Switching of RNA splicing regulators in immature neuroblasts: a key step in adult neurogenesis

Reviewer #1 (Public Review):

In this study, the authors use prospective sorting and microarray analyses, extended by single-cell RNA sequencing, in the neural stem cell niche of the subventricular zone (SVZ) to identify and refine a series of states along the continuum from quiescent neural stem cells to mature progeny. Of note, changes in the levels and subgroups of RNA splicing regulators are detailed across this continuum. Using in vitro proliferation and differentiation assays, coupled with in vivo engraftment of some prospectively sorted subsets, the authors argue that a stage they define as immature neuroblasts (iNBs) retain proliferative and multilineage differentiation capacity that is not seen in the mature neuroblast population, and is unexpected based on prior models for lineage progression in this system. This iNB stage is accompanied by a change in RNA splicing regulator expression, which is of interest due to the emerging roles for RNA processing and preferential translation within this niche.

These data complement several additional sc-RNAseq studies of this stem cell niche, and use a different, but similar, sorting strategy to isolate and profile subpopulations of stem/progenitor cells and neuroblast progeny. The claim that immature neuroblasts retain multipotency - the ability to generate glia and neurons - is surprising and somewhat controversial given that this has largely not been reported before under homeostatic conditions. Some factors to consider when interpreting these data are that the "immature neuroblast" populations are studied in some experiments using a transcriptional signature and a functional assay, namely the timing of reappearance of these cells after use of agents that kill rapidly dividing cells (in this case, radiation), leading to reconstitution of the lineage by previously quiescent stem cells. In a separate set of experiments, a tamoxifen-inducible labeling system is used in combination with cell-surface markers to prospectively isolate and study the differentiation potential of neuroblast populations that are assumed to be equivalent to those found in transcriptional experiments. It would be of interest in future to confirm that the exact sorted populations (using CD24/EGFR/DCX-CreERT2::CAG) have the same transcriptional profile as those studied in earlier experiments within the paper, and to confirm the purity of the sorted populations. Finally, while elegant use is made of engraftment of the sorted populations to study the differentiation and lineage potential of these immature neuroblasts, a remaining question is the relative abundance of each lineage (neurons/astrocytes/oligodendrocytes) produced by the engrafted cells - is production of glia rare, or common? Could this be due to factors such as alteration of lineage potential due to culture conditions, a disconnect between transcript expression and protein expression, or an incompletely purified starter population?

Overall, this manuscript presents an intriguing possible refinement of models for SVZ neurogenesis, and highlights the role of RNA splicing at specific stages in the lineage. It will be of interest to see if additional groups confirm these findings and whether multiplexed immunostaining, highly multiplexed flow cytometry, or other approaches focused at the proteomic level confirm and extend these findings, particularly given recent data in the developing brain that suggest transcript and protein levels are relatively poorly correlated in stem/progenitor populations.

A final point on terminology: "iNB", "A cells", and "D1/D2 cells" are all used in the manuscript to denote different stages along the continuum from TAP/C cells to mature neuroblasts; however, historically "D cells" refers to neuroblasts in the dentate gyrus, not those derived from the SVZ. In this case, the authors are exclusively studying SVZ-derived neuroblasts.

Reviewer #2 (Public Review):

Bernou et al use a FACS-based method to sort different cells along the neurogenesis trajectory. They identify cells that are LeX+EGFR+CD24+ which they call i-NBs. The authors suggest these cells proliferate performing neurosphere assays, and that they can make all NSC-derived differentiated cell types through transplantation into mice. They performed microarrays on the different cell subtypes, which led them to their interest in RNA splicing proteins. They additionally performed single-cell analyses to try to identify the cluster of i-NBs compared to other cell types. Further, they performed an irradiation experiment to initiate quiescence exit and depletion of the dividing cell types to create a directionality in the progression through cell types. Comparison with other published sequencing datasets of the same cell type revealed that the i-NBs were most similar to Mitotic TAPs. The authors use their single cell sequencing data to observe expression changes of the RNA splicing factors in different clusters. They also suggest that the i-NB population is heterogeneous in their DCX mRNA levels, with a high group and a low group that have different characteristics. They erroneously use a DCX-Cre-ERT2 line to identify GFP+ or GFP- cells to transplant, and find no GFP+ cells at the end of 5 weeks after transplantation, and draw the conclusion that the high DCX cells don't have the same NSC potential. The authors propose they have identified a new cell type, and that there should be a rewrite of the SVZ neurogenesis cascade to include this population.

Summary of response
This manuscript postulates the identification of a new cell type in the adult neurogenesis cascade. However, all of the author's analyses point to this population of sorted cells being the late mitotic TAPs on their way to becoming neuroblasts. This would suggest that these cells are in the trajectory between TAPs and NBs, so a pivot point, but not a unique cell type in its own. In their sequencing analyses, cell cycle becomes the defining factor of the clustering. Indeed, their cell type as compared to other datasets suggests this population is a mitotic TAP, which is supported by their own transcriptome data (Fig S2) showing that i-NBs are just further in mitosis than the TAPs.