**4. Discussion**

In this study, a novel StarTrack approach was adopted to address the ontogeny of di fferent cell types in the adult rodent OB, taking into account the identity of their progenitor cells and their location in the LV. We focused on the genetic profile of progenitor cells in order to define the heterogeneity of the NSCs that give rise to neural cells in the adult OB and the origin of these cells. Specific progenitor cells were targeted at E12 to track their adult cell progeny in the OB. StarTrack is a powerful tool to examine the fate and the clonal relationship between cells derived from specific progenitors in vivo, under both physiological and pathological conditions [15–17,21,25,27–33]. This tool also allows progenitor cells to be tracked in vivo, avoiding genetic manipulation of the animals or viral injections. In our particular case, NSCs in the LV were targeted by IUE, avoiding targeting progenitor cells at other sites in which these promoters may be active, such as pericytes in the case of the NG2 promoter [34–36]. NG2-hyPBase-driven integration of the StarTrack mix via IUE targets those neural progenitors in the LV with an active NG2-promoter, thereby limiting the developmental spatio-temporal parameters of the study (reviewed by Shimogory and Ogawa [37]).

The heterogeneity of NSC pools is an issue that has ye<sup>t</sup> to be resolved [20,38]. The past two decades have witnessed an accumulation of abundant evidence regarding such heterogeneity, not only in the OB, but also in the dorsal cortex [39], hippocampus [40], and cerebellum [30]. Moreover, recent data show the bipotent capacity of postnatal NSCs to generate OB interneurons and glia in the cortex and striatum [21]. NSCs generating OB cells follow highly patterned and complex behavior during embryogenesis [41,42], and at post-natal stages [13,41]. Single cell analysis provides new insight into the development of the forebrain and the changes it undergoes in the adult. Clonal and transcriptomic analyses make it possible to explore the huge heterogeneity of neural cells [24,32,43,44]. Indeed, the heterogeneity of cortical progenitor cells was recently reported, showing the cell lineage of cortical pyramidal cells restricted to either the deep or superficial layers [39], suggesting that the heterogeneity of neocortical progenitor cells is greater than that previously thought [45–47]. Currently, the extent of NSC heterogeneity still remains to be defined as such, and further studies and improved methods will be required to fully specify the cell diversity in the telencephalon.

Thus, it is relevant to target certain sites in order to study specific cell progeny. In particular, the origin of glial cells is not as well understood as that of neurons, and it has been suggested that glial cells travel from the LV to OB via the RMS [48]. The data presented here show that glial and mitral cells progenitors are located in the OV, as previously reported [49]. Nevertheless, it is important to consider those cells in the adult OB that were not targeted by our strategy due to the location of their progenitors, as the ensheathing cells migrating from the olfactory placode to the olfactory nerve layer of the OB [50]. While our data showed the importance of embryonic progenitor location more than the cell identity at E12, neonatal progenitor microdomains may also exist in which specific OB cells originate [13,41].

In summary, the results presented here open a window to explore the genomic profile of neural progenitor cells in more detail. Moreover, they highlight the importance of further studies at the single-cell level to define the heterogeneity of NSC populations and their progeny in the OB. Such studies will help us to better understand this complex brain structure.

**Author Contributions:** Conceptualization, L.L.-M.; methodology, R.S.-G., M.F.-O., A.C.O.-S.; formal analysis, R.S.-G.; investigation, R.S.-G.; writing—original draft preparation, L.L.-M., R.S.-G.; writing—review and editing, L.L.-M., R.S.-G., M.F.-O., A.C.O.-S; project administration, L.L.-M.; funding acquisition, L.L.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Economy and Competitivity (MINECO), gran<sup>t</sup> number BFU2016-75207-R.

**Acknowledgments:** We are very grateful to both the Imaging and Microscopy Facility at the Instituto Cajal and the Microscopy and Image Analysis Services at the National Hospital for Paraplegics, Toledo.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
