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Article

Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System

by
Elzava Yuslimatin Mujizah
1,
Satoshi Kuwana
2,
Kenjiroo Matsumoto
3,
Takuma Gushiken
1,
Naoki Aoyama
1,
Hiroyuki O. Ishikawa
4,
Takeshi Sasamura
1,
Daiki Umetsu
1,
Mikiko Inaki
5,
Tomoko Yamakawa
6,*,
Martin Baron
7,* and
Kenji Matsuno
1,*
1
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka 560-0043, Japan
2
Graduate School of Arts and Sciences, University of Tokyo, Meguro 153-8902, Japan
3
Institute for Glyco-Core Research, Gifu University, Gifu 501-1193, Japan
4
Graduate School of Science, Chiba University, Chiba 263-8522, Japan
5
School of Science, Graduate School of Science, University of Hyogo, Ako 678-1297, Japan
6
Department of Industrial Engineering, Chemistry, Bioengineering and Environmental Science Course, National Institute of Technology, Ibaraki College, Hitachinaka 312-8508, Japan
7
School of Biological Sciences, Manchester Academic Health Science Centre, University of Manchester, Manchester M13 9PL, UK
*
Authors to whom correspondence should be addressed.
Biomolecules 2024, 14(9), 1062; https://doi.org/10.3390/biom14091062
Submission received: 17 June 2024 / Revised: 7 August 2024 / Accepted: 14 August 2024 / Published: 26 August 2024
(This article belongs to the Section Molecular Medicine)
Browse Figures
Review Reports Versions Notes

Abstract

:
The role of Drosophila numb in regulating Notch signaling and neurogenesis has been extensively studied, with a particular focus on its effects on the peripheral nervous system (PNS). Previous studies based on a single loss-of-function allele of numb, numb1, showed an antineurogenic effect on the peripheral nervous system (PNS), which revealed that the wild-type numb suppresses Notch signaling. In the current study, we examined whether this phenotype is consistently observed in loss-of-function mutations of numb. Two more numb alleles, numbEY03840 and numbEY03852, were shown to have an antineurogenic phenotype in the PNS. We also found that introducing a wild-type numb genomic fragment into numb1 homozygotes rescued their antineurogenic phenotype. These results demonstrated that loss-of-function mutations of numb universally induce this phenotype. Many components of Notch signaling are encoded by maternal effect genes, but no maternal effect of numb was observed in this study. The antineurogenic phenotype of numb was found to be dependent on the Enhancer of split (E(spl)), a downstream gene of Notch signaling. We found that the combination of E(spl) homozygous and numb1 homozygous suppressed the neurogenic phenotype of the embryonic central nervous system (CNS) associated with the E(spl) mutation. In the E(spl) allele, genes encoding basic helix-loop-helix proteins, such as m5, m6, m7, and m8, remain. Thus, in the E(spl) allele, derepression of Notch activity by numb mutation can rescue the neurogenic phenotype by increasing the expression of the remaining genes in the E(spl) complex. We also uncovered a role for numb in regulating neuronal projections. Our results further support an important role for numb in the suppression of Notch signaling during embryonic nervous system development.

1. Introduction

Cell signaling is critical to a variety of biological processes. The Notch-mediated cell signaling pathway is responsible for a wide range of functions, including cell fate determination and patterning, through direct cell-cell contact throughout the postnatal animal [1,2]. Thus, in humans, aberrant activity of Notch signaling leads to the development of various diseases [3]. The major steps in the cascade leading to the activation of Notch signaling have been elucidated. Notch and the two major ligand families, Delta and Serrate, are single transmembrane proteins [1,2]. Interaction of the extracellular domain of Notch with ligands presented on the surface of adjacent cells causes a conformational change in Notch [4]. These conformational changes make the negative regulatory region of the Notch extracellular domain susceptible to the proteolytic activity of Kuzbanian/ADAM10 [4,5,6,7]. This proteolytic cleavage then leads to another cleavage of the Notch transmembrane domain by γ-secretase, leading to the release of the Notch intracellular domain (NICD) [4,5,6,7,8]. NICD then translocates to the nucleus, where it acts as a co-activator of transcription and activates the expression of target genes such as Enhancer of split (E(spl)) [5,6,7]. The mechanisms of this signaling cascade and the factors that comprise and regulate the pathway are evolutionarily conserved [4,8].
Various other proteins have been identified as regulators of Notch signaling. These proteins modulate Notch signaling activity through, for example, glycosylation, transport, and degradation to fine-tune Notch activity in ways that are crucial for normal development and homeostasis [4,9,10,11]. The numb gene was first identified in Drosophila and is known to be involved in the development of the peripheral nervous system (PNS) [12,13], and Numb family proteins are evolutionarily conserved from Drosophila to mammals [12,14]. Numb has a conserved phosphotyrosine-binding (PTB) domain near its N-terminal region responsible for its membrane localization [15,16]. In the C-terminal region of Numb, there are regions similar to the Shc PTB domain and an NPF (asparagine-proline-phenylalanine) motif [17]. The NPF motif can interact with the Eps homology (EH) domain in Eps15 [15,17]. These regions play an important role in linking Numb to other proteins involved in asymmetric cell division and Notch signaling inhibition [15,16,17].
The molecular mechanisms by which wild-type numb represses Notch signaling have been reported in a variety of developmental contexts [6,16,18]. During asymmetric cell division, Sanpodo (Spdo), a four-transmembrane protein homologous to the actin-related protein tropomodulin, enhances Notch signaling when numb is not present [19]. Numb, together with Adaptor protein 1 (AP-1), a protein important for recycling and protein sorting, form a complex that regulates Spdo recycling and prevents Spdo from returning to the cell surface [20,21].
In Drosophila, embryos homozygous for a loss-of-function mutant of numb (numb1) were previously shown to have fewer peripheral neurons than wild type embryos [12]. This phenotype was considered an “antineurogenic” phenotype, suggesting an overactive Notch signaling [22]. This idea established numb as a negative regulator of Notch signaling [16,23]. However, following these analyses, the role of numb in the development of the embryonic nervous system has not been studied intensively. Furthermore, these studies on the embryonic nervous system have used almost exclusively the numb1 allele, which did not produce detectable Numb protein on a Western blot [12]. Thus, it was unclear whether suppression of Notch signaling is a universal phenomenon for numb loss-of-function alleles. Therefore, to clarify this, we examined the phenotypes of alternative numb alleles in the embryonic nervous system. The results showed that various numb alleles exhibited the antineurogenic phenotype, suggesting that Notch signaling is generally upregulated in numb mutants. Genome rescue experiments also revealed that this antineurogenic phenotype is caused by the loss of function of the numb gene. Notch signaling activates a variety of target genes in the embryo [24]. Among these downstream cascades, the loss-of-function mutation of numb induced the antineurogenic phenotype mediated through Notch-dependent E(spl) activation.

2. Materials and Methods

2.1. Fly Stocks

The genotypes of the Drosophila lines used in this study are as follows: numb1, a null allele of numb (BDSC 4096) [12]; numbEY03840 (DGGR 114573) [25,26] and numbEY03852 (DGGR 114568) [26], both P-element insertion alleles of numb gene; fTRG_25, a line with a fosmid genomic fragment in which the wild-type numb gene is fused in-frame with a DNA fragment encoding superfolder GFP at its 5′-end, fTRG_25 (FlyFos015836(pRedFlp-Hgr) (numb [41193]::2 XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT) (VDRC 318011) [27]; Df(3)X10, a mδ-m3 deletion at the E(spl) locus [28]; ovoD FRT40A [29]; Df(2L)N22-3 (DGGR 105876) [30] and Df(2L)gamma7 (DGGR 108755) [31], two chromosomal deletion lines of numb. Canton-S was used as a wild type control strain. All flies were raised at 25 °C in a standard Drosophila culture medium.

2.2. Preparation of Fly Food

Fly food was prepared according to the following recipe for roughly 2400 food vials. A pot was filled with 9 L water, 108 g agar, and 1800 g sugar. We placed the pot on the stove and stirred until the agar and glucose dissolved. We kneaded 1150 g of corn flour, 250 g of corn grit, 720 g of brewer’s yeast, and 324 g of rice bran well with 4.5 L of water until uniform, then added them to a pot. Additionally, 4.5 L of hot water was added. Once it boiled, we reduced the heat and stirred well for 20 min to prevent burning, then turned off the heat and stirred until the mixture cooled to below 75 °C. Subsequently, 54 mL propionic acid, 90 mL butyl p-hydroxybenzoate, 54 mL propionic acid, and 90 mL butyl p-hydroxybenzoate were added. About 7 mL of food was dispensed in each vial.

2.3. Generation of numb Maternal and Zygotic Mutant Embryos

FLP-FRT dominant female sterility technique was used to produce numb germline clones [32]. y w hs-FLP; numbEY03840, FRT40A/CyO hb-lacZ, or y w hs-FLP; numbEY03852, FRT40A/CyO hb-lacZ female flies were mated with hs-FLP/+; ovoD, FRT40A/CyO hb-lacZ males. These flies were transferred to new vials daily. Late second or early third instar larvae from these crosses were heat-shocked at 37 °C for 1 h, incubated at 18 °C for 2 h, heat-shocked again at 37 °C for 1 h, and incubated at 25 °C until adults emerged. Emerged females were crossed with y w hs-FLP/+; numbEY03840, FRT40A/CyO hb-lacZ males or y w hs-FLP/+; numbEY03852, FRT40A/CyO hb-lacZ males to obtain embryos that were numb homozygous and lacking maternal contribution, designated as numbM/Z. The mated flies were placed in collection tubes containing grape agar medium and yeast paste, from which numbM/Z embryos were collected.

2.4. Immunohistochemical Staining

The embryonic stage was determined according to previously described gut morphology [33]. Homozygous mutant embryos were identified based on the absence of balancer chromosomes, CyO (CyO hb-lacZ), and TM6B (TM6B ubi-GFP) expressing lacZ and GFP, respectively. The recovered embryos were decolorized with 50% bleach and fixed in a 1:1 mixture of heptane and 4% paraformaldehyde for 30 min with shaking. The embryos were shaken vigorously in a 1:1 mixture of methanol and heptane to remove the vitelline membrane. Embryos were washed in 100% methanol and stored at −20 °C until immunohistochemical staining. The primary antibodies used for immunostaining were rat anti-embryonic lethal aberration (Elav) (DSHB 7E8A10, 1:500 dilution) [34], mouse anti-Fasciclin II (FasII) (DSHB 1D4, 1:50 dilution) [35], rat anti-Hunchback (Hb) (1:300 dilution) [36], chicken anti-β-galactosidase (β-gal) (Abcam 134435, 1:500 dilution), and rabbit anti-GFP (MBL 598, 1:500 dilution) [37]. Fluorescent Alexa488, Cy3, and Cy5-conjugated secondary antibodies were used at 1:500 dilution (all from Jackson Immunoresearch, West Grove, PA, USA). Embryos were mounted in 50% glycerol and 0.025% n-propyl gallate in PBS.

2.5. Scoring the Neurogenic and Antineurogenic Phenotypes of numb Mutants

Each experiment was conducted as at least biological triplicates (three independent crosses) to calculate the percentages of phenotypes in the nervous system of numb mutants. We added up the number of embryos obtained from biological replicates.

2.6. Confocal Microscope

The embryos were observed using a LSM700 confocal laser microscope (Zeiss). Images were acquired from the apical surface to a depth of 30 µm and constructed as Z-stack photographs (1 μm each). The obtained images were analyzed using LSM Image Browser ZEN 2012 (Zeiss, Jena, Germany) and ImageJ software (Version 13.0.6, NIH, Bethesda, MD, USA) [38].

3. Results

3.1. Strong Loss-of-Function Mutations in numb Universally Induce an Antineurogenic Phenotype in the PNS of Embryos

The majority of studies on the role of the numb gene in embryonic development have been conducted using the null allele called numb1 in Drosophila [12,13,18,25,39,40]. Pioneering studies reported that embryos homozygous for numb1 show reduced peripheral nervous system (PNS) neurons compared to wild type embryos [12]. This phenotype has been termed the antineurogenic phenotype, suggesting an overactivation of Notch signaling [6,22]. In the present study, we analyzed various numb mutant alleles, including numb1, numbEY03840, and numbEY03852, to confirm the generality of the antineurogenic phenotype with strong loss-of-function mutations in numb [12,25,26]. The objective of examining additional numb alleles was to verify the universality of the phenotypes and rule out the possibility that the phenotypes in numb1 were due to a second-site mutation or specific allele effect. Ensuring the phenotypes across different alleles confirms that they are due to the loss of numb functions but not caused by genetic background. We confirmed that numb1 homozygous embryos exhibit an antineurogenic phenotype, which was previously reported [12]. In wild type and numb1 homozygous embryos, differentiated neurons were detected by immunostaining with an anti-Elav antibody at stage 14 (Figure 1A–C) [34]. Analysis of these embryos resulted in reduced neurons in the PNS compared to the wild type (Figure 1B,C). To investigate whether such an antineurogenic phenotype is universally associated with the numb mutant allele, we here examined the PNS phenotype of embryos homozygous for the other numb alleles, numbEY03840 and numbEY03852, known as P-element insertion mutations [25,26]. The results showed that numbEY03840 and numbEY03852 exhibited an antineurogenic phenotype (Figure 1D,E). However, the phenotype of numbEY03852 was mild compared to numb1 and numbEY03840 phenotypes, suggesting that numb EY03852 is a hypomorphic allele of numb (Figure 1C–E). The antineurogenic phenotype of numbEY03840 was as severe as numb1, a pre-existing null allele of numb, suggesting that numbEY03840 is also a null allele of numb based on genetic criteria (Figure 1C,D) [12]. On the other hand, heterozygotes of numb1, numbEY03852, or numbEY03840 showed normal CNS and PNS, demonstrating that the antineurogenic phenotype is recessive (Figure 1F–H). The finding that numbEY03840 is a null allele of numb was further supported by severe antineurogenic phenotype in transheterozygous embryos of numbEY03840 and chromosomal deletion mutants lacking the numb locus, Df(2L)N22-3 (DGGR 105876) or Df(2L)gamma7 (DGGR 108755) (Figure 1I,J). Transheterozygotes of numb1 and numbEY03840 also showed the antineurogenic phenotype in the PNS, suggesting that the antineurogenic phenotype is caused by mutations in the numb gene (Figure 1C,K) [12]. These results collectively demonstrated that loss-of-function mutations in numb generally cause an antineurogenic phenotype in the embryonic PNS.
To further confirm this idea, we introduced a genomic fragment containing the wild-type numb locus (FlyFos015836(pRedFlp-Hgr)(numb[41193]::2XTY1-SGFP-V5-preTEV-BLRP-3XFLAG)dFRT) inserted into the 3rd chromosome, designated as fTRG_25, into numb1 homozygotes and investigated whether the antineurogenic phenotype of the PNS is rescued [27]. It was previously shown that a copy of fTRG_25 is sufficient to rescue the lethality associated with numb1 homozygotes [27]. We here revealed that introducing this genomic fragment into numb1 homozygotes effectively rescued the antineurogenic phenotype of the PNS (Figure 2A–C). Therefore, we conclude that the antineurogenic phenotype in the PNS of numb mutants is caused by the loss of function of the numb gene.

3.2. Maternal numb Does Not Contribute to Notch Signal Downregulation

Various genes encoding components of the Notch signaling pathway are known to have maternal functions in embryonic nervous system development [41,42,43,44,45]. Therefore, it is possible that numb may have maternal effects, as discussed in previous studies [12,46,47,48]. To test this possibility, we created homozygous embryos for numb mutations without its maternal contribution (numbM/Z embryos). For this purpose, we employed an FLP/FRT-based method to create germline clones using two numb alleles, numbEY03840 or numbEY03852 [29,49]. We observed neurons in wild type and numbM/Z embryos of numbEY03840 or numbEY03852 using anti-Elav antibody staining (Figure 3A–C). If numb has maternal effects, a more severe antineurogenic phenotype should be observed in numbM/Z embryos than in numb homozygous embryos receiving a maternal numb gene product supply. However, abnormalities of these numbM/Z embryos in the nervous system, including the antineurogenic phenotype of the PNS, were nearly equivalent to those of numbEY03840 or numbEY03852 homozygotes (Figure 1C,D and Figure 3B,C). These results suggest that numb has no maternal influence on the development of the embryonic nervous system.

3.3. The Antineurogenic Phenotype of the numb Mutant Was Dependent on E(spl), a Target Gene of Notch Signaling

The antineurogenic phenotype in numb mutants suggested that wild-type numb represses Notch signaling [18,50,51,52]. However, it is not known what downstream cascade of Notch signaling is involved in this repression. For example, various Notch signaling target genes, such as Enhancer of split (E(spl)), tramtrack (ttk), and single-minded (sim), are activated before and after neurogenesis in the embryo [53,54]. E(spl) is known to play a major role in the development of the CNS and PNS of the embryo [28]. Therefore, wild-type numb was postulated to suppress neuron formation in the PNS by upregulating Notch signaling-dependent E(spl) activation. The E(spl) locus contains multiple genes, such as m8, m7, m5, m3, mβ, mγ, and mδ, encoding basic helix-loop-helix transcription factors [55]. Df(3)X10 mutants have a deletion spanning mδ–m3 at the E(spl) locus [28,55]. In Df(3)X10 homozygotes, the E(spl) function is significantly reduced, but the E(spl) function remains as the genome contains m8, m7, m5, and m4 [55]. However, as previously reported, Df(3)X10 homozygous embryos show severe neuronal hyperplasia, called the neurogenic phenotype, and in this study, this phenotype was also detected by anti-Elav antibody staining (Figure 4C) [28,55]. To analyze the genetic interaction between numb and E(spl), we combined numb1 and Df(3)X10 and observed embryonic phenotypes in the PNS and CNS. The double heterozygotes of numb1 and Df(3)X10 did not show detectable defects in these tissues (Figure 4D). However, the double homozygotes of numb1 and Df(3)X10 showed no neurogenic phenotype in the CNS or antineurogenic phenotype in the PNS (Figure 4G). The overall structure of the CNS and PNS in these embryos was not much different from that of the wild type (Figure 4A,G). However, potential changes in the number of neurons may not be detected using this experimental procedure. Nevertheless, we revealed that the double homozygotes of numb1 and Df(3)X10 showed some irregularities in the nervous system structure compared to the wild type (Figure 4A,G). These results revealed an antagonistic genetic interaction between numb and E(spl). However, numb1/+; Df(3)X10/Df(3)X10 embryos showed a strong neurogenic phenotype comparable to Df(3)X10/Df(3)X10 (Figure 4C,F). Furthermore, numb1/numb1; Df(3)X10/+ embryos showed an antineurogenic phenotype comparable to numb1/numb1 (Figure 4B,E). Thus, numb1 and Df(3)X10 did not show dominant suppression of the neurogenic and antineurogenic phenotypes associated with E(spl) and numb mutations, respectively. However, since numb and E(spl) mutually suppress neurogenic and antineurogenic phenotypes, our results suggest that numb mutants enhance Notch activation, which can upregulate the expression of remaining E(spl) genes to compensate for the missing ones and rescue the Df(3)X10 mutant phenotype (Figure 4H).

3.4. Notch Signaling Is Not Upregulated during Lateral Inhibition in the CNS in numb Mutants

Our results so far indicate that numb suppresses the neurogenic phenotype of the E(spl) mutant in the CNS, suggesting that numb mutations upregulate Notch signaling in the CNS and the PNS (Figure 4G,H). Therefore, we examined whether lateral inhibition is enhanced in the CNS of numb mutants at embryonic stage 9 when neuroblast differentiation begins. Neuroblast identity is determined by several transcription factors, such as Hunchback (hb) [56,57]. Hence, hb is a marker of neuroblasts whose number is regulated by Notch signaling via lateral inhibition [58,59].
To observe neuroblasts, we detected Hunchback (Hb) protein, a marker of neuroblasts, by immunostaining with anti-Hb antibody in embryos at stage 9 (Figure 5A,B). The results showed that neuroblasts in the neuroectoderm of numb1 homozygous embryos were largely comparable to the wild type (Figure 5A,B). To quantitatively analyze the numbers of neuroblasts, we counted the number of Hb-positive nuclei in these embryos (Figure 5C). The average number of neuroblasts did not significantly differ between numb1 homozygous and wild type embryos (Figure 5C). This result is consistent with the observation that the CNS observed by anti-Elav antibody staining appeared normal in numb1 homozygotes (Figure 1B).

3.5. numb Mutations Impaired Neuronal Projection in the PNS

We examined the neuronal projections of numb mutants to determine if the lack of numb function causes abnormalities other than an antineurogenic phenotype in the development of the nervous system. The intersegmental nerve (ISN), crucial for the Drosophila PNS, is formed by pioneering motor neurons extending axons from the CNS and joining sensory axons. Cell adhesion molecules, particularly Fasciclin II (FasII), play a vital role in axon fasciculation by mediating homophilic interactions [60]. FasII is a member of the immunoglobulin superfamily that predominantly localizes to neurites [60,61]. Thus, FasII is a suitable marker for observing neurite projection. In this study, we used anti-FasII antibody staining to analyze the structure of neurite projections in the embryonic CNS and PNS. The results showed that numb1, numbEY03840, or numbEY03852 homozygotes had no detectable abnormalities in FasII-positive axons in the CNS (Figure 6B–D).
While no abnormalities were detected in FasII-positive axons in the CNS, the PNS showed significant disorganization (Figure 7B–D). In numb1, numbEY03840, or numbEY03852 homozygotes, FasII-positive neurite projection of the ISN axons became disorganized and occasionally shorter than the wild type (Figure 7A–D). To quantitatively analyze these defects, we obtained the percentages of the abnormally shorter ISN axons (Table 1). The shorter ISNs were observed more frequently in the numb1, numbEY03840, or numbEY03852 homozygotes compared with the wild type (Table 1). This indicates that numb is essential for proper axonal projections in the PNS. At this stage, however, the mechanism regarding how the pathways of these axons are disrupted in numb mutants remains unclear.

4. Discussion

The importance of numb in the downregulation of Notch signaling has been studied in a variety of species [16,62]. In Drosophila, only the numb1 allele has been used in early studies that revealed an antineurogenic phenotype in numb1 homozygous PNS [12]. Because of this, it was not clear whether such antineurogenic phenotypes were universally associated with loss-of-function mutations in numb. In this study, we addressed this question using two other numb alleles, numbEY03840 and numbEY03852. The results revealed that the loss-of-function mutants of numb universally exhibit an antineurogenic phenotype in the PNS (Figure 1C–E). To further confirm this finding, we examined whether the antineurogenic phenotype of numb1 homozygotes could be rescued by the introduction of a genomic fragment containing the wild-type numb locus [27]. In embryos homozygous for numb1 and carrying this genomic fragment, the antineurogenic phenotype in the PNS was suppressed (Figure 2C). Furthermore, as reported previously, these embryos survived to adulthood, indicating that the recessive lethality associated with the numb1 mutant was rescued by this genomic fragment [27]. It is unclear what anomalies in the numb1 mutant are responsible for the rescue of such recessive lethality. However, based on these results, we conclude that numb loss-of-function mutations universally induce an antineurogenic phenotype in the PNS [22].
It has been suggested that the maternal numb may compensate for the deletion of the zygotic numb and provide sufficient functions for a part of normal development during early embryogenesis [12,46,47,48]. In this study, we tested this possibility using the numbEY03840 allele, which was genetically suggested to be a null mutant. Previous studies have shown that numb has a maternal-specific alternative-splicing product [12,63,64]. Since numbEY03840 has a P-element inserted in an exon common to maternal and zygotic transcripts, this insertion likely disrupts both maternal and zygotic numb function in numbEY03840 mutation, suggesting that numbEY03840 is null maternally and zygotically [63,65,66]. In the present study, embryos of zygotes and maternal mutants of numbEY03840 exhibited an antineurogenic phenotype comparable to that of zygotic homozygotes of numbEY03840 (Figure 1D and Figure 3B). These results suggest that numb has no maternal effect, or if it does, it is negligible.
During embryonic development, Notch signaling exerts its function through the activation of various downstream target genes such as E(spl), ttk, and sim [53]. In this study, we found that numb and E(spl) mutually suppress antineurogenic and neurogenic phenotypes, respectively (Figure 4G). Df(3)X10 is a deletion mutant of E(spl) that lacks several genes in the E(spl) complex, such as m3, mβ, mγ, and mδ. These genes encode basic helix-loop-helix proteins and are known to be involved in lateral inhibition through Notch signaling [67,68]. Df(3)X10 homozygotes showed a strong neurogenic phenotype, but as previously reported, other genes of the E(spl) complex, such as m5, m6, m7, and m8, remain in Df(3)X10 [55]. Wild-type numb suppresses E(spl) activation by downregulating Notch. Thus, in Df(3)X10 homozygotes, derepression of Notch activity by numb mutation can rescue the neurogenic phenotype by increasing the expression of the remaining genes in the E(spl) complex.
Importantly, numb1 suppressed the neurogenic phenotype in the CNS of Df(3)X10. This suggests that wild-type numb suppresses Notch signaling-mediated lateral inhibition in the neuroectoderm. Therefore, we considered the potential role of numb in the neuroectoderm and examined the expression of the hb gene, a marker for neuroblasts, in numb1 homozygotes. However, the number of neuroblasts in numb1 homozygotes was equivalent to that of wild type (Figure 5C). This result agrees with the previous finding that the number of neuroblasts in the CNS was not affected in the numb1 homozygote [69]. Thus, in wild type embryos where the transcription of the E(spl) complex was activated at the normal level through Notch signaling, the absence of numb did not result in the hyperactivation of lateral inhibition in the neuroectoderm.
Since cell-fate determination to neurons is impaired in numb mutants, we speculated that such defects might lead to abnormalities other than neuronal differentiation. To test this possibility, we compared the neurite projection in numb mutants and wild type embryos. In Drosophila, FasII is often used as a marker for neurite projections of motor neurons [70,71]. In particular, anti-FasII antibody staining is useful for analyzing neurite structure. We found that the neurites of PNS motoneurons in numb mutants are disorganized and occasionally shorter (Figure 7B–D). These results suggest that wild-type numb is required for proper projection of the motor neurons. Numb interacts with endocytic components and affects the trafficking and localization of guidance receptors during axon guidance [52]. Thus, the loss of numb functions may impair axonal responses to guidance cues, leading to the observed ISN abnormalities. However, the defect in neurite projection may be due to the lack of some neurons in the numb1 mutant. Therefore, future studies need to elucidate the molecular and cellular mechanisms by which numb regulates neurite projection.

Author Contributions

Conceptualization: T.Y., M.B. and K.M. (Kenji Matsuno); Data curation, E.Y.M. and K.M. (Kenji Matsuno); Investigation: E.Y.M., S.K., K.M. (Kenjiroo Matsumoto), T.G. and N.A.; Methodology: E.Y.M. and S.K.; Supervision, H.O.I., T.S., D.U. and M.I.; Writing—original draft: E.Y.M. and K.M. (Kenji Matsuno); Writing—review and editing: T.Y. and M.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by a Grant-in-Aid for Early Career Scientists (JP18K14697) from the Japan Society for the Promotion of Science (JSPS), Biotechnology and Biological Sciences Research Council (BBSRC) (BB/V014218/1), the Next-Generation Researcher Development Project (JISEDAI) from Osaka University, Teraura Sayoko Scholarship, and INPEX Scholarship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are grateful to Anette Preiss (University of Hohenheim) for her generous gift of the Df(3)X10 line and to Norbert Perrimon (Harvard Medical School) for providing the ovoD line. We also thank Kyoto Stock Center (DGGR), Bloomington Drosophila Stock Center (BDSC), and Vienna Drosophila Resource Center (VDRC) for Drosophila stocks, as well as the Developmental Studies of Hybridoma Bank (DSHB) and National Institute of Genetics (NIG) for antibodies.

Conflicts of Interest

The authors declare no conflict of interest.

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Biomolecules 14 01062 g001
Figure 1. numb loss-of-function alleles exhibited an antineurogenic phenotype in the PNS. A schematic diagram of the CNS (orange) and PNS (green) in a Drosophila embryo at stage 14 (A). Embryos of wild type (B), numb1 homozygote (C), numbEY03840 homozygote (D), numbEY03852 homozygote (E), numb1 heterozygote (F), numbEY03840 heterozygote (G), numbEY03852 heterozygote (H), numbEY03840/Df(2L)N22-3 transheterozygote (I), numbEY03840/Df(2L)gamma7 transheterozygote (J), and numb1/numbEY03840 transheterozygote (K) were stained with an anti-Elav antibody. Lateral views of the embryos are shown. In these embryos, neurons were shown in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
Figure 1. numb loss-of-function alleles exhibited an antineurogenic phenotype in the PNS. A schematic diagram of the CNS (orange) and PNS (green) in a Drosophila embryo at stage 14 (A). Embryos of wild type (B), numb1 homozygote (C), numbEY03840 homozygote (D), numbEY03852 homozygote (E), numb1 heterozygote (F), numbEY03840 heterozygote (G), numbEY03852 heterozygote (H), numbEY03840/Df(2L)N22-3 transheterozygote (I), numbEY03840/Df(2L)gamma7 transheterozygote (J), and numb1/numbEY03840 transheterozygote (K) were stained with an anti-Elav antibody. Lateral views of the embryos are shown. In these embryos, neurons were shown in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
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Figure 2. The antineurogenic phenotype of numb1 homozygous was rescued by introducing a genomic fragment containing the wild-type numb locus. Embryos of wild type (A), numb1 homozygote (B), and numb1; fTRG_25/+ (C) were stained with an anti-Elav antibody. fTRG_25 carries the wild-type genomic fragment of the numb gene. Lateral views of the embryos are shown. In these embryos, neurons were observed in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. In C, 10% of embryos showed an antineurogenic phenotype. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
Figure 2. The antineurogenic phenotype of numb1 homozygous was rescued by introducing a genomic fragment containing the wild-type numb locus. Embryos of wild type (A), numb1 homozygote (B), and numb1; fTRG_25/+ (C) were stained with an anti-Elav antibody. fTRG_25 carries the wild-type genomic fragment of the numb gene. Lateral views of the embryos are shown. In these embryos, neurons were observed in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. In C, 10% of embryos showed an antineurogenic phenotype. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
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Figure 3. Embryos homozygous for numb mutants and lacking its maternal contribution showed an antineurogenic phenotype comparable to zygotic homozygotes for the numb alleles in the PNS. Embryos of wild type (A), numbEY03840 homozygote lacking maternal contribution of numb (numbEY03840 M/Z) (B), and numbEY03852 homozygote lacking maternal contribution of numb (numbEY03852 M/Z) (C) were stained with an anti-Elav antibody. Lateral views of the embryos are shown. In these embryos, neurons were observed in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
Figure 3. Embryos homozygous for numb mutants and lacking its maternal contribution showed an antineurogenic phenotype comparable to zygotic homozygotes for the numb alleles in the PNS. Embryos of wild type (A), numbEY03840 homozygote lacking maternal contribution of numb (numbEY03840 M/Z) (B), and numbEY03852 homozygote lacking maternal contribution of numb (numbEY03852 M/Z) (C) were stained with an anti-Elav antibody. Lateral views of the embryos are shown. In these embryos, neurons were observed in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm.
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Figure 4. numb and E(spl) mutually suppressed the neurogenic and antineurogenic phenotypes, respectively. Embryos of wild type (A), numb1 homozygote (B), Df(3)X10 homozygote (C), numb1/+; Df(3)X10/+ (D), numb1/numb1; Df(3)X10/+ (E), numb1/+; Df(3)X10/Df(3)X10 (F), and numb1/numb1; Df(3)X10/Df(3)X10 (G) were stained with an anti-Elav antibody. Df(3)X10 is a deletion mutant of the E(spl) complex. Lateral views of the embryos are shown. In these embryos, neurons were shown in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm. A schematic diagram of Notch signaling analyzed here is shown (H).
Figure 4. numb and E(spl) mutually suppressed the neurogenic and antineurogenic phenotypes, respectively. Embryos of wild type (A), numb1 homozygote (B), Df(3)X10 homozygote (C), numb1/+; Df(3)X10/+ (D), numb1/numb1; Df(3)X10/+ (E), numb1/+; Df(3)X10/Df(3)X10 (F), and numb1/numb1; Df(3)X10/Df(3)X10 (G) were stained with an anti-Elav antibody. Df(3)X10 is a deletion mutant of the E(spl) complex. Lateral views of the embryos are shown. In these embryos, neurons were shown in white. The percentage of embryos showing the phenotype presented is shown in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm. A schematic diagram of Notch signaling analyzed here is shown (H).
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Figure 5. numb1 homozygotes showed a similar number of neuroblasts. Wild type (A) and numb1 homozygous (B) embryos were stained with an anti-Hb antibody. Ventral views of the embryos are shown. Neuroblasts were observed to be white in these embryos. The percentage of embryos showing the presented phenotype is in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 μm. The average numbers of Hb-positive nuclei are shown (C). Standard deviations are shown as bars at the top of each bar. The t-test showed no significant differences between the wild type and numb1 embryos. The numbers of embryos examined are shown at the bottom of each bar.
Figure 5. numb1 homozygotes showed a similar number of neuroblasts. Wild type (A) and numb1 homozygous (B) embryos were stained with an anti-Hb antibody. Ventral views of the embryos are shown. Neuroblasts were observed to be white in these embryos. The percentage of embryos showing the presented phenotype is in % at the bottom right. The number of embryos examined is indicated by “n”. The scale bar represents 25 μm. The average numbers of Hb-positive nuclei are shown (C). Standard deviations are shown as bars at the top of each bar. The t-test showed no significant differences between the wild type and numb1 embryos. The numbers of embryos examined are shown at the bottom of each bar.
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Figure 6. Axons of motor neurons in the CNS were normal in numb homozygote mutants. Axons of CNS motoneurons stained with an anti-FasII antibody in wild type (A), numb1 homozygous (B), numbEY03840 homozygous (C), and numbEY03852 homozygous (D) embryos. Ventral views of the embryos are shown. In these embryos, axons were shown in white. The percentage of embryos showing the presented phenotype is in the lower right corner as %. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm. Axon tracts are indicated by white arrowheads.
Figure 6. Axons of motor neurons in the CNS were normal in numb homozygote mutants. Axons of CNS motoneurons stained with an anti-FasII antibody in wild type (A), numb1 homozygous (B), numbEY03840 homozygous (C), and numbEY03852 homozygous (D) embryos. Ventral views of the embryos are shown. In these embryos, axons were shown in white. The percentage of embryos showing the presented phenotype is in the lower right corner as %. The number of embryos examined is indicated by “n”. The scale bar represents 25 µm. Axon tracts are indicated by white arrowheads.
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Figure 7. Axons of motor neurons in the PNS were disorganized in numb homozygotes. Axons of PNS motoneurons stained with an anti-FasII antibody in wild type (A), numb1 homozygous (B), numbEY03840 homozygous (C), and numbEY03852 homozygous (D) embryos. Lateral views of the embryos are shown. In these embryos, axons were shown in white. The number of embryos examined is indicated by “n”. The scale bar represents 25 μm. Abnormally short ISNs are indicated by white asterisks.
Figure 7. Axons of motor neurons in the PNS were disorganized in numb homozygotes. Axons of PNS motoneurons stained with an anti-FasII antibody in wild type (A), numb1 homozygous (B), numbEY03840 homozygous (C), and numbEY03852 homozygous (D) embryos. Lateral views of the embryos are shown. In these embryos, axons were shown in white. The number of embryos examined is indicated by “n”. The scale bar represents 25 μm. Abnormally short ISNs are indicated by white asterisks.
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Table 1. The percentage of abnormally short axons in wild type and numb mutants.
Table 1. The percentage of abnormally short axons in wild type and numb mutants.
Genotype% of Abnormally Short Axons
Wild type0% (n = 10)
numb138% (n = 10)
numbEY0384025% (n = 10)
numbEY038524% (n = 10)
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Mujizah, E.Y.; Kuwana, S.; Matsumoto, K.; Gushiken, T.; Aoyama, N.; Ishikawa, H.O.; Sasamura, T.; Umetsu, D.; Inaki, M.; Yamakawa, T.; et al. Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System. Biomolecules 2024, 14, 1062. https://doi.org/10.3390/biom14091062

AMA Style

Mujizah EY, Kuwana S, Matsumoto K, Gushiken T, Aoyama N, Ishikawa HO, Sasamura T, Umetsu D, Inaki M, Yamakawa T, et al. Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System. Biomolecules. 2024; 14(9):1062. https://doi.org/10.3390/biom14091062

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Mujizah, Elzava Yuslimatin, Satoshi Kuwana, Kenjiroo Matsumoto, Takuma Gushiken, Naoki Aoyama, Hiroyuki O. Ishikawa, Takeshi Sasamura, Daiki Umetsu, Mikiko Inaki, Tomoko Yamakawa, and et al. 2024. "Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System" Biomolecules 14, no. 9: 1062. https://doi.org/10.3390/biom14091062

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