**1. Introduction**

The mammalian olfactory system is composed of the olfactory epithelium (OE), olfactory bulb (OB), and olfactory cortex (OC). The rodent OB is organized into six layers that contain distinct cell populations and that are essentially made up of two types of neurons, interneurons, and projection/output neurons [1,2]. Using the Golgi method, Santiago Ramón y Cajal described the layers in the OB and its components more than a century ago (Figure 1A reproduces an original drawing of Cajal [3]; reviewed in [4]). His morphological studies on the OB provided the basis to define the neurons present in this structure and when UbC-StarTrack strategy is compared, the cells labeled in the adult OB following in utero electroporation (IUE) are similar to those drawn by Cajal (Figure 1A–F). Mitral cells (Figure 1(Ae),C) are the first cell type to be born in the rodent OB, between E10 and E13, and with a neurogenic peak around E11 [5]. The axonal projections of mitral cells form the lateral olfactory tract (LOT) and they establish direct contacts with the OC [6,7].

Olfactory interneurons (periglomerular and granule cells) are a diverse group of cells located within the glomerular layer (GL) and granular cell layer (GcL; Figure 1D–E). These interneurons arise from progenitors located within the ganglionic eminences that migrate tangentially to their destination in the OB [8,9]. Neural stem cells (NSCs) in the subventricular zone (SVZ) also give rise to olfactory interneurons during postnatal life, and these progenitors are determined between E13 and E15 [9]. The different kinds of interneurons are generated from embryonic to postnatal stages [10–13], and their temporal origin defines the interneuronal diversity [14]. Glial cells are also widespread in the different layers of the OB, those found in each layer arising from different or the same progenitors (Figure 1F). For example, some astrocytes surrounding a single glomerulus have

been shown to be clonally-related [15]. In the embryo, glial progenitor cells are located in the most rostral part of the lateral ventricle (LV), which corresponds to the olfactory ventricle (OV) [15–17]. This complex organization and connectivity of the cells that populate the OB are largely determined during embryonic development [5,18,19], albeit with an additional contribution postnatally [20,21].

**Figure 1.** (**A**) Original drawing by Cajal of an olfactory bulb (OB) section from the brain of a perinatal cat [3] showing the glomerular layer (*A*); external plexiform layer (*B*); mitral cell layer (MCL; *C*); internal plexiform layer (*D*); granule cell layer and white matter (*E*); (*a,b*) terminal axonal arborizations of olfactory sensory neurons; (c) dendritic arborizations from tufted (*d*) and mitral cells (e) that form the glomerulus; (*f–h*) axonal projections from tufted and mitral cells); (*I–J*) granule cells; (*K*) short axon cells of the granule cell layer (Cajal Legacy, Instituto Cajal-CSIC, Madrid, Spain). (**B–F**) Adult OB neural cells labeled after in utero electroporation (IUE) of UbC-StarTrack constructs into the E12 mouse embryo lateral ventricle (LV). (**B**) Coronal section of the mouse OB in which UbC-StarTrack labelling shows the different cells that compose the layers described by Cajal. (**C**) Detail of the MCL, with projection neurons and glial cells labeled with UbC-EGFP-StarTrack. Detail of labeled periglomerular (**D**) and granule cells (**E**). (**F**) UbC-StarTrack labeled glia widely spread across the different OB layers. GcL, granular cell layer; IPL, internal plexiform layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.

To date, different approaches have been used to assess the diversity of OB progenitor pools during development, including the use of fluorescent and lipophilic tracers, viral vectors, immunostaining, and the generation of specific mouse lines. Nevertheless, the heterogeneity of progenitor cells has ye<sup>t</sup> to be fully defined, and more recent single-cell transcriptomic analyses have shed new light on the diversity and potential of progenitor cells [22,23]. Moreover, single-cell lineage tracing revealed the fate potential and lineage progression of some progenitors [24–26].

Here, in order to decipher the heterogeneity of progenitor cells, using UbC-StarTrack lineage tracing approaches under the specific regulation of different promoters, we targeted specific progenitors by IUE to analyze the fate potential of NSCs in the adult brain. The determination of specific cell types in the OB can be influenced by either the molecular profile by their progenitors, the age of the embryo, and/or the location of the labeled progenitors. The data we obtained here confirm that some degree of diversity is present in the pool of OB progenitor cells, highlighting the need of performing further single-cell analyses to define the progenitor cell identities required to generate the complex OB cytoarchitecture. We demonstrate that the origin, fate, and targeting of progenitors must be taken into consideration when studying OB heterogeneity.

#### **2. Materials and Methods**

## *2.1. Mouse Line*

C57BL/6 mice were housed at the animal facility of the Cajal Institute. All procedures were carried out in accordance with the guidelines of the European Union on the use and welfare of experimental animals (2010/63/EU) and those of the Spanish Ministry of Agriculture (RD 1201/2005 and L 32/2007). All the experiments were approved by the CSIC Bioethical Committee (PROEX 223/16). The day of visualization of the vaginal plug was considered as embryonic day (E0) and the day of birth as postnatal day (P0). In addition, mice were considered adults from P30 onwards. In all the experiments, a minimum of *n* = 3 animals was considered.
