, and dysplastic kidneys), obtained from spontaneous abortions and pregnancy terminations. Samples were classified into developmental phases Ph2–Ph4 according to established morphological criteria. Immunofluorescence staining was used to visualize MTNR1A and MTNR1B expression. Quantitative analysis was performed using ImageJ, measuring the fluorescence area percentage. Statistical comparisons were conducted using a two-way ANOVA. Results: In control kidneys, MTNR1A expression was predominantly observed in glomeruli and interstitial cells and showed a descending trend across developmental stages, whereas MTNR1B was localized to glomeruli and strongly to the apical membranes of tubules, particularly distal tubules, without substantial developmental variation. CAKUT phenotypes exhibited higher expression of both receptors compared to controls. Significant phase-dependent differences in MTNR1A expression were observed in horseshoe, duplex, and dysplastic kidneys. MTNR1B expression decreased across developmental stages in dysplastic kidneys and differed significantly between Ph3 and Ph4 in duplex kidneys. At Ph3, duplex kidneys showed the highest MTNR1B expression. Conclusions: Altered developmental expression patterns of MTNR1A and MTNR1B in CAKUT suggest an association between melatonin signaling and abnormal human kidney development.

Journal of Developmental Biology doi: 10.3390/jdb14020017

Authors: Shota Inoue Hiroaki Hatano Ikko Kawashima Kunimasa Ohta

Ribosomes are widely recognized as large intracellular macromolecular complexes responsible for protein synthesis. However, in recent years, numerous studies have revealed that ribosomal proteins possess non-canonical functions beyond translation, including roles in cell fate regulation, development, and disease. This review outlines emerging concepts surrounding the extracellular functions of ribosomes, with a particular focus on ribosome-induced cellular plasticity and transdifferentiation. Our studies have demonstrated that the incorporation of exogenous ribosomes reprograms somatic cells into a multipotent state and promotes differentiation into multiple lineages. These findings represent an alternative perspective to the conventional view of ribosomes as merely translational components. Furthermore, we discuss the biological significance of factors secreted by ribosome-incorporated cells by integrating the paracrine hypothesis with ribosome-mediated cell fate conversion. Finally, we explore the potential applications of ribosomes in regenerative medicine and cell-cultured food production. By redefining ribosomes as active regulators of cellular identity, this review provides a conceptual framework for understanding ribosome-driven cell fate regulation and its potential applications in sustainable biotechnology.

Journal of Developmental Biology doi: 10.3390/jdb14020016

Authors: Bing Jia Mohamed Milad Hannah C. Boehler Adam Guerra Joshua Mowry Jessica Hiley James Kasen Lisonbee Michael Hafen Troy Camarata

Mammals and reptiles possess a metanephric kidney as the terminal renal organ for homeostasis of solutes and waste products. The development of the metanephric kidney has primarily been studied in mammalian model systems. Little is known about the conservation of metanephric kidney formation in non-mammalian species such as reptiles. Uniquely, reptiles maintain kidney progenitor cell populations throughout life and continually develop new nephrons, the functional unit of the kidney. The red-eared slider turtle, Trachemys scripta elegans, was utilized to investigate the conservation of reptilian metanephric kidney development. The nephron progenitor cell (NPC) marker, Six2, was detected in whole-mount turtle kidneys in a similar pattern to mammals. However, there were differences in progenitor cell niche morphology where turtle NPC populations formed distinct elongated rows instead of the rosette-like morphology found in the mouse. The pattern of NPC populations in the embryonic turtle kidney was maintained in the adult turtle. Whole-genome bisulfite sequencing was performed on cortical tissue containing the NPC populations from adult turtle kidneys and compared to those of adult mice. Significant conservation of gene methylation was detected in adult cortical tissue between the two species, although unique signatures were detected in turtle samples related to DNA repair and β-catenin signaling. This suggests a high level of conservation of metanephric kidney development at the genetic level.

Journal of Developmental Biology doi: 10.3390/jdb14020015

Authors: Veronika S. Frolova Denis A. Nikishin

Serotonin is a critical morphogen in early development, yet the mechanisms regulating its homeostasis in the preimplantation embryo remain unclear, particularly under conditions of maternal antidepressant exposure. Here, we investigated embryonic serotonergic autonomy using mouse models of pharmacological transport blockade (maternal fluoxetine treatment) and in vitro treatment with the monoamine oxidase inhibitor pargyline. We employed immunofluorescence, RT-qPCR, and live-cell imaging to assess metabolic flux, gene expression, and physiological health. We demonstrate that monoamine oxidase functions as a metabolic firewall, progressively maturing from zygote to blastocyst to degrade excess amines. Paradoxically, maternal serotonin transporter blockade triggered significant intracellular serotonin hyper-accumulation in blastocysts, associated with a trend toward a compensatory upregulation of the biosynthetic gene Ddc. While this serotonin overload did not compromise morphology, mitochondrial function, or pluripotency marker expression, it induced a robust epigenetic response. Excess serotonin promoted elevated H3Q5ser immunoreactivity in both nuclear and cytoplasmic compartments via a transglutaminase-dependent mechanism. These findings reveal that the preimplantation embryo possesses a resilient, autonomous serotonergic system capable of compensatory synthesis. However, environmental fluctuations are chemically recorded via transglutaminase-mediated serotonylation, representing an epigenetic mark that warrants further long-term study within the Developmental Origins of Health and Disease (DOHaD) framework.

Journal of Developmental Biology doi: 10.3390/jdb14010014

Authors: Diyana Vladova

Bone tissue is among the most commonly transplanted tissues worldwide. The treatment of critical-sized bone defects remains a significant challenge, as there is currently no universally accepted experimental model or therapeutic standard. Recent advances in fundamental cell biology are driving a paradigm shift in approaches to bone regeneration, highlighting the transformative potential of biofabrication technologies that integrate tissue engineering with personalized regenerative strategies. Three-dimensional (3D) bioprinting technology enables precise control over the architecture and spatial distribution of cellular and biologically active components, facilitating the creation of complex, personalized bone constructs. Central to this process are bioinks and biomaterials that mimic the extracellular matrix (ECM) and provide an optimal microenvironment for cellular function. Despite the substantial body of accumulated data, a comprehensive theoretical framework for functional bone biofabrication has not yet been fully established, emphasizing both the challenges and the innovative potential of the field. This integrative review synthesizes current knowledge on bone biology—from embryogenesis and cell–matrix interactions to molecular and neural regulation—and links it to the opportunities offered by biofabrication. Particular attention is given to bioinks as mediators between cell biology and engineering sciences, as well as to strategies for creating biomimetic ECM, optimizing scaffold design, and guiding future research toward clinically translatable bone regeneration.

Journal of Developmental Biology doi: 10.3390/jdb14010013

Authors: Ana Beatriz Matos Laura Jesus Castro Torcato Martins

Transcriptional regulation is pivotal for developmental processes and cell fate specification in homeostasis. One particularly relevant group of transcription factors is the sine oculis homeobox (SIX) family, which is involved in a wide range of molecular processes from development to tissue maintenance. Within this family, distinct subfamilies exhibit specific DNA-binding preferences and can function as transcriptional activators or repressors. In this review, we focus on the Optix/SIX3–SIX6 subfamily and discuss their roles as transcriptional regulators, as well as the consequences of their deregulation for neuronal and ocular development and for the maintenance of tissue homeostasis. We further examine how SIX3 can act either as a tumour suppressor or as a marker of poor prognosis in different cancer types. Moreover, we summarize recent findings on the role of SIX3 in pancreatic β cells and highlight emerging evidence that SIX2 also contributes to β-cell identity and regulatory stability. Downregulation of SIX2 and SIX3 alters gene regulatory programs associated with β-cell homeostasis and contributes to type 2 diabetes. As accumulating evidence links members of the SIX family to cancer and metabolic disease, it is crucial to characterize how these transcription factors regulate cell identity, with important implications for disease mechanisms and therapeutic strategies.

Journal of Developmental Biology doi: 10.3390/jdb14010012

Authors: Amalia S. Parra Christopher A. Johnston

Development and maintenance of complex tissues depends on a number of coordinated steps from early development through adulthood. These processes are fundamentally controlled by highly regulated gene expression patterns. Although critical contributors during development, intrinsic changes in gene expression alone cannot fully explain the complicated pathways that control tissue homeostasis. Rather, tissues are continuously exposed to extrinsic factors that also influence essential cellular processes. These external environmental factors are collectively known as the exposome. Notably, how different exposures impact gene expression and protein function, as well as how certain exposures lead to disease states, is not well understood. To understand how internal and external factors influence organismal development and homeostasis, it is necessary to consider how genetic and nongenetic components interact to direct critical biochemical pathways. Doing so presents new avenues for precision medicine, understanding disease progression, identifying biological threats, and improving biological security concerns. In this review, we present recent advances in exposure biology, focusing on how these innovations can help identify novel biomarkers to better understand changing exposome components. We also discuss the need to integrate technologies and exposure research to better identify and predict threats.

Journal of Developmental Biology doi: 10.3390/jdb14010011

Authors: Takumi Satoh

Extant birds and the earliest dinosaurs may share fundamental metabolic features essential for aerobic exercise, suggesting that the extraordinary physical performance typical of avian species originated when dinosaurs first appeared during the Carnian Pluvial Episode (CPE). This physiological adaptation is complemented by hyperactive mitochondria that exhibit high oxygen consumption and low reactive oxygen species production. Molecular genomics of fossils, the so-called “Jurassic Genome,” indicates that these early dinosaurs possessed compact genomes, 50–60% the size of the human genome, and small cells, implying a highly stringent metabolic regime. We suggest that hyperactive mitochondria, closely associated with compact genomes and small cells, drive theropod adaptation to the hot, dry, and hypoxic environments of the Late Triassic period, ultimately enabling their ecological dominance. Early dinosaurs such as Herrerasaurus are hypothesized to have possessed advanced physiological traits shared with modern birds, including hyperactive mitochondria, compact genomes, small cells, and a developing air-sac system. Collectively, these features most likely may have contributed to exceptional metabolic capacity, locomotor performance, and adaptation to the harsh environment of the CPE.

Journal of Developmental Biology doi: 10.3390/jdb14010010

Authors: Manuel Escalona Rosa Roy

The Neuroblastoma Breakpoint Family (NBPF) consists of 23 genes, 9 of which are pseudogenes, and is characterized by extensive duplication events and species-specific diversification in Homo sapiens, as well as by the presence of a unique protein domain known as Olduvai (also referred to as DUF1220 or the NBPF domain). Previous studies have attempted to define subfamilies based on the presence of HLS triplet domains; however, this classification has become increasingly unclear with the identification of additional NBPF members. The family remains poorly understood, and the functions of many genes are still unknown, although several have been hypothesized to play key roles in cell proliferation and developmental processes, particularly in neural and skeletal tissues. In this study, we systematically analyzed all available data on the NBPF gene family using the PRISMA-S methodology to infer the biological functions in which these genes may be involved. We also generated multiple phylogenetic trees to support the creation of coherent subfamilies and to correlate the origin of each subfamily with homologous genes in our last common ancestor with the Pan genus, providing what we believe to be one of the most comprehensive phylogenetic reconstructions including all currently annotated NBPF members. Through comparative genomic and phylogenetic analyses, we propose that the NBPF may have originated from a duplication of the PDE4DIP gene, with NBPF26 representing the ancestral member from which the remaining NBPF genes diverged via lineage-specific segmental duplications. In this systematic review and comparative genomic study, we present the first integrative synthesis of our knowledge of the NBPF, encompassing its evolutionary origins, structural dynamics, expression across tissues, and clinical associations.

Journal of Developmental Biology doi: 10.3390/jdb14010009

Authors: Masayo Nagata Satoru Hayano Ziyi Wang Takahiro Kosami Hiroshi Kamioka

Craniofacial development is orchestrated by a finely regulated interplay of numerous genes and signaling pathways. Palatogenesis proceeds through a complex, stepwise process, in which endogenous mechanical stresses within tissues have been implicated. However, the impact of exogenous fluidic flow mechanical stress derived from maternal movement on palatal development remains unclear. In this study, we investigated the effect of exogenous fluidic flow mechanical stress on palatal morphogenesis, focusing on the horizontal outgrowth of palatal shelves after elevation. Palatal tissues dissected from mouse embryos were subjected to organ culture with or without mechanical loading (loaded and unloaded groups, respectively). Stress magnitude was quantified by calculating wave energy, and morphometric and molecular analyses were performed. Compared with the unloaded group, palatal shelves in the loaded group showed significant increases in thickness and volume, accompanied by enhanced cell proliferation, nuclear translocation of YAP and β-catenin, and upregulation of the osteogenic markers Osterix and Osteocalcin. No significant difference in apoptosis was observed. These findings indicate that exogenous mechanical stress promotes cell proliferation and osteogenic differentiation through the Hippo and WNT/β-catenin pathways in palate explants. Our results suggest that moderate maternal movement-induced mechanical stress contributes to normal palatogenesis, providing new insights into the mechanisms underlying cleft palate.

Journal of Developmental Biology doi: 10.3390/jdb14010008

Authors: Pierre Thiébaud Emilie Simon François Moisan Sandrine Fedou Hamid-Reza Rezvani Nadine Thézé

VESTIGIAL-LIKE proteins constitute a family of evolutionarily conserved proteins that act as cofactors in regulating gene expression through their binding to TEAD transcription factors. Among the four members of this family in vertebrates, VESTIGIAL-LIKE 4 has emerged as a tumor suppressor that competes with YAP in binding TEADs, thus inhibiting the HIPPO pathway downstream of YAP. Nevertheless, very few studies have addressed its function during early vertebrate development. Here, we used gain- and loss-of-function strategies to investigate the role of vestigial-like 4 during Xenopus laevis development. Our data show that vestigial-like 4 is a key regulator of neurogenesis and neural crest formation. In embryos depleted of vestigial-like 4, neurogenesis is severely impaired, and neither neurog1 nor neurod1 is able to stimulate neurogenesis. Vestigial-like 4 is also required for neural crest formation through pax3 and sox9 regulation, and this property does not necessarily require its interaction with tead. Collectively, our findings demonstrate that vestigial-like 4 is an important regulator of neurogenesis and neural crest formation. Although vestigial-like 4 can bind to tead proteins in the embryo, its function does not depend solely on this interaction, suggesting a complex level of regulation with which vestigial-like 4 regulates early steps in development and differentiation.

Journal of Developmental Biology doi: 10.3390/jdb14010007

Authors: Nadya V. Ilicheva Olga I. Podgornaya

Lampbrush chromosomes (LBCs) are a feature of amphibian oocytes and are typically associated with high levels of transcription during active oocyte growth. However, their state during winter hibernation has not been studied. Here, we investigated LBCs in early vitellogenic oocytes (early stage 4) of the grass frog Rana temporaria during winter hibernation. We found that the chromosomes retained their lampbrush morphology, and the phosphorylated form of RNA polymerase II resided on the lateral loops. Transcription on the lateral loops was reduced but detectable at cold conditions and significantly increased when the oocytes were transferred at room temperature. Satellite S1a transcripts were detected at the lateral loops of the chromosomes by RNA FISH. The possible significance of maintaining chromosomes in the lampbrush form during hibernation is discussed.

Journal of Developmental Biology doi: 10.3390/jdb14010006

Authors: Alexey S. Anisimov Dmitry S. Bogolyubov Irina O. Bogolyubova

Cumulus cells (CCs) are a distinct population of granulosa cells (GCs) that surround the developing and ovulated mammalian oocyte. The features of their structural organization and the expression pattern of key genes significantly affect oocyte viability. Changes in the functional activity of the nucleus are often expressed in changes in the structure of nuclear bodies (NBs), including Cajal bodies (CBs). The diagnostic protein of CBs is coilin, which maintains their structural integrity. Using fluorescent and electron microscopy, we examined maternal aging-associated changes in coilin pattern in mouse CCs. We found that older mice had a decrease in the number of coilin-positive bodies, while external transcriptome data analysis revealed no significant changes in Coil and Smn1 gene expression. We hypothesized that the age-related dynamics of coilin-containing bodies are determined not by changes in the expression level of key components of these bodies, but by age-related changes in CC metabolism. Considering that CCs are a by-product of IVF protocols, making them available for analysis in sufficient quantities, age-related changes in the number and size of coilin-positive NBs in CCs may serve as a promising biomarker for assessing ovarian functional aging.

Journal of Developmental Biology doi: 10.3390/jdb14010005

Authors: Kohei Kawaguchi Takuya Kawaguchi Juma Obayashi Yasuji Seki Kunihide Tanaka Kei Ohyama Junki Koike Shigeyuki Furuta Kevin C. Pringle Hiroaki Kitagawa

Obstructive uropathy (OU) during fetal development induces a fetal cystic dysplastic kidney. The mechanisms of cyst formation and the onset of renal dysfunction remain unclear. Determining whether nephrogenic potential persists during fetal life may suggest whether early intervention could preserve renal development. We aimed to evaluate residual nephrogenic activity in fetal cystic dysplastic kidneys using β-catenin and CD10 immunostaining, and to assess whether the site of obstruction influences cystogenesis. After appropriate approval, 20 timed-gestation fetal lambs had OU created at 60 days. Males underwent urethral and urachal ligation (n = 8, 3 lost), and females underwent unilateral ureteric ligation (n = 8, 1 lost). Fetuses were sacrificed at 80 days (n = 6) and 140 days (term, n = 10), comparing kidneys with normal controls of the same gestational age using immunohistochemical staining for β-catenin and CD10. Developing fetal cystic dysplastic kidneys were identified at 80 days. β-catenin staining showed the absence of granular cytoplasmic expression in cystic regions, indicating arrested nephrogenesis. In male models, cysts originated exclusively from proximal tubules. Female models exhibited mixed proximal and distal tubular involvement. CD10 staining confirmed the loss of proximal tubular markers. Renal development remained arrested at term. Cyst formation disrupts renal development early in gestation, which persists until term. Differences in cystogenesis between the models suggest that the site of obstruction influences pathogenic mechanisms.

Journal of Developmental Biology doi: 10.3390/jdb14010004

Authors: Guillermo Diez-Pinel Alessandro Muratore Christiana Ruhrberg Giovanni Canu

Erythro-myeloid progenitors (EMPs) originate from the haemogenic endothelium in the yolk sac via an endothelial-to-haematopoietic transition (EHT) to generate blood and immune cells that support embryo development. Yet, the transitory nature of EHT and the limited availability of molecular markers have constrained our understanding of the origin, identity, and differentiation dynamics of EMPs. Here, we have refined the annotation of yolk sac haemato-vascular populations in publicly available single-cell RNA sequencing (scRNAseq) datasets from mouse embryos to identify novel molecular markers of haemogenic endothelium and EMPs. By sub-clustering key cell populations followed by pseudotime analysis, we refined cluster annotations and then reconstructed differentiation trajectories. Subsequent differential gene expression analysis between clusters identified novel cell surface markers for haemogenic endothelial cells (Fxyd5 and Scarf1) and EMPs (Fcer1g, Tyrobp, and Mctp1). Further, we have identified candidate signalling and metabolic pathways that may regulate yolk sac haematopoietic emergence and differentiation. The specificity of FXYD5, SCARF1, and FCER1G for haemogenic endothelium and EMPs was validated by immunostaining of the mouse yolk sac. These insights into the transcriptional dynamics in the yolk sac should support future investigation of EHT and haematopoietic differentiation during early mammalian development.

Journal of Developmental Biology doi: 10.3390/jdb14010003

Authors: Kazi Sarjana Safain Kendall C. Swanson Joel S. Caton

Maternal nutrition during gestation profoundly influences fetal growth, organogenesis, and long-term offspring performance through developmental programming. Among the molecular mechanisms responsive to maternal nutrient availability, one-carbon metabolism plays a central role by integrating folate, methionine, choline, and vitamin B12 pathways that regulate methylation, nucleotide synthesis, and antioxidant defense. These processes link maternal nutritional status to epigenetic remodeling, cellular proliferation, and redox balance during fetal development. Mitochondria act as nutrient sensors that translate maternal metabolic cues into bioenergetic and oxidative signals, shaping tissue differentiation and metabolic flexibility. Variations in maternal diet have been associated with shifts in fetal amino acid, lipid, and energy metabolism, suggesting adaptive responses to constrained intrauterine environments. This review focuses on the molecular interplay between one-carbon metabolism, mitochondrial function, and metabolomic adaptation in developmental programming of ruminant livestock. Understanding these mechanisms offers opportunities to design precision nutritional strategies that enhance fetal growth, offspring productivity, and long-term resilience in livestock production systems.

Journal of Developmental Biology doi: 10.3390/jdb14010002

Authors: Claudia B. Gil David Paz Briana E. Pinales Victoria L. Castro Claire E. Perucho Annalise Gonzales Giulio Francia Sepiso K. Masenga Antentor Hinton Anita M. Quintana

Mutations in the transcriptional co-factor HCFC1 cause methylmalonic aciduria and homocystinemia, cblX type (cblX) (MIM#309541), non-syndromic X-linked intellectual disability (XLID), and focal epilepsy. Zebrafish studies have revealed increased activation of the Akt/mTor signaling pathway after mutation of hcfc1a, one ortholog of HCFC1. mTOR hyperactivation is linked to seizures, and its inhibition alleviates epilepsy in other preclinical models. We hypothesized that mTor overactivity in hcfc1a mutant zebrafish increases seizure susceptibility and/or severity. We employed a two-concentration model of the seizure-inducing agent, pentylenetetrazol (PTZ), with or without pretreatment of the mTor inhibitor, torin1. Mutation of hcfc1a did not alter the response to PTZ at sub-optimal concentrations, and the pharmaceutical inhibition of mTor using the compound Torin1 reduced response to 1 µM PTZ, but only in a dose-dependent manner. Higher doses of mTor inhibition did not reduce the seizure response in mutant larvae but were effective in wildtype siblings. These data suggest that inhibition of mTor in an hcfc1a-deficient background leads to a reaction that differs from the traditional response observed in wildtype siblings. Collectively, we present a model that can be used to test dose–response and the development of combinatorial treatment approaches in a high-throughput manner.

Journal of Developmental Biology doi: 10.3390/jdb14010001

Authors: Attila Placido Sachslehner Julia Steinbinder Claudia Hess Veronika Mlitz Leopold Eckhart

The epithelial egg tooth is used by birds to open the eggshell for hatching. This ectodermal structure consists of a multilayered periderm and a hard cornified portion, the caruncle or actual egg tooth. Here, we determined the protein composition of the egg tooth of the chicken and compared the proteins to markers of other epithelia identified in previous studies. The egg tooth and the upper beak of chicken embryos of Hamburger and Hamilton (HH) stage 44 were subjected to mass spectrometry-based proteomics. We found that scaffoldin, a marker of the embryonic periderm and the feather sheath, was enriched in the egg tooth relative to the beak. Likewise, Epidermal Differentiation protein containing DPCC Motifs (EDDM) and Epidermal Differentiation protein starting with a MTF motif and rich in Histidine (EDMTFH), which had previously been characterized as markers of the subperiderm on embryonic scutate scales and the barbs of feathers, were also enriched in the egg tooth. The expression of EDDM and EDMTFH was confirmed RT-PCR analysis. Our data suggest that the epithelial egg tooth is related to the subperiderm and feathers, a hypothesis with potentially important implications for the evolution of the avian integument.

Journal of Developmental Biology doi: 10.3390/jdb13040045

Authors: Lana Shteynman Genevieve Monanian Gilberto Torres Giancarlo Sabetta Deborah M. Li Zhaosheng Jin Tiffany Angelo Bahaa E. Daoud Morgane Factor

Placenta Accreta Spectrum (PAS) disorders, including placenta accreta, increta, and percreta, are serious obstetric conditions characterized by abnormal placental adherence to the uterine wall. With increasing incidence, PAS poses significant risks, primarily through massive hemorrhage during or after delivery, often necessitating hysterectomy. Key risk factors include prior cesarean sections, uterine surgery, and placenta previa diagnosis. In this review, we will examine the pathophysiology of PAS, with a focus on the mechanisms underlying abnormal trophoblast invasion and defective decidualization. We will highlight the role of uterine scarring, extracellular matrix remodeling, dysregulated signaling pathways, and immune and vascular alterations in disrupting the maternal-fetal interface, ultimately predisposing to morbid placentation and delivery complications. We will also discuss the life-threatening complications of PAS, such as shock and multi-organ failure, which require urgent multidisciplinary intensive care, as well as the optimization of management through preoperative planning and intraoperative blood loss control to reduce maternal morbidity and mortality.

Journal of Developmental Biology doi: 10.3390/jdb13040044

Authors: Sophia Tsokkou Ioannis Konstantinidis Antonios Keramas Vasileios Anastasiou Alkis Matsas Maria Florou Alexandra Arvanitaki Emmanouela Peteinidou Theodoros Karamitsos George Giannakoulas Themistoklis Dagklis Theodora Papamitsou Antonios Ziakas Vasileios Kamperidis

Background. Gestational diabetes mellitus (GDM) induces maternal hyperglycemia, which may alter fetal cardiac structure and function, increasing short- and long-term cardiovascular risks. Purpose. To systematically review the evidence on the fetal cardiac structural and functional effects of GDM, to explore the diagnostic role of novel imaging and biochemical biomarkers, and to summarize the long-term cardiovascular complications associated with GDM. Materials and Methods. A systematic search of PubMed, Scopus, and Cochrane Library was conducted according to the PRISMA guidelines. All studies comparing cardiac outcomes in GDM and non-GDM pregnancies were included. Data on myocardial hypertrophy, diastolic and systolic function, imaging modalities, and biomarkers were extracted and qualitatively synthesized. Results. A total of twelve eligible studies were identified. Fetal cardiac hypertrophy and diastolic and early systolic dysfunction are common among GDM pregnancies and can be detected by dual-gate Doppler and speckle-tracking echocardiography. Abnormalities are observed in indices such as the myocardial performance index, E/A, E/e′ ratios, and global longitudinal and circumferential strain in fetuses and may persist in the neonatal period. Alterations may be more pronounced for the right ventricle compared to the left. Septal hypertrophy is associated with elevated umbilical cord pro-brain natriuretic peptide. The risk of early-onset cardiovascular disease in the progeny of diabetic mothers is 29% higher, as evidenced by population-based cohort data. Conclusions. GDM is linked to fetal cardiac remodeling and an increased long-term cardiovascular risk. Early detection and customized interventions to reduce adverse outcomes may be achieved by integrating advanced echocardiographic techniques and biomarkers into prenatal surveillance.

Journal of Developmental Biology doi: 10.3390/jdb13040043

Authors: Stefka Delimitreva Irina Chakarova

Observations of the processes of oogenesis, fertilization, and the earliest embryonic development have given us the opportunity to estimate the importance of chromosomal distribution errors for the success of mammalian reproduction. It is now known that in the large volume of oocytes, zygotes and the first embryonic cells, the rearrangement of chromatin is associated with a complex rearrangement of cytoskeletal structures, which creates specific problems. This review discusses two main issues critical to the success of early embryos: Why oocyte meiosis is too frequently wrong in chromosomal segregation? Why the first zygotic mitoses are too frequently wrong in chromosomal segregation? We concluded the following: (1) The main cytoskeletal defects that disturb oocyte meiosis are a problematic connection between cytoskeleton and nucleoskeleton, unsuccessful movement of the spindle to the oocyte periphery, unstable anchoring of the spindle to oolemma, and deviations in meiotic spindle morphology; (2) The main cytoskeletal defects that disturb pronuclear unification are nonfunctional male centriole, unsuccessful forming of microtubule aster around the sperm centrosome, problematic movement of the two pronuclei towards each other and inappropriate contacts between centrosomes, microtubules and nuclear pore complexes; (3) Cytoskeletal defects that disturb zygote mitosis are unsuccessful forming of bipolar mitotic spindle, non-synchronized congression of maternal and paternal chromosomes, and unsuccessful attachment of kinetochores to microtubules.

Journal of Developmental Biology doi: 10.3390/jdb13040042

Authors: Dong Zhang Xiaoyun Pang Zhenxing Yan Weitao Dong Zihao Fang Jincheng Yang Yanyan Wang Li Xue Jiahao Zhang Chen Xue Hongwei Duan Xianghong Du Yuxuan He

Autophagy has a potential regulatory effect on spermatogenesis and testicular development. Dynamic alterations in the testicular autophagy of prepubertal mice were analyzed, and the relationship between autophagy levels and testicular development was clarified using C57BL/6 mice aged 1, 2, 4, 6, and 8 weeks. Transmission electron microscopy was used to identify autophagic vacuoles. The expression of autophagy-related proteins and PI3K/AKT/mTOR signaling pathway-related proteins was determined using Western blotting. Localization of microtubule-associated protein light chain 3 (LC3) and sequestosome 1 (p62) in testicular tissues was determined using immunofluorescence and immunohistochemistry. Autophagic vacuoles in spermatogenic cells increased gradually from weeks 1 to 4, peaked at 2 weeks, decreased sharply at 6 weeks, and were undetectable at 8 weeks. The expression of Beclin 1 autophagy-related protein, LC3-II, and p62 was highest at 2 weeks among the five age groups, whereas LC3-II and p62 were mainly localized in spermatogonia and spermatocytes. Moreover, low mTOR expression and its increased expression were detected at 1–2 weeks and 2–8 weeks, respectively. These results show that testicular autophagic levels exhibit a dynamic pattern of “increase (1–2 weeks) followed by a decrease (2–8 weeks),” providing a reference in determining the relationship between autophagy levels and testicular development.

Journal of Developmental Biology doi: 10.3390/jdb13040041

Authors: Junkun Lai Xiaoyu Gao Guke Zhang Xiao Wu Yiqian Zhang Shunbo Wang Zhenfang Wu Zicong Li Zheng Xu

Somatic cell nuclear transfer (SCNT) or cloning technology is widely used in agriculture and biomedicine. However, the application of this technology is limited by the low developmental competence of cloned embryos or fetuses, which frequently exhibit abnormal development of trophoblast cells or placentas. The purpose of this study was to investigate the possible causes of the erroneous placental development of SCNT-derived pig fetuses. The placental transcriptomic and lipidomic profiles were compared between 30-day-old SCNT- and artificial insemination (AI)-produced pig fetuses. Differentially expressed lipid metabolites between two groups of placentas were selected to test their effects on porcine trophoblast cell activity. The results showed that SCNT placentas exhibit impaired lipid metabolism and function. The level of a metabolite, lysophosphatidylcholine (LPC), in the glycerophospholipid metabolism pathway was substantially increased in SCNT placentas, compared with AI placentas. The elevation in LPC content may lead to impaired placental development in cloned pig fetuses, as LPC inhibited the proliferation and migration of porcine trophoblast cells. This study discovers a main cause of erroneous development of cloned pig fetuses, which will be beneficial for understanding the regulation of SCNT embryo development, as well as developing new methods to improve the efficiency of pig cloning.

Journal of Developmental Biology doi: 10.3390/jdb13040040

Authors: Aki Shigenaka Eri Nitta Tadashi Nakagawa Makiko Nakagawa Toru Hosoi

Neurodevelopmental disorders (NDDs), including autism spectrum disorder, intellectual disability, and attention deficit hyperactivity disorder, are increasingly recognized as disorders of early brain construction arising from defects in neural stem and progenitor cell (NSPC) proliferation. NSPCs are responsible for generating the diverse neuronal and glial lineages that establish cortical architecture and neural circuitry; thus, their expansion must be tightly coordinated by intrinsic cell cycle regulators and extrinsic niche-derived cues. Disruption of these mechanisms—through genetic mutations, epigenetic dysregulation, or environmental insults—can perturb the balance between NSPC self-renewal and differentiation, resulting in aberrant brain size and connectivity. Recent advances using animal models and human pluripotent stem cell-derived brain organoids have identified key signaling pathways, including Notch, Wnt, SHH, and PI3K–mTOR, as central hubs integrating proliferative cues, while transcriptional and chromatin regulators such as PAX6, CHD8, SETD5, and ANKRD11 govern gene expression essential for proper NSPC cycling. Furthermore, prenatal exposure to teratogens such as Zika virus infection, valproic acid, or metabolic stress in phenylketonuria can recapitulate proliferation defects and microcephaly, underscoring the vulnerability of NSPCs to environmental perturbation. This review summarizes emerging insights into the molecular and cellular mechanisms by which defective NSPC proliferation contributes to NDD pathogenesis, highlighting convergence among genetic and environmental factors on cell cycle control. A deeper understanding of these pathways may uncover shared therapeutic targets to restore neurodevelopmental trajectories and mitigate disease burden.

Journal of Developmental Biology doi: 10.3390/jdb13040039

Authors: Rui Caetano Oliveira Sandra Ferreira Isabel Gonçalves Maria Fátima Martins

Despite looking monotonous, liver histology represents a highly complex structure of hepatocytes, bile ducts and vessels. This complex interaction and development originate in embryology and remain in adult life. In this manuscript, we highlight the features of liver embryology, translating the events into pathologic features and opening possibilities for disease understanding and research. We revisit liver embryology, from biliary to vascular processes, stressing some developing abnormalities with a focus on the histological findings. With this manuscript, we hope to increase the awareness of the importance of embryology in diseases, prompting its detailed study.

Journal of Developmental Biology doi: 10.3390/jdb13040038

Authors: Andrew S. Powers Petar Gajic Ethan Rittereiser Kavindra Dasrat Gregory A. Lnenicka

We examined whether an increase in synaptic activity resulted in an increase in quantal size at the neuromuscular junction (NMJ) of third-instar Drosophila larvae. Spontaneous miniature excitatory postsynaptic currents (mEPSCs) or miniature excitatory postsynaptic potentials (mEPSPs) were recorded before and after nerve stimulation. We found that prolonged (60 s) or brief (1.25 s) nerve stimulation produced an increase in quantal size; this appears to be a general property of these synapses since it was seen at all four muscle fibers (MFs) used in this study. The effect was examined along Is and Ib terminals by expressing GCaMP in the MF membrane and examining postsynaptic Ca2+ signals produced by spontaneous transmitter release. The activity-dependent increase in quantal size occurred at both Is and Ib terminals, and the increase in frequency and amplitude of quantal events at individual synaptic boutons was correlated. Both the increase in quantal size and frequency were found to be dependent upon an increase in postsynaptic Ca2+, based on studies in which MFs were preinjected with the Ca2+ chelator BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). To examine the effect of postsynaptic activity on glutamate sensitivity, we iontophoresed glutamate pulses at the NMJ and recorded the glutamate-evoked excitatory postsynaptic potentials (gEPSPs). Trains of glutamate pulses produced an increase in gEPSP amplitude; this potentiation was not seen when Ca2+ was eliminated from the bath or after inhibiting calmodulin or CaMKII. The activity-dependent increase in quantal size may result from an increase in postsynaptic sensitivity due to activation of CaMKII.

Journal of Developmental Biology doi: 10.3390/jdb13040037

Authors: Valeria Merico Paola Rebuzzini Mario Zanoni Maurizio Zuccotti Silvia Garagna

Estrogen metabolites (EMs) play a local regulatory role in mammalian ovarian function. Among them, 2-hydroxyestradiol (2-OHE2) exerts dose-dependent effects on reproductive physiology, supporting either normal ovarian processes or contributing to pathological conditions. Specifically, 2-OHE2 modulates ovarian vasculature and progesterone biosynthesis, and at 1–10 nM concentrations, it enhances in vitro developmental competence and blastocyst quality in mouse oocytes. Conversely, doses below 1 nM show no appreciable effects, suggesting the existence of a biological activity threshold. However, the impact of supra-physiological concentrations remains largely unexplored. In this study, we investigated the effects of increasing 2-OHE2 doses (0.05, 0.50, and 5.00 µM) on oocyte meiotic progression and quality. Exposure to 0.50 and 5.00 µM significantly impaired oocyte maturation, while only the highest dose notably reduced the percentage of embryos developing to the blastocyst stage. Morphometric analysis during the GV-to-MII transition revealed altered first polar body morphology, defective asymmetric division, and disruptions in cytoskeletal organization, including enlarged meiotic spindles, increased F-actin cap angles, and aberrant microtubule-organizing centers distribution. These structural alterations were paralleled by distinct changes in cytoplasmic movement velocity patterns observed through time-lapse imaging during meiotic resumption. Together, these findings demonstrate that supra-physiological exposure to 2-OHE2 compromises oocyte maturation and developmental competence by perturbing key cytoskeletal dynamics and cellular architecture necessary for successful meiosis and early embryogenesis.

Journal of Developmental Biology doi: 10.3390/jdb13040036

Authors: Jennifer L. Fenner Boyuan Wang Cheikhouna Ka Sujan Gautam Ryan C. Range

Wnt signaling is an ancient developmental mechanism that drives the initial specification and patterning of the primary axis in many metazoan embryos. Yet, it is unclear how exactly the various Wnt components interact in most Wnt-mediated developmental processes as well as in the molecular mechanism regulating adult tissue homeostasis. Recent work in invertebrate deuterostome sea urchin embryos indicates that three different Wnt signaling pathways (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) form an interconnected Wnt signaling network that specifies and patterns the primary anterior–posterior (AP) axis. Here, we detail our current knowledge of this critical regulatory process in sea urchin embryos. We also illustrate examples from a diverse group of metazoans, from cnidarians to vertebrates, that suggest aspects of the sea urchin AP Wnt signaling network are deeply conserved. We explore how the sea urchin is an excellent model to elucidate a detailed molecular understanding of AP axis specification and patterning that can be used for identifying unifying developmental principles across animals.

Journal of Developmental Biology doi: 10.3390/jdb13040035

Authors: Abdullah F. Idriss Edward J. Okello Roger G. Sturmey Miguel A. Velazquez

High non-esterified fatty acids (NEFAs) during negative energy balance in dairy cattle can impair reproduction. While their effects on oocyte maturation and preimplantation embryo development are known, their impact during fertilisation is largely unexplored. This study examined the effects of high NEFA exposure exclusively during in vitro fertilisation (IVF). Bovine oocytes were matured in vitro and fertilised under physiological or high NEFA concentrations. High NEFA concentrations decreased fertilisation, cleavage, and blastocyst rates. Reactive oxygen species production in zygotes was not affected, but blastocysts derived from the High-NEFA group had fewer cells. Spermatozoa exposed to high NEFA concentrations exhibited increased plasma membrane and acrosome damage, higher DNA fragmentation, and reduced mitochondrial membrane potential. The expression of H3K27me3, a repressive histone mark normally erased from fertilisation to embryonic genome activation, was higher in 2-cell than in 4-cell embryos on day 2 after IVF, but only in the High-NEFA group. This delayed H3K27me3 loss, along with increased DNA damage, could partially explain the reduced blastocyst formation observed. In conclusion, high NEFA concentrations can impair pre-implantation embryo development during zygote formation, potentially via effects on both the oocyte and spermatozoon. The latter warrants further investigation using an intracytoplasmic sperm injection model.

Journal of Developmental Biology doi: 10.3390/jdb13040034

Authors: Saadia Khilji Munerah Hamed Jihong Chen Qiao Li

We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the differentiation and fusion of skeletal myoblasts. We have also analyzed the genomic programs underlying rexinoid-enhanced myogenic differentiation to identify novel regulatory pathways. As such, we observed a significant upregulation of a transcript encoding a predicted transmembrane protein, Tmem182, during C2C12 myoblast differentiation. Despite the documentation of Tmem182 expression in skeletal muscles, its regulation had yet to be explored. Here, we show that Tmem182 gene expression is markedly augmented in early myoblast differentiation and further enhanced by RXR signaling. In addition, Tmem182 expression is specific to muscle tissues and related to muscle master regulator MyoD. We found that MyoD and histone acetyltransferase p300 are bound to the Tmem182 promoter, and Tmem182 expression is p300-dependent. Thus, our data display a putative epigenetic signature associated with p300 and histone acetylation in rexinoid-responsive locus activation and transcription of myogenic targets.

Journal of Developmental Biology doi: 10.3390/jdb13030033

Authors: Yan Jiao Jiapeng Liu Congge Li Yuexin Hu Sanjun Zhao

In recent years, assisted reproductive technology (ART) has developed rapidly with the delay in reproductive age and the rise in infertility rates. During ART, blastocyst quality is a key factor affecting the rate of implantation and clinical pregnancy, and blastocyst formation is dependent on the precise regulation of multiple signaling pathways in preimplantation embryo development. In this review, we systematically analyze the molecular mechanisms of the core pathways, including Hippo, Wnt/β-catenin, FGF, Nodal, and BMP, in blastocyst lineage differentiation and morphogenesis, and assess the feasibility of optimizing in vitro culture by targeting key signaling nodes, as well as provide theoretical support for constructing research models of preimplantation embryos.

Journal of Developmental Biology doi: 10.3390/jdb13030032

Authors: Latifa Kazzazy Flóra Huba Bálint Lóránt Hausz Dávid Mező Viktória Perey-Simon Bálint Jezsó Abdulrahman Seddik Zoran Marinović Judit Tóth Angéla Békési Beáta G. Vértessy Máté Varga

DNA repair is a multifaceted biological process that involves multiple pathways to counter the types of damage the genome encounters throughout life. In the past decade zebrafish became a popular model organism to study various aspects of vertebrate DNA repair, and the characterization of several mutant lines deficient in key players of the repair pathways has significantly contributed to our understanding of the roles the corresponding proteins play in the maintenance of genomic integrity. Interestingly, the base-excision repair (BER) pathway remained one of the less characterized DNA repair processes in fish. Here we provide a detailed characterization of zebrafish deficient in one of the key components of BER, the uracil-DNA glycosylase Unga. We show that while these fish are viable, they display an altered response to genotoxic stress and unga mutant males show an interesting form of subfertility.

Journal of Developmental Biology doi: 10.3390/jdb13030031

Authors: Samira Alam Danielle Duncan Sharmin Hasan

Vertebrate embryonic development relies on tightly regulated signaling pathways that guide morphogenesis, cell fate specification, and tissue organization. Among these, the Wnt signaling pathway plays a central role, orchestrating key developmental events. The non-canonical Wnt pathways, including the Planar Cell Polarity and Wnt/Ca2+ branches, are especially critical for regulating cytoskeletal dynamics during gastrulation. Recent studies highlight that these pathways interface with cytoskeletal effectors to control actin remodeling in response to extracellular cues. One such effector is Profilin, a small, evolutionarily conserved actin-binding protein that modulates actin polymerization and cellular architecture. Profilins, particularly Profilin1 and 2, are known to interact with Daam1, a formin protein downstream of PCP signaling, thereby linking Wnt signals to actin cytoskeletal regulation. Emerging evidence suggests that Profilins are active signaling intermediates that contribute to morphogenetic processes. Their context-dependent interactions and differential expression across species also suggest that they play specialized roles in development and disease. This review synthesizes the current understanding of Profilin’s role in non-canonical Wnt signaling, examining its molecular interactions and contributions to cytoskeletal control during development. By integrating data across model systems, we aim to clarify how Profilins function at the intersection of signaling and cytoskeletal dynamics, with implications for both developmental biology and disease pathogenesis.

Journal of Developmental Biology doi: 10.3390/jdb13030030

Authors: Brigite Cabrita Mary Enyioko Rui Gonçalo Martinho

Female gametogenesis is orchestrated by dynamic epigenetic modifications. In mammals, SETDB1, a histone H3K9 methyltransferase, is required for proper meiotic progression and early embryonic development. In Drosophila, the ortholog of SETDB1 plays a critical role in germ cell differentiation, transposon silencing, and the transcriptional repression of specific germline genes during oocyte fate determination. Moreover, Polycomb group (PcG) proteins in both mammals and Drosophila are essential for primary oocyte viability and meiosis, functioning through the silencing of early prophase I genes during later stages of prophase. While the repressive roles of epigenetic regulators in both Drosophila and mammalian oogenesis are well characterized, the functions of epigenetic activators remain less defined. Gene expression is controlled by the opposing activities of PcG and Trithorax group (TrxG) proteins, with the latter constituting a diverse family of chromatin remodelling factors that include H3K4 methyltransferases. In Drosophila, SET domain containing 1 (Set1)—the ortholog of mammalian SETD1A/B—acts as the primary regulator of global H3K4me2/3 levels. Set1 is critical for germline stem cell (GSC) self-renewal, functioning through both cell-autonomous and non-cell-autonomous mechanisms, with its depletion in the germline resulting in a progressive loss of GSC. More recently, Set1 has been implicated in germline cyst differentiation, although the mechanisms underlying this role remain poorly understood due to the complexity of the observed phenotypes. To investigate this, we analyzed ovaries from recently eclosed females in which Set1 and its highly conserved COMPASS partner, absent, small, or homeotic discs 2 (Ash2), were depleted—thus minimizing the confounding effects from GSC loss. We observed striking defects in both oocyte determination and Synaptonemal Complex (SC) integrity in one- to two-day-old females, within otherwise normal egg chambers. Interestingly, while defects in oocyte fate and oocyte–chromatin architecture were partially recovered in older egg chambers, SC integrity remained compromised. These findings suggest a critical window for SC assembly during germline cyst differentiation, after which this assembly cannot occur.

Journal of Developmental Biology doi: 10.3390/jdb13030029

Authors: Rajalakshmi Ramamoorthy Arianna K. Carden Hussain Hussain Brian Z. Druyan Ping Ping Chen Rima Hajjar Carmen Fernandez Nila Elumalai Amirah B. Rashed Karen Young Anna Rosa Speciale Emily M. West Staci Marbin Bradley Safro Ian J. Bishop Arumugam R. Jayakumar Luis Sanchez-Ramos Michael J. Paidas

Glucagon-like peptide 1 (GLP-1), a hormone derived from the proglucagon gene, regulates various physiological processes; however, its impact on pregnancy outcomes remains poorly understood. Assessing the effects of GLP-1 on neonates is vital as GLP-1 is increasingly administered during pregnancy. This study evaluates the effect of GLP-1 exposure on maternal complications and neonatal defects in mice. Pregnant female A/J mice received subcutaneous injections of recombinant GLP-1 (rGLP-1; 1000 nmol/kg) on embryonic day 1 (EP, early pregnancy) or day 15 (E15, late pregnancy). Maternal and neonatal body weights, morphology, and mortality were recorded, and mRNA sequencing was conducted to analyze gene expression in neonatal tissues. Maternal body weight decreased following rGLP-1 exposure, and pups born to both the early and late exposure groups experienced significant weight loss. Pups in the late exposure group exhibited uniform skin detachment and a dramatically higher mortality rate than those born to the early exposure group. Further, RT-PCR analysis confirms the significantly increased expression of selected genes in the skin and associated pathogenesis. RNA sequencing of pups’ skin, brain, lung, and liver tissues from the late exposure group showed altered gene expression. Since maternal weight loss, increased neonatal mortality, and altered gene expression have been observed, GLP-1 receptor agonists (GLP-1RAs) should be avoided during pregnancy.

Journal of Developmental Biology doi: 10.3390/jdb13030028

Authors: Rui Caetano Oliveira Paula Martins Maria de Fátima Martins

The development process of the heart and cardiovascular system is fundamental in human development and highly regulated by genetic factors. This process needs to be highly regulated to prevent malformations. Nevertheless, some heart defects may be identified, especially with modern imaging methodology. Atrial septal defects (ASDs) are particularly common. Understanding the mechanisms involved in ASD formation is fundamental for developing new treatment strategies. In this article, we explore cardiac development and embryology, with a focus on atrial septal defects and their clinical implications.

Journal of Developmental Biology doi: 10.3390/jdb13030027

Authors: Jackson Dann Frank Grützner

The stomach has been a highly conserved organ throughout vertebrate evolution; however, there are now over 20 lineages composed of monotremes, lungfish and teleost fish displaying a secondary loss of stomach function and morphology. This “agastric phenotype” has evolved convergently and is typified by a loss of gastric glands and gastric acid secretion and a near-to-complete loss of storage capacity of the stomach. All agastric species have lost the genes for gastric enzymes (Pga and Pgc) and proton pump subunits (Atp4a and Atp4b), and gastrin (Gast) has been lost in monotremes. As a key gastric hormone, the conservation of gastrin has not yet been investigated in the lungfish or agastric teleosts, and it is unclear how the loss of gastrin affects the evolution and selection of the native receptor (Cckbr), gastrin-releasing peptide (Grp) and gastrin-releasing peptide receptor (Grpr) in vertebrates. Furthermore, there are still many genes implicated in gastric development and function which have yet to be associated with the agastric phenotype. We analysed the evolution, selection and conservation of the gastrin pathway and a novel gastric gene repertoire (Gkn1, Gkn2, Tff1, Tff2, Vsig1 and Anxa10) to determine the correlation with the agastric phenotype. We found that the loss of gastrin or its associated genes does not correlate with the agastric phenotype, and their conservation is due to multiple pleiotropic roles throughout vertebrate evolution. We found a loss of the gastric gene repertoire in the agastric phenotype, except in the echidna, which retained several genes (Gkn1, Tff2 and Vsig1). Our findings suggest that the gastrin physiological pathway evolved differently in pleiotropic roles throughout vertebrate evolution and support the convergent evolution of the agastric phenotype through shared independent gene-loss events.

Journal of Developmental Biology doi: 10.3390/jdb13030026

Authors: Emilie Jones Jay Miguel Fonticella Kelly A. McLaughlin

Craniofacial development is a complex, highly conserved process involving multiple tissue types and molecular pathways, with perturbations resulting in congenital defects that often require invasive surgical interventions to correct. Remarkably, some species, such as Xenopus laevis, can correct some craniofacial abnormalities during pre-metamorphic stages through thyroid hormone-independent mechanisms. However, the full scope of factors mediating remodeling initiation and coordination remain unclear. This study explores the differential remodeling responses of craniofacial defects by comparing the effects of two pharmacological agents, thioridazine-hydrochloride (thio) and ivermectin (IVM), on craniofacial morphology in X. laevis. Thio-exposure reliably induces a craniofacial defect that can remodel in pre-metamorphic animals, while IVM induces a permanent, non-correcting phenotype. We examined developmental changes from feeding stages to hindlimb bud stages and mapped the effects of each agent on the patterning of craniofacial tissue types including: cartilage, muscle, and nerves. Our findings reveal that thio-induced craniofacial defects exhibit significant consistent remodeling, particularly in muscle, with gene expression analysis revealing upregulation of key remodeling genes, matrix metalloproteinases 1 and 13, as well as their regulator, prolactin.2. In contrast, IVM-induced defects show no significant remodeling, highlighting the importance of specific molecular and cellular factors in pre-metamorphic craniofacial correction. Additionally, unique neuronal profiles suggest a previously underappreciated role for the nervous system in tissue remodeling. This study provides novel insights into the molecular and cellular mechanisms underlying craniofacial defect remodeling and lays the groundwork for future investigations into tissue repair in vertebrates.

Journal of Developmental Biology doi: 10.3390/jdb13030025

Authors: Lei Huang Maowu Cao Xiangbin Zhu Na Li Can Huang Kunfu Ouyang Ze'e Chen

Background: Left ventricular non-compaction cardiomyopathy (LVNC) is a congenital heart disease characterized by abnormal prenatal development of the left ventricle that has an aberrantly thick trabecular layer and a thinner compacted myocardial layer. However, the underlying molecular mechanisms of LVNC regulated by mitochondrial phosphatase genes remain largely unresolved. Methods: We generated a mouse model with cardiac-specific deletion (CKO) of Ptpmt1, a type of mitochondrial phosphatase gene, using the αMHC-Cre, and investigated the effects of cardiac-specific Ptpmt1 deficiency on cardiac development. Morphological, histological, and immunofluorescent analyses were conducted in Ptpmt1 CKO and littermate controls. A transcriptional atlas was identified by RNA sequencing (RNA-seq) analysis. Results: We found that CKO mice were born at the Mendelian ratio with normal body weights. However, most of the CKO mice died within 24 h after birth, developing spontaneous ventricular tachycardia. Morphological and histological analysis further revealed that newborn CKO mice developed an LVNC phenotype, evidenced by a thicker trabecular layer and a thinner myocardium layer, when compared with the littermate control. We then examined the embryonic hearts and found that such an LVNC phenotype could also be observed in CKO hearts at E15.5 but not at E13.5. We also performed the EdU incorporation assay and demonstrated that cardiac cell proliferation in both myocardium and trabecular layers was significantly reduced in CKO hearts at E15.5, which is also consistent with the dysregulation of genes associated with heart development and cardiomyocyte proliferation in CKO hearts at the same stage, as revealed by both the transcriptome analysis and the quantitative real-time PCR. Deletion of Ptpmt1 in mouse cardiomyocytes also induced an increase in phosphorylated eIF2α and ATF4 levels, indicating a mitochondrial stress response in CKO hearts. Conclusions: Our results demonstrated that Ptpmt1 may play an essential role in regulating left ventricular compaction during mouse heart development.

Journal of Developmental Biology doi: 10.3390/jdb13030024

Authors: Mikhail A. Zhukovsky Si-Eun Sung Albrecht Ott

Axis definition plays a key role in the establishment of animal body plans, both in normal development and regeneration. The cnidarian Hydra can re-establish its simple body plan when regenerating from a random cell aggregate or a sufficiently small tissue fragment. At the beginning of regeneration, a hollow cellular spheroid forms, which then undergoes symmetry breaking and de novo body axis definition. In the past, we have published related work in a physics journal, which is difficult to read for scientists from other disciplines. Here, we review our work for readers not so familiar with this type of approach at a level that requires very little knowledge in mathematics. At the same time, we present a few aspects of Hydra biology that we believe to be linked to our work. These biological aspects may be of interest to physicists or members of related disciplines to better understand our approach. The proposed theoretical model is based on fluctuations of gene expression that are triggered by mechanical signaling, leading to increasingly large groups of cells acting in sync. With a single free parameter, the model quantitatively reproduces the experimentally observed expression pattern of the gene ks1, a marker for ‘head forming potential’. We observed that Hydra positions its axis as a function of a weak temperature gradient, but in a non-intuitive way. Supposing that a large fluctuation including ks1 expression is locked to define the head position, the model reproduces this behavior as well—without further changes. We explain why we believe that the proposed fluctuation-based symmetry breaking process agrees well with recent experimental findings where actin filament organization or anisotropic mechanical stimulation act as axis-positioning events. The model suggests that the Hydra spheroid exhibits huge sensitivity to external perturbations that will eventually position the axis.

Journal of Developmental Biology doi: 10.3390/jdb13030023

Authors: Junya Leim Nikko Adhitama Quang Dang Nong Pijar Religia Yasuhiko Kato Hajime Watanabe

The crustacean Daphnia magna produces genetically identical females and males by parthenogenesis. Males are produced in response to environmental cues including crowding and lack of food. For male development, the DM-domain containing transcription factor Doublesex1 (DSX1) is expressed spatiotemporally in male-specific traits and orchestrates male trait formation in both somatic and gonadal tissues. However, it remains unknown how the dsx1 gene is silenced in females to avoid male trait development. Heterochromatin Protein 1 (HP1) plays a crucial role in epigenetic gene silencing during developmental processes. Here we report the identification of four HP1 orthologs in D. magna. None of these orthologs exhibited sexually dimorphic expression, and among them, HP1-1 was most abundantly expressed during embryogenesis. The knock-down of HP1-1 in female embryos led to the derepression of dsx1 in the male-specific traits, resulting in the development of male characteristics, such as the elongation of the first antennae. These results suggest that HP1-1 silences dsx1 for female development while environmental cues unlock this silencing to induce male production. We infer the HP1-dependent formation of a sex-specific chromatin structure on the dsx1 locus is a key process in the environmental sex determination of D. magna.

Journal of Developmental Biology doi: 10.3390/jdb13030022

Authors: Nathan Zimmerman Aaron Marta Carly Baker Zeljka Korade Károly Mirnics Annemarie Shibata

Cholesterol homeostasis is necessary for normal vertebrate development. The disruption of cholesterol homeostasis can cause abnormal body and nervous system development and lead to dysfunctional behavior and increased mortality. Commonly prescribed psychopharmaceuticals can alter cholesterol synthesis and may disrupt early vertebrate development. A high-throughput vertebrate zebrafish model system was used to test the hypothesis that exposure to psychopharmaceutical medications alters cholesterol biosynthesis and disrupts gene transcription, early whole-body and brain development, and nervous system function, resulting in abnormal behavior. Exposure to cariprazine, aripiprazole, trazodone, and AY9944 increased 7-dehydrocholesterol levels compared to vehicle-treated zebrafish. Significant differences in disease-associated gene expression, brain structure, and functional behaviors were observed in psychopharmaceutical and AY9944-treated zebrafish compared to controls. These data reveal that the high-throughput zebrafish model system can discern psychopharmaceutical effects on cholesterol synthesis, gene transcription, and key features of early vertebrate development that influences behavior.

Journal of Developmental Biology doi: 10.3390/jdb13030021

Authors: Manashree S. Malpe Leon F. McSwain Heath M. Aston Karl A. Kudyba Chun Ng Megan P. Wright Cordula Schulz

The germline stem cells (GSCs) in the male gonad of Drosophila can increase their division frequency in response to a demand for more sperm caused by repeated mating. However, the molecules and mechanisms regulating and mediating this response have yet to be fully explored. Here, we present the results of a transcriptome analysis comparing expression from the testis tips from non-mated and mated males. An overlapping set of 18 differentially expressed genes (DEGs) from two independent wild-type (wt) strains revealed that the majority of the DEGs encode secreted proteins, which suggests roles for them in cell–cell interactions. Consistent with a role for secretion in regulating GSC divisions, knocking down Signal Recognition Particle (SRP) components within the germline cells using RNA Interference (RNAi), prevented the increase in GSC division frequency in response to mating. The major class of DEGs encodes polypeptides below the size of 250 amino acids, also known as small proteins. Upon reducing germline expression of small proteins, males no longer increased GSC division frequency after repeated mating. We hypothesize that mating induces cellular interactions via small proteins to ensure continued GSC divisions for the production of sperm.

Journal of Developmental Biology doi: 10.3390/jdb13020020

Authors: Thanh Khoa Nguyen Sophia Baker Julienne Angtuaco Liana Arceri Samuel Kaczor Bram Fitzsimonds Matthew R. Hawkins Rebecca A. Wingert

Endocannabinoid signaling plays a significant role in neurogenesis and nervous system physiology, but its roles in the development of other tissues are just beginning to be appreciated. Previous reports have shown the presence of the key endocannabinoid receptor Cannabinoid receptor 1 (CB1 or Cnr1) in multiciliated (MCC) tissues and its upregulation in kidney diseases, yet the relationship between Cnr1 and renal MCC development is unknown. Here, we report that Cnr1 is essential for cilia development across tissues and regulates renal MCCs via cyclic AMP (cAMP) signaling during zebrafish embryogenesis. Using a combination of genetic and pharmacological studies, we found that the loss of function, agonism and antagonism of cnr1 all lead to reduced mature renal MCC populations. cnr1 deficiency also led to reduced cilia development across tissues, including the pronephros, ear, Kupffer’s vesicle (KV), and nasal placode. Interestingly, treatment with the cAMP activator Forskolin (FSK) restored renal MCC defects in agonist-treated embryos, suggesting that cnr1 mediates cAMP signaling in renal MCC development. Meanwhile, treatment with the cAMP inhibitor SQ-22536 alone or with cnr1 deficiency led to reduced MCC populations, suggesting that cnr1 also mediates renal MCC development independently of cAMP signaling. Our findings indicate that cnr1 has a critical role in controlling renal MCC development both via cAMP signaling and an independent pathway, further revealing implications for ciliopathies and renal diseases.

Journal of Developmental Biology doi: 10.3390/jdb13020019

Authors: Shota Inoue Arif Istiaq Anamika Datta Mengxue Lu Shintaro Nakayama Kousei Takashi Nobushige Nakajo Shigehiko Tamura Ikko Kawashima Kunimasa Ohta

Previously, we reported that mammalian cells, specifically human dermal fibroblasts (HDFs), could be transdifferentiated by lactic acid bacteria (LAB). Later, we observed that HDFs incorporated LAB-derived ribosomes, forming the ribosome-induced cell clusters (RICs) and transdifferentiating into cells derived from all three germ layers. Based on this insight, we hypothesized that incorporating ribosomes into non-mammalian cells could reveal the universality of this mechanism and open the door to commercial applications. Our current study demonstrates that ribosome incorporation can transdifferentiate chick primary muscle-derived cells (CMCs) into adipocytes, osteoblasts, and chondrocytes. Furthermore, the culture medium supernatant from ribosome-incorporated CMCs was found to significantly enhance CMC’s proliferation. RNA-seq analysis revealed that RICs-CMC exhibit increased expression of genes related to multi-lineage cell growth. In addition, we developed a novel technological shift in meat production—the “CulNet System”—which replicates organ interactions within mechanical systems for cell-cultured meat production. While significant efforts are still required to implement this technology in a cost-effective manner, we believe that combining the “CulNet System” with ribosome-incorporated multipotent cells that have prolonged culture capability could substantially improve the scalability and cost-effectiveness of cultured chicken meat production. This report highlights a promising approach for cell-culture-based meat production, offering a sustainable alternative to traditional methods.

Journal of Developmental Biology doi: 10.3390/jdb13020018

Authors: Attila Placido Sachslehner David A. D. Parry Leopold Eckhart

Keratins and keratin-associated proteins (KRTAPs) are the main components of mammalian nails and hair. Comparative genomics and gene expression studies have revealed that keratins are conserved in all vertebrates, whereas KRTAPs exist only in mammals. Recently, we found hair keratin-like cysteine-rich keratins in jawless vertebrates with confirmed expression in the cornified epithelial teeth of the sea lamprey (Petromyzon marinus). Here, we report that KRTAP-like proteins are also present in the horny teeth of the lamprey. Mass spectrometry-based proteomics identified proteins that share features with KRTAPs, such as high contents of cysteine and tyrosine residues, which support intermolecular interactions, and abundant glycine residues, which endow the proteins with flexibility. Genes encoding KRTAP-like proteins are arranged in a cluster in P. marinus, and the presence of at least one KRTAP-like protein is conserved in phylogenetically diverse species of lamprey, including Lampetra fluviatilis, Lethenteron reissneri, Geotria australis, and Mordacia mordax. The KRTAP-like genes of lampreys contain two exons, whereas mammalian KRTAPs have only a single exon. Although KRTAPs and KRTAP-like proteins are products of independent evolution, their common expression in cornified skin appendages suggests that they fulfill similar functions.

Journal of Developmental Biology doi: 10.3390/jdb13020017

Authors: Reza Amanollahi Stacey L. Holman Ashley S. Meakin Monalisa Padhee Kimberley J. Botting-Lawford Song Zhang Severence M. MacLaughlin David O. Kleemann Simon K. Walker Jennifer M. Kelly Skye R. Rudiger I. Caroline McMillen Michael D. Wiese Mitchell C. Lock Janna L. Morrison

Assisted reproductive technology (ART)such as in vitro embryo culture (IVC), is widely used in human infertility treatments; however, its long-term effects on the cardiac health of offspring remain unclear. This study aimed to determine whether the effects of IVC on cardiac metabolism and associated signaling pathways persist after birth into adolescence. Embryos were either transferred to an intermediate ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) with methionine supplementation (IVCHS+M) for 6 days after mating. Naturally mated (NM) ewes were used as controls. Protein expression and hormone concentrations in the left ventricle (LV) were analyzed using Western blot and LC-MS/MS analyses, respectively. IVC was associated with sex-specific alterations in cardiac mitochondria, with males exhibiting reduced mitochondrial abundance. Cardiac protein expression of oxidative phosphorylation (OXPHOS) complexes 1 and 4 was reduced by IVC. Additionally, IVC reduced protein expression of PDK-4 and Mn-SOD in the IVCHS+M group, which may impact energy efficiency and defense against oxidative stress. These changes may predispose IVC offspring to cardiac oxidative stress and mitochondrial dysfunction, particularly in males. This study provides insights into the sex-dependent effects of IVC on cardiac health, emphasizing the importance of evaluating long-term cardiovascular risks associated with IVC protocols.

Journal of Developmental Biology doi: 10.3390/jdb13020016

Authors: Sanja Jovicic Ivan R. Nikolic Ljiljana Amidžić Vesna Ljubojevic Maja Barudzija Ranko Skrbic

Background: The process of prenatal hematopoiesis occurs in various anatomical locations, including the placenta. The placenta is not merely a temporary hematopoietic reservoir, but it is one of the key sites for the synthesis of hematopoietic stem cells (HSCs). This study aimed to investigate the presence, distribution, and immunoprofiles of HSCs in the human placenta during different gestational periods. Materials and Methods: Placental samples of different gestational ages (first, second, and third trimesters) were analyzed using classical hematoxylin and eosin staining and immunohistochemical staining for CD34, CD117, and CD41 markers, with HSC quantification through numerical areal density (NA). Results: Highly immunoreactive CD34 HSCs were present in placentas throughout gestation, while highly immunoreactive CD117 and CD41 HSCs were observed during the first two trimesters. In the first trimester, HSCs were found within the lumen of blood vessels and as individual cells in the mesenchyme of chorionic villi. With advancing gestation, the number of HSCs in the mesenchyme of chorionic villi increased. Conclusions: Immunoreactive CD34, CD117, and CD41 cells are present in significant proportions in various parts of the placenta throughout gestation, indicating that the placenta provides a substantial proportion of HSCs for hematopoiesis.

Journal of Developmental Biology doi: 10.3390/jdb13020015

Authors: Darian J. Gamble Samantha Lopez Melody Yazdi Toni Castro-Torres Thomas P. Lozito

Lizards are distinguished as the only amniotes, and closest relatives of mammals, capable of multilineage epimorphic regeneration. Tail blastemas of green anole lizards (Anolis carolinensis) consist of col3a1+ fibroblastic connective tissue cells enclosed in krt5+ wound epidermis (WE), both of which are required for regeneration. Blastema and WE formation are known to be closely associated with phagocytic cell populations, including macrophages and osteoclasts. However, it remains unclear what specific phagocytic cell types are required to stimulate regeneration. Here, we explicitly assess the roles of osteoclast activity during blastema and WE formation in regenerating lizard tails. First, probe sequencing was performed at regenerative timepoints on fibroblasts isolated based on col3a1 expression toward establishing pathways involved in stimulating blastema formation and subsequent tail regrowth. Next, treatments with osteoclast inhibitor zoledronic acid (ZA) were used to assess the roles of osteoclast activity in lizard tail regeneration and fibroblast signaling. ZA treatment stunted lizard tail regrowth, suggesting osteoclast activity was required for blastema formation and regeneration. Transcriptomic profiling of fibroblasts isolated from ZA-treated and control lizards linked inhibition of osteoclast activity with limitations in fibroblasts to form pro-regenerative extracellular matrix and support WE formation. These results suggest that crosstalk between osteoclasts and fibroblasts regulates blastema and WE formation during lizard tail regeneration.

Journal of Developmental Biology doi: 10.3390/jdb13020014

Authors: Audra W. Harl Verónica M. Negrón-Pérez Jacob W. Stewart George A. Perry Alan D. Ealy Michelle L. Rhoads

It is not yet understood why cows that exhibit estrus and ovulate are more likely to become pregnant than those that ovulate but do not exhibit estrus during a fixed-time artificial insemination (FTAI) protocol. The objective of this work was to determine whether the follicular fluid from cows that exhibit estrus contributes to the increased likelihood of pregnancy. Lactating crossbred cows were subjected to an FTAI estrous synchronization protocol. Estrous behavior was observed and recorded prior to transvaginal follicle aspiration from cows that did (estrus, n = 7) or did not exhibit estrus (non-estrus, n = 6). Follicular fluid (25%) was then added to in vitro maturation media for the maturation of oocytes (n = 1489) from slaughterhouse ovaries. Cleavage rates were not affected by the estrous status of the cows from which the follicular fluid was collected. Blastocyst rates, however, were greater following maturation in the presence of follicular fluid from estrus cows compared to non-estrus cows (p ≤ 0.01). This difference in blastocyst rates was not related to blastocyst cell numbers (inner cell mass, trophoblast, and total), as they did not differ between estrus and non-estrus animals. This study demonstrates that the follicular fluid, and thus, the follicular environment just prior to ovulation does indeed contribute to improved pregnancy rates following FTAI.

Journal of Developmental Biology doi: 10.3390/jdb13020013

Authors: Christina L. Greene Geoffrey Traeger Akshay Venkatesh David Han Mark W. Majesky

Compartment boundaries divide the embryo into segments with distinct fates and functions. In the vascular system, compartment boundaries organize endothelial cells into arteries, capillaries, and veins that are the fundamental units of a circulatory network. For vascular smooth muscle cells (SMCs), such boundaries produce mosaic patterns of investment based on embryonic origins with important implications for the non-uniform distribution of vascular disease later in life. The morphogenesis of blood vessels requires vascular cell movements within compartments as highly-sensitive responses to changes in fluid flow shear stress and wall strain. These movements underline the remodeling of primitive plexuses, expansion of lumen diameters, regression of unused vessels, and building of multilayered artery walls. Although the loss of endothelial compartment boundaries can produce arterial–venous malformations, little is known about the consequences of mislocalization or the failure to form SMC-origin-specific boundaries during vascular development. We propose that the failure to establish a normal compartment boundary between cardiac neural-crest-derived SMCs of the 6th pharyngeal arch artery (future ductus arteriosus) and paraxial-mesoderm-derived SMCs of the dorsal aorta in mid-gestation embryos leads to aortic coarctation observed at birth. This model raises new questions about the effects of fluid flow dynamics on SMC investment and the formation of SMC compartment borders during pharyngeal arch artery remodeling and vascular development.

Journal of Developmental Biology doi: 10.3390/jdb13020012

Authors: Lorenzo Alibardi

Molecules involved in the activation of regeneration in reptiles are almost unknown. MARCK-like proteins are indicated to activate regeneration in some amphibians and fish, and it would be important to know whether this is a general process also present in other vertebrates. To address this problem, the present study reports the immunolocalization of a MARCK-like protein in injured tissues of a lizard. Bioinformatics and immunofluorescence after 5BrdU administration, and detection of MARCK-like proteins, have been performed on regenerating tail and limb of the lizard Podarcis muralis. Transcriptome data indicate up-regulation of MARCKS and MARCK-like1 expression in the initial regenerating tail and limb blastemas, supporting their involvement in the activation of regeneration in both appendages. Immunofluorescence for 5BrdU shows numerous proliferating cells in the blastemas of both appendages. Immunolocalization of a MARCK-like protein, using an antibody generated against a homologous protein from the axolotl, shows that the wound epidermis, nerves, and myotubes accumulate most of the protein in the limb and tail. MARCK-like immunolabeling is also detected in the regenerating spinal cord of the tail. The study indicates that, although the limb later turns into a scar, the MARCK-like protein is also up-regulated in this appendage, like in the regenerating tail. These results indicate that the initial reaction to an injury in lizards, an amniote representative, includes some triggering processes observed in amphibians and fish (anamniotes), with the activation of MARCK-like genes and proteins. This suggests that a MARCK-like-dependant mechanism for tissue repair is likely activated during the initial phases of vertebrate wound healing.

Journal of Developmental Biology doi: 10.3390/jdb13020011

Authors: Gracy X. Rosario Samuel Brown Subhradip Karmakar Mohammad A. Karim Rumi Nihar R. Nayak

The proliferation of trophoblast stem (TS) cells and their differentiation into multiple lineages are pivotal for placental development and functions. Various transcription factors (TFs), such as CDX2, EOMES, GATA3, TFAP2C, and TEAD4, along with their binding sites and cis-regulatory elements, have been studied for their roles in trophoblast cells. While previous studies have primarily focused on individual enhancer regions in trophoblast development and differentiation, recent attention has shifted towards investigating the role of super-enhancers (SEs) in different trophoblast cell lineages. SEs are clusters of regulatory elements enriched with transcriptional regulators, forming complex gene regulatory networks via differential binding patterns and the synchronized stimulation of multiple target genes. Although the exact role of SEs remains unclear, they are commonly found near master regulator genes for specific cell types and are implicated in the transcriptional regulation of tissue-specific stem cells and lineage determination. Additionally, super-enhancers play a crucial role in regulating cellular growth and differentiation in both normal development and disease pathologies. This review summarizes recent advances on SEs’ role in placental development and the pathophysiology of placental diseases, emphasizing the potential for identifying SE-driven networks in the placenta to provide valuable insights for developing therapeutic strategies to address placental dysfunctions.

Journal of Developmental Biology doi: 10.3390/jdb13020010

Authors: Ting-Xin Jiang Ping Wu Ang Li Randall B. Widelitz Cheng-Ming Chuong

How to elicit and harness regeneration is a major issue in wound healing. Skin injury in most amniotes leads to repair rather than regeneration, except in hair and feathers. Feather follicles are unique organs that undergo physiological cyclic renewal, supported by a dynamic stem cell niche. During normal feather cycling, growth-phase proximal follicle collar bulge stem cells adopt a ring configuration. At the resting and initiation phases, these stem cells descend to the dermal papilla to form papillary ectoderm and ascend to the proximal follicle in a new growth phase. Plucking resting-phase feathers accelerates papillary ectoderm cell activation. Plucking growth-phase feathers depletes collar bulge stem cells; however, a blastema reforms the collar bulge stem cells, expressing KRT15, LGR6, Sox9, integrin-α6, and tenascin C. Removing the follicle base and dermal papilla prevents feather regeneration. Yet, transplanting an exogenous dermal papilla to the follicle base can induce re-epithelialization from the lower follicle sheath, followed by feather regeneration. Thus, there is a stepwise regenerative strategy using stem cells located in the collar bulge, papillary ectoderm, and de-differentiated lower follicle sheath to generate new feathers after different levels of injuries. This adaptable regenerative mechanism is based on the hierarchy of stem cell regenerative capacity and underscores the remarkable resilience of feather follicle regenerative abilities.

Journal of Developmental Biology doi: 10.3390/jdb13010009

Authors: Guy Sion Domenic C. D’Amore

Stressors such as injuries, embryonic instability during development, and higher levels of stress hormones such as testosterone can result in increases in fluctuating asymmetry in reptiles and other vertebrates. Digit asymmetry, digit ratio variability, and skull trait asymmetry such as eye and jaw size have been correlated with stress level in both snakes and lizards. Teeth asymmetry has also been used as a biomarker for stress and brain laterality. Body size is correlated with many potential stressors, yet there has been little research on how body size in reptiles relates to asymmetry. We investigate teeth asymmetry within the lizard family Varanidae, a clade with a diverse range of sizes consisting of the largest living lizard, Varanus komodoensis. Using a landmark/semi-landmark analysis, we derived Centroid Size for 671 pairs of teeth from 13 varanid species, and asymmetry was derived for each pair. Right-biased asymmetry was significantly greater in the upper tooth row, but breaking up tooth positions into further sections did not yield a significant difference. We found a significant positive linear correlation between body size and right-biased teeth directional asymmetry within Varanus, but only when excluding V. komodoensis. This significant correlation may result from fewer potential predators and more potential food items, thus resulting in less overall stress. When analyzed separately, V. komodoensis individuals with <180 mm head length demonstrated a positive, yet non-significant, trend along a similar trajectory to their congenerics with a high goodness of fit. On the other hand, individuals > 180 mm showed a high degree of scatter, with several specimens having pronounced left-biased asymmetry. We suspect that this dramatic change was due to a combination of ontogenetic niche shift, bigger home ranges, a greater susceptibility to negative anthropogenic influences, and/or a male bias in the bigger specimens sampled, but a larger sample size is required to determine if there is statistical significance in these intra-specific trends. Body asymmetry can reflect brain laterality, which may be a potential driver for the teeth asymmetry seen here.

Journal of Developmental Biology doi: 10.3390/jdb13010008

Authors: Vladimira Foteva Joshua J. Fisher Yixue Qiao Roger Smith

Molybdenum is an essential trace element sourced during pregnancy from the maternal diet. Studies regarding molybdenum have primarily focused on overexposure in animal and cell culture studies. The effects of molybdenum supplementation on placental function are unknown. An immortalised trophoblast cell line was used to examine the placental cellular response to molybdenum in its bioavailable form as molybdate. Cells of the extravillous trophoblast first-trimester cell line HTR8-SVneo were cultured in complete cell media in the presence of 10 nM to 1 mM of ammonium molybdate or sodium molybdate. Following the addition of the molybdate salts, cell growth, viability, and several gene pathways were monitored. Sodium molybdate salt in doses from 10 nM to 1 mM did not affect cell growth or viability. Exposure to ammonium molybdate at a 1 mM concentration significantly decreased cell growth and viability (p < 0.05). Gene pathways involving molybdoenzyme expression, molybdenum cofactor synthesis, antioxidant response, and angiogenesis were affected following supplementation, although these effects differed depending on the dose and molybdate salt utilised. Molybdoenzyme activity was not affected by supplementation in a dose-dependent manner. The results indicate sodium molybdate is a more appropriate salt to use in vitro, as ammonium molybdate exposure reduced cell viability and growth and downregulated the expression of antioxidant genes NFE2L2 (p < 0.01), SOD1 (p < 0.001) and SOD2 (p < 0.001), suggestive of an inflammatory response. Sodium molybdate affected gene, protein, and activity levels of molybdoenzyme, antioxidant, and angiogenic molecules in vitro. This work demonstrates that sodium molybdate supplementation has pleiotropic effects in vitro and is well tolerated by placental cells at a range of nanomolar and micromolar concentrations.

Journal of Developmental Biology doi: 10.3390/jdb13010007

Authors: Daisy (Jihyung) Ko Tess Kelly Lacey Thompson Jasmene K. Uppal Nasim Rostampour Mark Adam Webb Ning Zhu George Belev Prosanta Mondal David M. L. Cooper Julia C. Boughner

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Journal of Developmental Biology doi: 10.3390/jdb13010006

Authors: Jaromír Vašíček Andrej Baláži Mária Tirpáková Marián Tomka Peter Chrenek

Cryopreservation is a widely used method for the long-term preservation of reproductive or somatic cells. It is known that this storage method may negatively affect cell viability, proliferation, differentiation, etc. However, there is a lack of information about whether cryostorage can alter the content of intracellular minerals. Therefore, we focused this study on the analysis of the mineral composition of living cells before and after long-term cold storage. Briefly, three different primary cell lines were established from rabbits as follows: endothelial progenitor cells from peripheral blood (EPCs), endothelial progenitor cells from bone marrow (BEPCs), and mesenchymal stem cells from adipose tissue (AT-MSCs), which were cultured until passage 3 prior to cryopreservation in liquid nitrogen. Samples from freshly cultured and frozen–thawed cells were mineralized and analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES) for the content of minerals (macro: Ca, Na, K, and Mg, and micro: Zn, Fe, Cu, Al, Co, Mn, Sr, and Ni). After cryopreservation, we found significantly decreased content of K in frozen–thawed EPCs (p < 0.01) and BEPCs (p < 0.0001) and Ca in AT-MSCs (p < 0.05), while Na was increased in frozen–thawed BEPCs (p < 0.05). Concentrations of Fe and Al were reduced significantly in frozen–thawed EPCs (both p < 0.0001) and AT-MSCs (p < 0.001 and p < 0.0001, respectively). On the contrary, Fe and Al were elevated in frozen–thawed BEPCs (p < 0.0001 and p < 0.01, respectively) together with Ni (p < 0.0001). In addition, decreased Zn (p < 0.05) was observed in cryopreserved AT-MSCs. In conclusion, the ICP-OES technique might be used to analyze the basic elemental composition of animal cells in fresh or frozen–thawed conditions. Nevertheless, additional studies are needed to reveal the possible impact of cryopreservation on cell fate by changing the content of intracellular minerals.

Journal of Developmental Biology doi: 10.3390/jdb13010005

Authors: Mohamed I. Elashry Victoria C. Schneider Manuela Heimann Sabine Wenisch Stefan Arnhold

Skeletal muscle plays a pivotal role in physical activity, protein storage and energy utilization. Skeletal muscle wasting due to immobilization, aging, muscular dystrophy and cancer cachexia has negative impacts on the quality of life. The deletion of myostatin, a growth and differentiation factor-8 (GDF-8) augments muscle mass through hyperplasia and hypertrophy of muscle fibers. The present study examines the impact of myostatin deletion using CRISPR/Cas9 editing on the myogenic differentiation (MD) of C2C12 muscle stem cells. A total of five myostatin loci were targeted using guided RNAs that had been previously cloned into a vector. The clones were transfected in C2C12 cells via electroporation. The cell viability and MD of myostatin-edited clones (Mstn−/−) were compared with C2C12 (Mstn+/+) using a series of assays, including MTT, sulforhodamine B, immunocytochemistry, morphometric analysis and RT-qPCR. The clones sequenced showed evidence of nucleotides deletion in Mstn−/− cells. Mstn−/− cells demonstrated a normal physiological performance and lack of cytotoxicity. Myostatin depletion promoted the myogenic commitment as evidenced by upregulated MyoD and myogenin expression. The number of MyoD-positive cells was increased in the differentiated Mstn−/− clones. The Mstn−/− editing upregulates both mTOR and MyH expression, as well as increasing the size of myotubes. The differentiation of Mstn−/− cells upregulates ActRIIb; in contrast, it downregulates decorin expression. The data provide evidence of successful CRISPR/Cas9-mediated myostatin deletion. In addition, targeting myostatin could be a beneficial therapeutic strategy to promote MD and to restore muscle loss. In conclusion, the data suggest that myostatin editing using CRISPR/Cas9 could be a potential therapeutic manipulation to improve the regenerative capacity of muscle stem cells before in vivo application.

Journal of Developmental Biology doi: 10.3390/jdb13010004

Authors: Sofia M. Perez Helena S. Augustineli Matthew R. Marcello

The nematode C. elegans is a proven model for identifying genes involved in human disease, and the study of C. elegans reproduction, specifically spermatogenesis and fertilization, has led to significant contributions to our understanding of cellular function. Approximately 70 genes have been identified in C. elegans that control spermatogenesis and fertilization (spe and fer mutants). This review focuses on eight genes that have human orthologs with known pathogenic phenotypes. Using C. elegans to study these genes has led to critical developments in our understanding of protein domain function and human disease, including understanding the role of OTOF (the ortholog of C. elegans fer-1) in hearing loss, the contribution of the spe-39 ortholog VIPAS39 in vacuolar protein sorting, and the overlapping functions of spe-26 and KLHL10 in spermatogenesis. We discuss the cellular function of both the C. elegans genes and their human orthologs and the impact that C. elegans mutants and human variants have on cellular function and physiology. Utilizing C. elegans to understand the function of the genes reviewed here, and additional understudied and undiscovered genes, represents a unique opportunity to understand the function of variants that could lead to better disease diagnosis and clinical decision making.

Journal of Developmental Biology doi: 10.3390/jdb13010003

Authors: Sunjidmaa Zolzaya Dai Ihara Munkhsoyol Erkhembaatar Shinsuke Ochiai Ayaka Isa Mariko Nishibe Jean-Pierre Bellier Takahiro Shimizu Satoshi Kikkawa Ryo Nitta Yu Katsuyama

Human genome studies have suggested that strawberry notch homologue 1 (SBNO1) is crucial for normal brain development, with mutations potentially contributing to neurodevelopmental disorders. In a previous study, we observed significant developmental abnormalities in the neocortex of Sbno1 as early as one week after birth. In the present study, we conducted an extensive analysis of Sbno1 postnatal expression in the brain of C57BL/6 mice using a newly developed in-house polyclonal antibody against Sbno1. We found that Sbno1 is expressed in all neurons, with certain neuronal populations exhibiting distinct dynamic changes (both temporal and spatial) in expression level. These findings suggest that the neuronal expression of Sbno1 is developmentally regulated after birth. They also indicate that while Sbno1 may play a general role across all neurons, it may also serve more specialized functions in certain neuronal types and/or for certain cellular activities related to particular neuronal pathways.

Journal of Developmental Biology doi: 10.3390/jdb13010002

Authors: Lorenzo Alibardi

The present, brief review paper summarizes previous studies on a new interpretation of the presence and absence of regeneration in invertebrates and vertebrates. Broad regeneration is considered exclusive of aquatic or amphibious animals with larval stages and metamorphosis, where also a patterning process is activated for whole-body regeneration or for epimorphosis. In contrast, terrestrial invertebrates and vertebrates can only repair injury or the loss of body parts through a variable “recovery healing” of tissues, regengrow or scarring. This loss of regeneration likely derives from the change in genomes during land adaptation, which included the elimination of larval stages and intense metamorphosis. The terrestrial conditions are incompatible with the formation of embryonic organs that are necessary for broad regeneration. In fact, no embryonic organ can survive desiccation, intense UV or ROS exposition on land, and rapid reparative processes without embryonic patterning, such as recovery healing and scarring, have replaced broad regeneration in terrestrial species. The loss of regeneration in land animals likely depends on the alteration of developmental gene pathways sustaining regeneration that occurred in progenitor marine animals. Terrestrial larval stages, like those present in insects among arthropods, only metamorphose using small body regions indicated as imaginal disks, a terrestrial adaptation, not from a large restructuring process like in aquatic-related animals. These invertebrates can reform body appendages only during molting, a process indicated as regengrow, not regeneration. Most amniotes only repair injuries through scarring or a variable recovery healing, occasionally through regengrow, the contemporaneous healing in conjunction with somatic growth, forming sometimes new heteromorphic organs.

Journal of Developmental Biology doi: 10.3390/jdb13010001

Authors: Mirco Galiè

Since its first conceptualization over a century ago, the mesenchymal phenotype has traditionally been viewed as either a transient phase between successive epithelial stages or as a feature of cell types primarily devoted to structural support. However, recent findings in cancer research challenge this limited view, demonstrating that mesenchymal traits and hybrid mesenchymal/epithelial states can mark cancer cells with stem cell properties. By analyzing publicly available single-cell transcriptome datasets from early embryonic stages and adult tissues, this study aims to extend this concept beyond pathological contexts, suggesting that a partial or fully mesenchymal phenotype may represent the morphological expression of undifferentiated and multipotent states in both the developing embryo and adult organs.

Journal of Developmental Biology doi: 10.3390/jdb12040034

Authors: John P. Marquart Qian Nie Tessa Gonzalez Angie C. Jelin Ulrich Broeckel Amy J. Wagner Honey V. Reddi

(1) Background: The exact etiology for gastroschisis, the most common abdominal defect, is yet to be known, despite the rising prevalence of this condition. The leading theory suggests an increased familial risk, indicating a possible genetic component possibly in the context of environmental risk factors. This systematic review aims to summarize the studies focused on the identification of a potential genetic etiology for gastroschisis to elucidate the status of the field. (2) Methods: Following the PRISMA-ScR method, Pubmed and Google Scholar were searched, and eligible publications were mined for key data fields such as study aims, cohort demographics, technologies used, and outcomes in terms of genes identified. Data from 14 human studies, with varied cohort sizes from 40 to 1966 individuals for patient vs. healthy controls, respectively, were mined to delineate the technologies evaluated. (3) Results: Our results continue the theory that gastroschisis is likely caused by gene–environment interactions. The 14 studies utilized traditional methodologies that may not be adequate to identify genetic involvement in gastroschisis. (4) Conclusions: The etiology of gastroschisis continues to remain elusive. A combination of omics and epigenetic evaluation studies would help delineate a possible genetic etiology for gastroschisis.

Journal of Developmental Biology doi: 10.3390/jdb12040033

Authors: Katrin Domsch

Gene regulation depends on the interaction between chromatin-associated factors, such as transcription factors (TFs), which promote chromatin loops to ensure tight contact between enhancer and promoter regions. So far, positive interactions that lead to gene activation have been the main focus of research, but regulations related to blocking or inhibiting factor binding are also essential to maintaining a defined cellular status. To understand these interactions in greater detail, I investigated the possibility of the muscle differentiation factor Mef2 to prevent early Hox factor binding, leading to the proper timing of regulatory processes and the activation of differentiation events. My investigations relied on a collection of publicly available genome-wide binding data sets of Mef2 and Ubx (as the Hox factor), Capture-C interactions, and ATAC-seq analysis in Mef2 mutant cells. The analysis indicated that Mef2 can form possible chromatin loops to Ubx-bound regions. These regions contain low-affinity Ubx binding sites, and the chromatin architecture is independent of Mef2’s function. High levels of Ubx may disrupt the loops and allow specific Ubx bindings to regulate defined targets. In summary, my investigations highlight that the use of many publicly available data sets enables computational approaches to make robust predictions and, for the first time, suggest a molecular function of Mef2 as a preventer of Hox binding, indicating that it may act as a timer for muscle differentiation.

Journal of Developmental Biology doi: 10.3390/jdb12040032

Authors: Paige L. Snider Elizabeth A. Sierra Potchanant Catalina Matias Donna M. Edwards Jeffrey J. Brault Simon J. Conway

Barth syndrome (BTHS) is a rare, infantile-onset, X-linked mitochondriopathy exhibiting a variable presentation of failure to thrive, growth insufficiency, skeletal myopathy, neutropenia, and heart anomalies due to mitochondrial dysfunction secondary to inherited TAFAZZIN transacetylase mutations. Although not reported in BTHS patients, male infertility is observed in several Tafazzin (Taz) mouse alleles and in a Drosophila mutant. Herein, we examined the male infertility phenotype in a BTHS-patient-derived D75H point-mutant knockin mouse (TazPM) allele that expresses a mutant protein lacking transacetylase activity. Neonatal and adult TazPM testes were hypoplastic, and their epididymis lacked sperm. Histology and biomarker analysis revealed TazPM spermatogenesis is arrested prior to sexual maturation due to an inability to undergo meiosis and the generation of haploid spermatids. Moreover, TazPM testicular mitochondria were found to be structurally abnormal, and there was an elevation of p53-dependent apoptosis within TazPM seminiferous tubules. Immunoblot analysis revealed that TazPM gamete genome integrity was compromised, and both histone γ-H2Ax and Nucleoside diphosphate kinase-5 protein expression were absent in juvenile TazPM testes when compared to controls. We demonstrate that Taz-mediated transacetylase activity is required within mitochondria for normal spermatogenesis, and its absence results in meiotic arrest. We hypothesize that elevated TazPM spermatogonial apoptosis causes azoospermia and complete infertility.

Journal of Developmental Biology doi: 10.3390/jdb12040031

Authors: Fumiko Yamamoto Takeshi Yokoyama Yan Su Masataka G. Suzuki

The classic model of sex determination in insects suggests that they do not have sex hormones and that sex is determined in a cell-autonomous manner. On the other hand, there is accumulating evidence that the development of secondary sexual traits is controlled in a non-cell-autonomous manner through external factors. To evaluate the degrees of the cell-autonomous and non-cell-autonomous regulation of secondary sexual trait development, we analyzed the dynamics of the sexually dimorphic transcriptome in gynandromorphic individuals of the mo mutant strain in the silkworm Bombyx mori. The silkworm possesses a female heterogametic sex-determination system (ZZ = male/ZW = female), where the master regulatory gene for femaleness, Feminizer (Fem), is located in the W chromosome. As a secondary sexual trait, we focused on the fat body, which shows remarkable differences between the sexes during the last instar larval stage. A comparison of the transcriptomes between the fat bodies of male and female larvae identified 232 sex-differentially expressed genes (S-DEGs). The proportions of ZZ and ZW cells constituting the fat body of the gynandromorphic larvae were calculated according to the expression level of the Fem. Based on the obtained values, the expression level of each S-DEG was estimated, assuming that the levels of S-DEG expression were determined according to the proportion of ZZ and ZW cells. The estimated expression levels of 207 out of 232 S-DEGs were strongly correlated with the corresponding S-DEG expression level of the gynandromorphic fat body, determined by RNA-seq. These results strongly suggest that most of the sexually dimorphic transcriptome in the fat body is regulated in a cell-autonomous manner.

Journal of Developmental Biology doi: 10.3390/jdb12040030

Authors: Daniel Halloran Venu Pandit Kelechi Chukwuocha Anja Nohe

During aging, disruptions in various signaling pathways become more common. Some older patients will exhibit irregular bone morphogenetic protein (BMP) signaling, which can lead to osteoporosis (OP)—a debilitating bone disease resulting from an imbalance between osteoblasts and osteoclasts. In 2002, the Food and Drug Administration (FDA) approved recombinant human BMP-2 (rhBMP-2) for use in spinal fusion surgeries as it is required for bone formation. However, complications with rhBMP-2 arose and primary osteoblasts from OP patients often fail to respond to BMP-2. Although patient samples are available for study, previous medical histories can impact results. Consequently, the C57BL/6 mouse line serves as a valuable model for studying OP and aging. We find that BMP receptor type Ia (BMPRIa) is upregulated in the bone marrow stromal cells (BMSCs) of 15-month-old mice, consistent with prior data. Furthermore, conjugating BMP-2 with Quantum Dots (QDot®s) allows effective binding to BMPRIa, creating a fluorescent tag for BMP-2. Furthermore, after treating BMSCs with methyl-β-cyclodextrin (MβCD), a disruptor of cellular endocytosis, BMP signaling is restored in 15-month-old mice, as shown by von Kossa assays. MβCD has the potential to restore BMPRIa function, and the BMP signaling pathway offers a promising avenue for future OP therapies.

Journal of Developmental Biology doi: 10.3390/jdb12040029

Authors: Kai Kitamura Kyoko Saito Takeshi Homma Aimi Fuyuki Sawa Onouchi Shouichiro Saito

Prosaposin is a glycoprotein widely conserved in vertebrates, and it acts as a precursor for saposins that accelerate hydrolysis in lysosomes or acts as a neurotrophic factor without being processed into saposins. Neurogenesis in the olfactory neuroepithelia, including the olfactory epithelium (OE) and the vomeronasal epithelium (VNE), is known to occur throughout an animal’s life, and mature olfactory neurons (ORNs) and vomeronasal receptor neurons (VRNs) have recently been revealed to express prosaposin in the adult olfactory organ. In this study, the expression of prosaposin in the rat olfactory organ during postnatal development was examined. In the OE, prosaposin immunoreactivity was observed in mature ORNs labeled using olfactory marker protein (OMP) from postnatal day (P) 0. Immature ORNs showed no prosaposin immunoreactivity throughout the examined period. In the VNE, OMP-positive VRNs were mainly observed in the basal region of the VNE on P10 and showed an adult-like distribution from P20. On the other hand, prosaposin immunoreactivity was observed in VRNs from P0, suggesting that not only mature VRNs but also immature VRNs express prosaposin. This study raises the possibility that prosaposin is required for the normal development of the olfactory organ and has different roles in the OE and the VNE.

Journal of Developmental Biology doi: 10.3390/jdb12040028

Authors: Venera Nikolova Maya Markova Ralitsa Zhivkova Irina Chakarova Valentina Hadzhinesheva Stefka Delimitreva

Oocyte meiotic maturation includes large-scale chromatin remodeling as well as cytoskeleton and nuclear envelope rearrangements. This review addresses the dynamics of key cytoskeletal proteins (tubulin, actin, vimentin, and cytokeratins) and nuclear envelope proteins (lamin A/C, lamin B, and the nucleoporin Nup160) in parallel with chromatin reorganization in maturing mouse oocytes. A major feature of this reorganization is the concentration of heterochromatin into a spherical perinucleolar rim called surrounded nucleolus or karyosphere. In early germinal vesicle (GV) oocytes with non-surrounded nucleolus (without karyosphere), lamins and Nup160 are at the nuclear envelope while cytoplasmic cytoskeletal proteins are outside the nucleus. At the beginning of karyosphere formation, lamins and Nup160 follow the heterochromatin relocation assembling a new spherical structure in the GV. In late GV oocytes with surrounded nucleolus (fully formed karyosphere), the nuclear envelope gradually loses its integrity and cytoplasmic cytoskeletal proteins enter the nucleus. At germinal vesicle breakdown, lamin B occupies the karyosphere interior while all the other proteins stay at the karyosphere border or connect to chromatin. In metaphase oocytes, lamin A/C surrounds the spindle, Nup160 localizes to its poles, actin and lamin B are attached to the spindle fibers, and cytoplasmic intermediate filaments associate with both the spindle fibers and the metaphase chromosomes.

Journal of Developmental Biology doi: 10.3390/jdb12040027

Authors: Samuel Liu Kellianne D. Alexander Michael M. Francis

As nervous systems mature, neural circuit connections are reorganized to optimize the performance of specific functions in adults. This reorganization of connections is achieved through a remarkably conserved phase of developmental circuit remodeling that engages neuron-intrinsic and neuron-extrinsic molecular mechanisms to establish mature circuitry. Abnormalities in circuit remodeling and maturation are broadly linked with a variety of neurodevelopmental disorders, including autism spectrum disorders and schizophrenia. Here, we aim to provide an overview of recent advances in our understanding of the molecular processes that govern neural circuit remodeling and maturation. In particular, we focus on intriguing mechanistic insights gained from invertebrate systems, such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. We discuss how transcriptional control mechanisms, synaptic activity, and glial engulfment shape specific aspects of circuit remodeling in worms and flies. Finally, we highlight mechanistic parallels across invertebrate and mammalian systems, and prospects for further advances in each.

Journal of Developmental Biology doi: 10.3390/jdb12040026

Authors: Ling Sun Yan Wang Mo Yang Zhuang-Ju Xu Juan Miao Ying Bai Tao Lin

The purpose of this study was to investigate the influence of blastocyst formation timing on the quality of porcine embryos derived from parthenogenetic activation. Newly formed blastocysts at days 6, 7, and 8 of culture [termed formation 6, 7, and 8 blastocysts (F6, F7, and F8 blastocysts)] were obtained, and a series of parameters related to the quality of blastocysts, including apoptosis incidents, DNA replication, pluripotent factors, and blastocyst hatching capacity, were assessed. Delayed blastocyst formation (F7 and/or F8 blastocysts) led to increased levels of ROS, DNA damage, and apoptosis while decreasing the mitochondrial membrane potential, DNA replication, Oct4 levels, and numbers of Sox2-positive cells. F7 blastocysts showed a significantly reduced hatching rate compared to F6 blastocysts; however, F8 blastocysts were unable to develop to the hatching stage. Collectively, our findings suggest a negative correlation between delayed blastocyst formation and blastocyst quality.

Journal of Developmental Biology doi: 10.3390/jdb12030025

Authors: Aaron N. Johnson

Myofibers are highly specialized contractile cells of skeletal muscles, and dysregulation of myofiber morphogenesis is emerging as a contributing cause of myopathies and structural birth defects. Myotubes are the myofiber precursors and undergo a dramatic morphological transition into long bipolar myofibers that are attached to tendons on two ends. Similar to axon growth cones, myotube leading edges navigate toward target cells and form cell–cell connections. The process of myotube guidance connects myotubes with the correct tendons, orients myofiber morphology with the overall body plan, and generates a functional musculoskeletal system. Navigational signaling, addition of mass and volume, and identification of target cells are common events in myotube guidance and axon guidance, but surprisingly, the mechanisms regulating these events are not completely overlapping in myotubes and axons. This review summarizes the strategies that have evolved to direct myotube leading edges to predetermined tendon cells and highlights key differences between myotube guidance and axon guidance. The association of myotube guidance pathways with developmental disorders is also discussed.

Journal of Developmental Biology doi: 10.3390/jdb12030024

Authors: Jiaxin Yang Wenjing Sun Guizhong Cui

The NR2F family, including NR2F1, NR2F2, and NR2F6, belongs to the nuclear receptor superfamily. NR2F family members function as transcription factors and play essential roles in the development of multiple organs or tissues in mammals, including the central nervous system, veins and arteries, kidneys, uterus, and vasculature. In the central nervous system, NR2F1/2 coordinate with each other to regulate the development of specific brain subregions or cell types. In addition, NR2F family members are associated with various cancers, such as prostate cancer, breast cancer, and esophageal cancer. Nonetheless, the roles of the NR2F family in the development and diseases of the lung have not been systematically summarized. In this review, we mainly focus on the lung, including recent findings regarding the roles of the NR2F family in development, physiological function, and cancer.

Journal of Developmental Biology doi: 10.3390/jdb12030023

Authors: Nuwanthika Wathuliyadde Katherine E. Willmore Gregory M. Kelly

Craniometaphyseal Dysplasia (CMD) is a rare skeletal disorder that can result from mutations in the ANKH gene. This gene encodes progressive anksylosis (ANK), which is responsible for transporting inorganic pyrophosphate (PPi) and ATP from the intracellular to the extracellular environment, where PPi inhibits bone mineralization. When ANK is dysfunctional, as in patients with CMD, the passage of PPi to the extracellular environment is reduced, leading to excess mineralization, particularly in bones of the skull. Zebrafish may serve as a promising model to study the mechanistic basis of CMD. Here, we provide a detailed analysis of the zebrafish Ankh paralogs, Ankha and Ankhb, in terms of their phylogenic relationship with ANK in other vertebrates as well as their spatiotemporal expression patterns during zebrafish development. We found that a closer evolutionary relationship exists between the zebrafish Ankhb protein and its human and other vertebrate counterparts, and stronger promoter activity was predicted for ankhb compared to ankha. Furthermore, we noted distinct temporal expression patterns, with ankha more prominently expressed in early development stages, and both paralogs also being expressed at larval growth stages. Whole-mount in situ hybridization was used to compare the spatial expression patterns of each paralog during bone development, and both showed strong expression in the craniofacial region as well as the notochord and somites. Given the substantial overlap in spatiotemporal expression but only subtle patterning differences, the exact roles of these genes remain speculative. In silico analyses predicted that Ankha and Ankhb have the same function in transporting PPi across the membrane. Nevertheless, this study lays the groundwork for functional analyses of each ankh paralog and highlights the potential of using zebrafish to find possible targeted therapies for CMD.

Journal of Developmental Biology doi: 10.3390/jdb12030022

Authors: Anna Fazzio Angela Caponnetto Carmen Ferrara Michele Purrello Cinzia Di Pietro Rosalia Battaglia

Extracellular vesicles represent a large heterogeneous class of near and long-distance intercellular communication mediators, released by both prokaryotic and eukaryotic cells. Specifically, the scientific community has shown growing interest in exosomes, which are nano-sized vesicles with an endosomal origin. Not so long ago, the physiological goal of exosome generation was largely unknown and required more investigation; at first, it was hypothesized that exosomes are able to remove excess, reject and unnecessary constituents from cells to preserve cellular homeostasis. However, thanks to recent studies, the central role of exosomes in regulating cellular communication has emerged. Exosomes act as vectors in cell–cell signaling by their cargo, proteins, lipids, and nucleic acids, and influence physiological and pathological processes. The findings on exosomes are widespread in a large spectrum of biomedical applications from diagnosis and prognosis to therapies. In this review, we describe exosome biogenesis and the current methods for their isolation and characterization, emphasizing the role of their cargo in female reproductive processes, from gametogenesis to implantation, and the potential involvement in human female disorders.

Journal of Developmental Biology doi: 10.3390/jdb12030021

Authors: Cody J. Hatchett M. Kristen Hall Abel R. Messer Ruth A. Schwalbe

The attachment of sugar to proteins and lipids is a basic modification needed for organismal survival, and perturbations in glycosylation cause severe developmental and neurological difficulties. Here, we investigated the neurological consequences of N-glycan populations in the spinal cord of Wt AB and mgat1b mutant zebrafish. Mutant fish have reduced N-acetylglucosaminyltransferase-I (GnT-I) activity as mgat1a remains intact. GnT-I converts oligomannose N-glycans to hybrid N-glycans, which is needed for complex N-glycan production. MALDI-TOF MS profiles identified N-glycans in the spinal cord for the first time and revealed reduced amounts of complex N-glycans in mutant fish, supporting a lesion in mgat1b. Further lectin blotting showed that oligomannose N-glycans were more prevalent in the spinal cord, skeletal muscle, heart, swim bladder, skin, and testis in mutant fish relative to WT AB, supporting lowered GnT- I activity in a global manner. Developmental delays were noted in hatching and in the swim bladder. Microscopic images of caudal primary (CaP) motor neurons of the spinal cord transiently expressing EGFP in mutant fish were abnormal with significant reductions in collateral branches. Further motor coordination skills were impaired in mutant fish. We conclude that identifying the neurological consequences of aberrant N-glycan processing will enhance our understanding of the role of complex N-glycans in development and nervous system health.

Journal of Developmental Biology doi: 10.3390/jdb12030020

Authors: De-Li Shi

The formation of embryonic axes is a critical step during animal development, which contributes to establishing the basic body plan in each particular organism. Wnt signaling pathways play pivotal roles in this fundamental process. Canonical Wnt signaling that is dependent on β-catenin regulates the patterning of dorsoventral, anteroposterior, and left–right axes. Non-canonical Wnt signaling that is independent of β-catenin modulates cytoskeletal organization to coordinate cell polarity changes and asymmetric cell movements. It is now well documented that components of these Wnt pathways biochemically and functionally interact to mediate cell–cell communications and instruct cellular polarization in breaking the embryonic symmetry. The dysfunction of Wnt signaling disrupts embryonic axis specification and proper tissue morphogenesis, and mutations of Wnt pathway genes are associated with birth defects in humans. This review discusses the regulatory roles of Wnt pathway components in embryonic axis formation by focusing on vertebrate models. It highlights current progress in decoding conserved mechanisms underlying the establishment of asymmetry along the three primary body axes. By providing an in-depth analysis of canonical and non-canonical pathways in regulating cell fates and cellular behaviors, this work offers insights into the intricate processes that contribute to setting up the basic body plan in vertebrate embryos.

Journal of Developmental Biology doi: 10.3390/jdb12030019

Authors: Chihiro Iwaya Sunny Yu Junichi Iwata

Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.

Journal of Developmental Biology doi: 10.3390/jdb12030018

Authors: Luca Ambrosio Jordy Schol Clara Ruiz-Fernández Shota Tamagawa Kieran Joyce Akira Nomura Elisabetta de Rinaldis Daisuke Sakai Rocco Papalia Gianluca Vadalà Vincenzo Denaro

The intervertebral disc (IVD) is the largest avascular organ of the human body and plays a fundamental role in providing the spine with its unique structural and biomechanical functions. The inner part of the IVD contains the nucleus pulposus (NP), a gel-like tissue characterized by a high content of type II collagen and proteoglycans, which is crucial for the disc’s load-bearing and shock-absorbing properties. With aging and IVD degeneration (IDD), the NP gradually loses its physiological characteristics, leading to low back pain and additional sequelae. In contrast to surrounding spinal tissues, the NP presents a distinctive embryonic development since it directly derives from the notochord. This review aims to explore the embryology of the NP, emphasizing the pivotal roles of key transcription factors, which guide the differentiation and maintenance of the NP cellular components from the notochord and surrounding sclerotome. Through an understanding of NP development, we sought to investigate the implications of the critical developmental aspects in IVD-related pathologies, such as IDD and the rare malignant chordomas. Moreover, this review discusses the therapeutic strategies targeting these pathways, including the novel regenerative approaches leveraging insights from NP development and embryology to potentially guide future treatments.

Journal of Developmental Biology doi: 10.3390/jdb12020017

Authors: Fayhaa Khan Lenore Pitstick Jessica Lara Rosa Ventrella

Lung epithelial development relies on the proper balance of cell proliferation and differentiation to maintain homeostasis. When this balance is disturbed, it can lead to diseases like cancer, where cells undergo hyperproliferation and then can undergo migration and metastasis. Lung cancer is one of the deadliest cancers, and even though there are a variety of therapeutic approaches, there are cases where treatment remains elusive. The rho-associated protein kinase (ROCK) has been thought to be an ideal molecular target due to its role in activating oncogenic signaling pathways. However, in a variety of cases, inhibition of ROCK has been shown to have the opposite outcome. Here, we show that ROCK inhibition with y-27632 causes abnormal epithelial tissue development in Xenopus laevis embryonic skin, which is an ideal model for studying lung cancer development. We found that treatment with y-27632 caused an increase in proliferation and the formation of ciliated epithelial outgrowths along the tail edge. Our results suggest that, in certain cases, ROCK inhibition can disturb tissue homeostasis. We anticipate that these findings could provide insight into possible mechanisms to overcome instances when ROCK inhibition results in heightened proliferation. Also, these findings are significant because y-27632 is a common pharmacological inhibitor used to study ROCK signaling, so it is important to know that in certain in vivo developmental models and conditions, this treatment can enhance proliferation rather than lead to cell cycle suppression.

Journal of Developmental Biology doi: 10.3390/jdb12020016

Authors: Shiori Ikeda Keita Sato Hirofumi Fujita Hitomi Ono-Minagi Satoru Miyaishi Tsutomu Nohno Hideyo Ohuchi

The mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10+/− heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10+/− mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10+/− mice. We found that more than half of the Fgf10+/− mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10+/− embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period.

Journal of Developmental Biology doi: 10.3390/jdb12020015

Authors: Nicholas S. Tolwinski

In this Special Issue, titled “Drosophila—A Model System for Developmental Biology”, we present a series of articles and reviews looking at the diverse ways that researchers are using the humble fruit fly, also known as the vinegar fly, to tackle the many aspects of development and homeostasis [...]

Journal of Developmental Biology doi: 10.3390/jdb12020014

Authors: Raiane Cristina Fratini de Castro Tiago William Buranello Kaiana Recchia Aline Fernanda de Souza Naira Caroline Godoy Pieri Fabiana Fernandes Bressan

The generation of mature gametes and competent embryos in vitro from pluripotent stem cells has been successfully achieved in a few species, mainly in mice, with recent advances in humans and scarce preliminary reports in other domestic species. These biotechnologies are very attractive as they facilitate the understanding of developmental mechanisms and stages that are generally inaccessible during early embryogenesis, thus enabling advanced reproductive technologies and contributing to the generation of animals of high genetic merit in a short period. Studies on the production of in vitro embryos in pigs and cattle are currently used as study models for humans since they present more similar characteristics when compared to rodents in both the initial embryo development and adult life. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine.

Journal of Developmental Biology doi: 10.3390/jdb12020013

Authors: Kaiva Zile Zarina Mara Pilmane

Background: Little is known about morphogenetic changes in the umbilical cord during the maturation process. Extracellular matrix remodeling, angiogenesis, progenitor activity, and immunomodulation are represented by specific markers; therefore, the aim of this study was to determine the expression of matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases-2 (TIMP2), CD34, vascular endothelial growth factor (VEGF), and human β-defensin 2 (HBD2) in preterm and full-term human umbilical cord tissue. Methods: Samples of umbilical cord tissue were obtained from 17 patients and divided into two groups: very preterm and moderate preterm birth umbilical cords; late preterm birth and full-term birth umbilical cords. Routine histology examination was conducted. Marker-positive cells were detected using the immunohistochemistry method. The number of positive structures was counted semi-quantitatively using microscopy. Statistical analysis was carried out using the SPSS Statistics 29 program. Results: Extraembryonic mesenchyme cells are the most active cell producers, expressing MMP2, TIMP2, VEGF, and HBD2 at notable levels in preterm and full-term umbilical cord tissue. Statistically significant differences in the expression of CD34, MMP2, and TIMP2 between the two patient groups were found. The expression of VEGF was similar in both patient groups, with the highest number of VEGF-positive cells seen in the extraembryonic mesenchyme. The expression of HBD2 was the highest in the extraembryonic mesenchyme and the amniotic epithelium, where mostly moderate numbers of HBD2-positive cells were detected. Conclusions: Extracellular matrix remodeling in preterm and term umbilical cords is strongly regulated, and tissue factors MMP2 and TIMP2 take part in this process. The expression of VEGF is not affected by the umbilical cord’s age; however, individual patient factors can affect the production of VEGF. Numerous CD34-positive cells in the endothelium of the umbilical arteries suggest a significant role of progenitor cells in very preterm and moderate preterm birth umbilical cords. Antimicrobial activity provided by HBD2 is essential and constant in preterm and full-term umbilical cords.

Journal of Developmental Biology doi: 10.3390/jdb12020012

Authors: Sandeep Kacker Varuneshwar Parsad Naveen Singh Daria Hordiichuk Stacy Alvarez Mahnoor Gohar Anshu Kacker Sunil Kumar Rai

The planar cell polarity (PCP) system is essential for positioning cells in 3D networks to establish the proper morphogenesis, structure, and function of organs during embryonic development. The PCP system uses inter- and intracellular feedback interactions between components of the core PCP, characterized by coordinated planar polarization and asymmetric distribution of cell populations inside the cells. PCP signaling connects the anterior–posterior to left–right embryonic plane polarity through the polarization of cilia in the Kupffer’s vesicle/node in vertebrates. Experimental investigations on various genetic ablation-based models demonstrated the functions of PCP in planar polarization and associated genetic disorders. This review paper aims to provide a comprehensive overview of PCP signaling history, core components of the PCP signaling pathway, molecular mechanisms underlying PCP signaling, interactions with other signaling pathways, and the role of PCP in organ and embryonic development. Moreover, we will delve into the negative feedback regulation of PCP to maintain polarity, human genetic disorders associated with PCP defects, as well as challenges associated with PCP.

Journal of Developmental Biology doi: 10.3390/jdb12020011

Authors: Sophia F. Ehlers Dominique Manikowski Georg Steffes Kristina Ehring Fabian Gude Kay Grobe

During their biosynthesis, Sonic hedgehog (Shh) morphogens are covalently modified by cholesterol at the C-terminus and palmitate at the N-terminus. Although both lipids initially anchor Shh to the plasma membrane of producing cells, it later translocates to the extracellular compartment to direct developmental fates in cells expressing the Patched (Ptch) receptor. Possible release mechanisms for dually lipidated Hh/Shh into the extracellular compartment are currently under intense debate. In this paper, we describe the serum-dependent conversion of the dually lipidated cellular precursor into a soluble cholesteroylated variant (ShhC) during its release. Although ShhC is formed in a Dispatched- and Scube2-dependent manner, suggesting the physiological relevance of the protein, the depalmitoylation of ShhC during release is inconsistent with the previously postulated function of N-palmitate in Ptch receptor binding and signaling. Therefore, we analyzed the potency of ShhC to induce Ptch-controlled target cell transcription and differentiation in Hh-sensitive reporter cells and in the Drosophila eye. In both experimental systems, we found that ShhC was highly bioactive despite the absence of the N-palmitate. We also found that the artificial removal of N-terminal peptides longer than eight amino acids inactivated the depalmitoylated soluble proteins in vitro and in the developing Drosophila eye. These results demonstrate that N-depalmitoylated ShhC requires an N-peptide of a defined minimum length for its signaling function to Ptch.

Journal of Developmental Biology doi: 10.3390/jdb12020010

Authors: Mengyao Zhao Gerile Subudeng Yufen Zhao Shaoyu Hao Haijun Li

Gap junctional connection (GJC) in the cumulus–oocyte complex (COC) provides necessary support for message communication and nutrient transmission required for mammalian oocyte maturation. Cyclic adenosine monophosphate (cAMP) is not only a prerequisite for regulating oocyte meiosis, but also the key intercellular factor for affecting GJC function in COCs. However, there are no reports on whether cAMP regulates connexin 37 (Cx37) expression, one of the main connexin proteins, in sheep COCs. In this study, the expression of Cx37 protein and gene in immature sheep COC was detected using immunohistochemistry and PCR. Subsequently, the effect of cAMP on Cx37 expression in sheep COCs cultured in a gonadotropin-free culture system for 10 min or 60 min was evaluated using competitive ELISA, real-time fluorescent quantitative PCR (RT-qPCR), and Western blot. The results showed that the Cx37 protein was present in sheep oocytes and cumulus cells; the same results were found with respect to GJA4 gene expression. In the gonadotropin-free culture system, compared to the control, significantly higher levels of cAMP as well as Cx37 gene and protein expression were found in sheep COCs following treatment in vitro with Forskolin and IBMX (100 μM and 500 μM)) for 10 min (p < 0.05). Compared to the controls (at 10 or 60 min), cAMP levels in sheep COCs were significantly elevated as a result of Forskolin and IBMX treatment (p < 0.05). Following culturing in vitro for 10 min or 60 min, Forskolin and IBMX treatment can significantly promote Cx37 expression in sheep COCs (p < 0.05), a phenomenon which can be counteracted when the culture media is supplemented with RP-cAMP, a cAMP-specific competitive inhibitor operating through suppression of the protein kinase A (PKA). In summary, this study reports the preliminary regulatory mechanism of cAMP involved in Cx37 expression for the first time, and provides a novel explanation for the interaction between cAMP and GJC communication during sheep COC culturing in vitro.

Journal of Developmental Biology doi: 10.3390/jdb12010009

Authors: Noopur Naik Mansi Patel Rwik Sen

Developmental biology is intricately regulated by epigenetics and metabolism but the mechanisms are not completely understood. The situation becomes even more complicated during diseases where all three phenomena are dysregulated. A salient example is COVID-19, where the death toll exceeded 6.96 million in 4 years, while the virus continues to mutate into different variants and infect people. Early evidence during the pandemic showed that the host’s immune and inflammatory responses to COVID-19 (like the cytokine storm) impacted the host’s metabolism, causing damage to the host’s organs and overall physiology. The involvement of angiotensin-converting enzyme 2 (ACE2), the pivotal host receptor for the SARS-CoV-2 virus, was identified and linked to epigenetic abnormalities along with other contributing factors. Recently, studies have revealed stronger connections between epigenetics and metabolism in COVID-19 that impact development and accelerate aging. Patients manifest systemic toxicity, immune dysfunction and multi-organ failure. Single-cell multiomics and other state-of-the-art high-throughput studies are only just beginning to demonstrate the extent of dysregulation and damage. As epigenetics and metabolism directly impact development, there is a crucial need for research implementing cutting-edge technology, next-generation sequencing, bioinformatics analysis, the identification of biomarkers and clinical trials to help with prevention and therapeutic interventions against similar threats in the future.

Journal of Developmental Biology doi: 10.3390/jdb12010008

Authors: Lorenzo Alibardi

The present brief manuscript summarizes the main points supporting recently proposed hypotheses explaining the different distributions of regenerative capacity among invertebrates and vertebrates. The new hypotheses are based on the evolution of regeneration from marine animals to the terrestrial animals derived from them. These speculations suggest that animals that were initially capable of broad regeneration in the sea underwent epigenetic modifications during terrestrial adaptation that determined the loss of their regenerative abilities in sub-aerial conditions. These changes derived from the requirements of life on land that include variable dry and UV-exposed conditions. Terrestrial conditions do not allow for organ regeneration, especially in arthropods and amniotes. Nematodes, the other main metazoan group unable of regeneration, instead evolved eutely (a fixed number of body cells), a process which is incompatible with regeneration. All these changes involved gene loss, modification and new gene interactions within the genomes of terrestrial adapting animals that gave rise to sophisticated invertebrates and vertebrates adapted to living on land but with low cellular plasticity.

Journal of Developmental Biology doi: 10.3390/jdb12010007

Authors: Hiromasa Hasegawa Katsumitsu Shimada Takanaga Ochiai Yasuo Okada

Hyperplastic dental follicles (HDFs) represent odontogenic hamartomatous lesions originating from the pericoronal tissues and are often associated with impacted or embedded teeth. These lesions may occasionally feature unique calcifying bodies, known as calcifying whorled nodules (CWNs), characterized by stromal cells arranged in a whorled or spiral fashion. CWNs are typically observed in multiple calcifying hyperplastic dental follicles or regional odontodysplasia. In our study, we examined 40 cases of HDFs, including nine instances with characteristics of CWNs, referred to as calcifying hyperplastic dental follicles (CHDFs), which are infrequently accompanied by odontodysplasia. The median ages of the HDFs and CHDFs were 16 (ranging from 3 to 66) and 15 (ranging from 11 to 50) years, respectively. The lower third molars were the most frequently affected by HDSFs and CHDFs, followed by the upper canines. A histological examination was conducted on all 40 cases, with an immunohistochemical analysis performed on 21 of them. Among the cases with CWN, nine affected a single embedded tooth, with one exception. CWNs exhibited diverse calcifications featuring sparse or entirely deposited psammoma bodies, and some displayed dentinoid formation. Immunohistochemically, the stromal cells of HDFs were frequently positive for CD56 and nestin. By contrast, CWNs were negative for CD56 but positive for nestin as well as hairy and enhancer split 1 (HES1), with a few dentin sialoprotein (DSP)-positive calcified bodies. Our results revealed that hamartomatous CHDFs can impact multiple and single-embedded teeth. CWNs composed of nestin and HES1-positive ectomesenchymal cells demonstrated the potential to differentiate into odontoblasts and contribute to dentin matrix formation under the influence of HES1. This study is the first report documenting odontoblastic differentiation in HDFs. The rare occurrence of HDFs and CHDFs contributes to limited comprehension. To prevent misdiagnosis, a better understanding of these conditions is necessary.

Journal of Developmental Biology doi: 10.3390/jdb12010006

Authors: Evgeniya Kananykhina Andrey Elchaninov Galina Bolshakova

A characteristic feature of repair processes in mammals is the formation of scar tissue at the site of injury, which is designed to quickly prevent contact between the internal environment of the organism and the external environment. Despite this general pattern, different organs differ in the degree of severity of scar changes in response to injury. One of the areas in which regeneration after wounding leads to the formation of a structure close to the original one is the abdominal skin of laboratory rats. Finding out the reasons for such a phenomenon is essential for the development of ways to stimulate full regeneration. The model of skin wound healing in the abdominal region of laboratory animals was reproduced in this work. It was found that the wound surface is completely epithelialized on the abdomen by 20 days, while on the back—by 30 days. The qPCR method revealed higher expression of marker genes of skin stem cells (Sox9, Lgr6, Gli1, Lrig1) in the intact skin of the abdomen compared to the back, which corresponded to a greater number of hairs with which stem cells are associated on the abdomen compared to the back. Considering that some stem cell populations are associated with hair, it can be suggested that one of the factors in faster regeneration of abdominal skin in the rat is the greater number of stem cells in this area.

Journal of Developmental Biology doi: 10.3390/jdb12010005

Authors: Stephania A. Cormier Claudia Kappen

Hox genes encode transcription factors whose roles in patterning animal body plans during embryonic development are well-documented. Multiple studies demonstrate that Hox genes continue to act in adult cells, in normal differentiation, in regenerative processes, and, with abnormal expression, in diverse types of cancers. However, surprisingly little is known about the regulatory mechanisms that govern Hox gene expression in specific cell types, as they differentiate during late embryonic development, and in the adult organism. The murine Hoxc8 gene determines the identity of multiple skeletal elements in the lower thoracic and lumbar region and continues to play a role in the proliferation and differentiation of cells in cartilage as the skeleton matures. This study was undertaken to identify regulatory elements in the Hoxc8 gene that control transcriptional activity, specifically in cartilage-producing chondrocytes. We report that an enhancer comprising two 416 and 224 bps long interacting DNA elements produces reporter gene activity when assayed on a heterologous transcriptional promoter in transgenic mice. This enhancer is distinct in spatial, temporal, and molecular regulation from previously identified regulatory sequences in the Hoxc8 gene that control its expression in early development. The identification of a tissue-specific Hox gene regulatory element now allows mechanistic investigations into Hox transcription factor expression and function in differentiating cell types and adult tissues and to specifically target these cells during repair processes and regeneration.

Journal of Developmental Biology doi: 10.3390/jdb12010004

Authors: Karin Brigit Holthaus Leopold Eckhart

The epidermal differentiation complex (EDC) is a cluster of genes that encode protein components of the outermost layers of the epidermis in mammals, reptiles and birds. The development of the stratified epidermis from a single-layered ectoderm involves an embryo-specific superficial cell layer, the periderm. An additional layer, the subperiderm, develops in crocodilians and over scutate scales of birds. Here, we review the expression of EDC genes during embryonic development. Several EDC genes are expressed predominantly or exclusively in embryo-specific cell layers, whereas others are confined to the epidermal layers that are maintained in postnatal skin. The S100 fused-type proteins scaffoldin and trichohyalin are expressed in the avian and mammalian periderm, respectively. Scaffoldin forms the so-called periderm granules, which are histological markers of the periderm in birds. Epidermal differentiation cysteine-rich protein (EDCRP) and epidermal differentiation protein containing DPCC motifs (EDDM) are expressed in the avian subperiderm where they are supposed to undergo cross-linking via disulfide bonds. Furthermore, a histidine-rich epidermal differentiation protein and feather-type corneous beta-proteins, also known as beta-keratins, are expressed in the subperiderm. The accumulating evidence for roles of EDC genes in the development of the epidermis has implications on the evolutionary diversification of the skin in amniotes.