Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis

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1. 

   Version published to 10.7554/elife.67436 on eLife

   Jul 14, 2021
2. 

   eLife

   Apr 7, 2021

   Author Response:

   Reviewer #1 (Public Review):

   Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the …

   Author Response:

   Reviewer #1 (Public Review):

   Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the manuscript is well written and clear, and the figures are organized and polished. The datasets generated in this manuscript will be a great resource for the field of adult neurogenesis. However, the arguments and supporting data used to assign dorsal/ventral identities to B cells and A cells could be strengthened, and more rigorous data analysis could result in new biological insights into stem and progenitor cell heterogeneity in the V-SVZ.

   We thank the Reviewer for their feedback on our manuscript. As suggested by Reviewer #1, we are performing additional analyses in the following areas:

   1. Performing additional analyses to further strengthen the dorsal/ventral scRNA-Seq B cell marker analysis and its relationship to our sNucRNA-Seq B data.

   2. Performing additional analyses to identify potential novel biological insights into stem & progenitor cell heterogeneity and text edits to discuss how differentially-expressed sets of genes among B cells and A cells are related to biological processes and/or signaling pathways.

   Reviewer #2 (Public Review):

   The paper is well written, and the data are well analyzed and presented. My concerns centre on terminology and alternative explanations of some of the data, which the authors might deal with in the introduction or discussion.

   We thank Reviewer #2 for their positive reception of our manuscript and the data, and for the constructive suggestions, which we have addressed by changes to the manuscript and in our responses below:

   1. I am slightly confused about some of the data shown in Figure 1. If B cells are defined as GFAP expressing cells, then why do only 25% of the B cells in the plot in Figure 1C express GFAP? I may be missing something here, as other readers may as well. Similarly in the same panel, only 25% of astrocytes seem to be expressing GFAP or GFP driven by a GFAP promotor.

   Importantly, among all cells captured in our scRNA-Seq, only B cells (51.86%), a subpopulation of parenchymal astrocytes (25%) and a small subpopulation of ependymal cells (E cells) had GFAP expression. This is consistent with immunocytochemical staining (Ponti et al. 2013) and other studies of scRNA-Seq expression (Xie et al. 2020). Similarly, Gfp (under the control of hGFAP promoter) is not expected to be expressed in all B cells (here 31.08% of B cells are Gfp+).

   Note that previous work has shown that B cells express different levels of GFAP protein, and some B1 cells were negative (Ponti et al. 2013). This supports the notion that this intermediate filament is a good marker of the V-SVZ primary progenitors, but also present in a subpopulation of parenchymal astrocytes and ependymal cells. However, a negative signal for GFAP does not imply that a cell is not a B cell. This highlights the importance of our clustering analysis revealing additional genes associated with B cells. Our analysis suggests that a combination of Gfap, Thrombospondin 4, Slc1a3 (GLAST) and S100a6 provide a better marker combination to identify B cells.

   The reason for the variability among B cells in the expression of GFAP remains unknown. It could be associated with the normal regulation of intermediate filaments as B cells transit the cell cycle or different stages of their activation or quiescence. It could also be linked to technical aspects of scRNA-Seq analysis: e.g gene dropout; detection limits; sequencing saturation. Since on our dot plot the actual proportion is only graphically shown, to clarify this issue in the text we have added the specific percentages and the following sentences:

   “A fraction of both populations expressed GFAP: 51.85% of B cells (clusters 5,13,14 & 22), 24.37% of parenchymal astrocytes (clusters 21, 26 & 29). This is consistent with previous reports (Chai et al. 2017; Xie et al. 2020; Ponti et al. 2013). Note that across all cells captured in our scRNAseq analysis, only B cells, parenchymal astrocytes or ependymal cells expressed GFAP. Among these three cell types, B cells had the highest average expression of GFAP (4.41 for B cells, 1.00 for astrocytes, 0.29767 for Ependymal cells, values in Pearson residuals). Other markers, like S100a6 (Kjell et al. 2020) (88.9% of B cells; 54% of parenchymal astrocytes and 80% of ependymal cells) and Thbs4 (Zywitza et al. 2018) (45% of B cells; 28.77% in parenchymal astrocytes, 2.88 % in ependymal cells) are also expressed preferentially in B cells and parenchymal astrocytes, but they alone do not distinguish these two cell populations.”

   1. The authors term the germinal zone of the adult mouse brain - the ventricular-subventricular zone. They should discuss the evidence that the adult germinal zone is made up of cells from both the ventricular zone and the sub ventricular zone in the late embryo, where those zones are described clearly on the basis of morphology. Many of the early embryonic neural stem cells are present in the ventricular zone before the sub ventricular zone has developed and continue to be present into the adult. If there is not clear mouse evidence that the progeny of embryonic sub ventricular cells are present in the adult germinal zone independent of embryonic ventricular zone progeny, then the authors might consider calling the zone - the adult ventricular zone, or alternatively terming the neurogenic area around the lateral ventricle the adult germinal zone or by a more straightforward descriptive term - the adult subependymal zone or the adult periventricular zone. Also, I think the first word in line 6 on page 3 should be neural rather than neuronal.

   We agree that the terminology in the field is confusing and multiple names have been used to describe the same region. In order to clarify that we are referring to the same adult periventricular germinal region, we have added a short sentence in the introduction to indicate that the V-SVZ is also referred by other authors as the SVZ, the subependyma or subependymal zone: We have added in the text: “This neurogenic region has also been referred to as the SVZ or the subependymal zone (Kazanis et al. 2017; Morshead et al. 1994)”.

   This reviewer argues that the adult V-SVZ should only be called V-SVZ if a lineage relationship could be established with the embryonic SVZ. To our knowledge there is no need to link the adult SVZ to the embryo, as this structure, like the embryonic SVZ, anatomically sits beneath the VZ (the area next to the ventricle). However, a lineage relationship does exist between the adult V-SVZ and the embryonic VZ, established in previous studies showing that PreB1 cells around E15.5 became quiescent and give rise to adult B cells in the V-SVZ (Fuentealba et al., 2015; Furutachi et al., 2015). In addition, developmental studies show a continuum in the gradual transformation of the embryonic periventricular germinal layers, including the SVZ. Importantly, B1 cells are derived from VZ radial glia (RG), maintain RG markers and retain RG-like interkinetic behavior establishing that functionally and anatomically a VZ is retained in the adult (Merkle et al., 2004; Mirzadeh et al., 2008). Therefore the adult periventricular epithelium is not made of a pure layer of ependymal cells with progenitor cells underneath, as previously thought. Moreover, recent work indicates that just like in the embryo, the more basal adult SVZ progenitors (B2 cells) can be derived from adult VZ progenitors (B1 cells) (Obernier et al. 2018). This transformation of apical to basal cells begins to occur in embryonic stages further suggesting equivalences between the adult and the embryonic progenitor cells. For all the above reasons we prefer to use the term V-SVZ.

   In line 6, page 3, We have changed neuronal cell types to “neural cell types”, as suggested.

   1. The authors refer to their molecularly described B cells as stem cells. Certainly, their lab and others have shown that adult olfactory bulb neurons are the progeny of those B cells, however the classic definition of stem cells (in the blood or intestine for example) require demonstration that single stem cells can make all of the differentiated cells in that tissue. Is their evidence that a single adult B1 cell can make astrocytes, neurons and oligodendrocytes? Indeed, what percentage of the single adult B cells characterized here on the bases of RNA expression can be shown to be multipoint for both macroglial and neuron lineages in vivo or in vitro? Perhaps progenitor or precursor cells might be a better term for a B cells that appears to give rise to neurons primarily.

   This is also an issue of definitions. We modified the text to refer to the primary progenitors in the V-SVZ as adult neural stem cells, or progenitor cells “NSPCs”. We agree that this needs to be clarified and in the introduction we modified one paragraph to indicate:

   “From the initial interpretation that adult NSPCs are multipotent and able to generate a wide range of neural cell types (Reynolds and Weiss 1992; van der Kooy and Weiss 2000; Morshead et al. 1994), more recent work suggest that the adult NSPCs in vivo are heterogeneous and specialized, depending on their location, for the generation of specific types of neurons, and possibly glia (Merkle et al. 2014; Fiorelli et al. 2015; Chaker, Codega, and Doetsch 2016; Merkle, Mirzadeh, and Alvarez-Buylla 2007; Tsai et al. 2012; Delgado et al. 2020).”

   Under normal in vivo conditions, a primitive state for NSCs capable of generating all neuronal and glial cell types of the CNS may only exist at very early stages of development and even their regional specification seems to occur very early (as early as E10.5; Fuentealba et al. 2015). Note that recent work in the hematopoietic system suggests that stem cells there also become restricted embryonically (Carrelha et al., 2018) and in adults their potential to generate lymphoid or myeloid lineages changes dramatically with age, yet at all these ages they are referred as HSCs. We are well aware of the work from the van der Kooy lab, suggesting the existence in the V-SVZ of rare “primitive” Oct4+/GFAP- cells that may be pluripotent and earlier in the lineage from B cells (Reeve et al., 2017). However, as indicated above lineage tracing from the embryo indicates that adult NSPC are specified in the embryo and are already in place and regionally specified between E11.5 and E15. We have investigated whether we could detect Oct4+/Gfap- cells in our datasets. However, we did not detect Oct4 expression in B cells or other cell types. We now indicate in the discussion:

   “It has been suggested that in the adult V-SVZ a more primitive population of Oct4+/GFAP- NSCs may be present and that these cells may be earlier in the lineage from the “definitive” GFAP+ B cells (Reeve et al. 2017). However, regionally specified NSPCs can be lineage traced to the embryo (Fuentealba et al. 2015; Furutachi et al. 2015), and we could not detect a population of Oct4+ cells in our datasets. We, however, cannot exclude that rare primitive OCT4+ NSPCs were not captured in our analysis for technical reasons.” ……. “This underscores the early embryonic regional specification of adult V-SVZ NSPCs and how these primary progenitors maintain a memory of their regions of origin.”

   1. This may be more than a semantic issue, as the rare clonal neurophere forming neural stem cells that do make all three neural cell types in vitro, and also maintain their AP and DV positional identity through clonal passaging in vitro (Hitoshi et al, 2002). However, Emx1 expressing cortical neural stem cells can be lineage traced as they migrate from the embryonic cortical germinal zone to the striata germinal zone in the perinatal period (Willaime-Morawek et al, 2006). Surprisingly, in their new striatal home the Emx1 lineage cortical neural stem cells will turn down Emx1 expression and turn up Dlx2 striatal germinal zone expression. The switch in positional identities of clonal neural stem cells can be seen also in vitro when the stem cells are co-cultured with an excess of cells from a different region and then regrown as clonal neural stem cells. This may suggested that Emx1 expressing neural stem cells (the clonal neurosphere forming cells), may switch their positional identities in vivo as they migrate into the striatal germinal zone, but the downstream neuron producing precursor B cells studied in this paper may maintain their Emx1 expression into the adult germinal zone. This raises an interesting issue concerning which cells in the neural stem cell lineage can be regionally re-specified.

   The interesting question about plasticity and respecification is not addressed by our current manuscript that focuses on the gene expression profile of unmanipulated cells from adult samples. However, regional re-specification is controversial. While work from van der Kooy lab suggests that striatal Emx1+ NSPCs originate in the pallium and migrate into the striatum in the perinatal brain (Willaime-Morawek et la., 2006), other studies suggest that rare Emx1 cells are already present in the developing LGE from embryonic stages as early as E12.5 (Gorski et al. 2002). In addition, we have labeled neonatal radial glial cells in the pallium, when this migration has been suggested to occur, and do not see migration of cells ventrally into the striatal wall. We have also transplanted dorsal NSPCs into ventral locations -- and vice versa -- and do not observe evidence of regional re-specification (Merkle, Mirzadeh, and Alvarez-Buylla 2007; Delgado et al. 2020).

   1. The authors nicely show dorsal versus ventral germinal zone lineages are marked by some of the same positional genes from B cells to A cells, suggesting complete dorsal versus ventral neurogenic lineages giving rise to different types of olfactory bulb neurons. Indeed, they nicely test this idea with dissection of the dorsal versus ventral germinal zones, followed by nuclear RNA sequencing. However, they don't discuss the broader issues concerning the embryological origins of the dorsal versus ventral germinal zones. Emx1 is one of the genes the authors use to mark dorsal lineages. The authors reference papers (Young et al, 2007; Willaime-Morawek et al, 2006;2008) that use Emx1 lineage tracing to show that certain classes of olfactory bulb neurons originate from embryonic cortical neural stem cells that migrate perinatally from the cortical germinal zone into the dorsal subcortical germinal zone. Could cortical versus subcortical embryonic origins of the dorsal versus ventral adult germinal zone explain the origin of different sets of adult olfactory bulb neurons? Further, the authors report that one of the GO terms for their dorsal lineages in cortical regionalization.

   This is a very interesting question that unfortunately we cannot answer. The dorsal domain includes both pallial and subpallial components, but the specific origin of B cells in this dorsal domain and the contribution of the pallium and subpallium remains unresolved.

   We went back to our data to try to find evidence of pallial vs. subpallial components in the dorsal B clusters (5 & 22). Indeed, there are some hints that cluster 22 may be more pallial and 5 more dorsal subpallial. However, when we try to confirm differential distribution of markers associated with these two dorsal subdomains anatomically, it is not possible to determine segregation, likely due to the intermixing of cells as the wedge is formed. We also looked for Dbx1, a relatively specific marker of the border region between pallium and subpallium that has been termed ventral pallium, but unfortunately our scRNA-Seq dataset did not capture this marker. Further, targeted lineage tracing of this region is required to determine the subdivisions of the dorsal V-SVZ. We have added as requested a short discussion on this issue:

   “The dorsal V-SVZ domain is likely further subdivided into multiple subdomains. In the current analysis we pooled together clusters B(5) and B(22) as dorsal. However, largely pallial marker Emx1 and dorsal lateral ganglionic eminence marker Gsx2 were differentially enriched in clusters B(22) and B(5), respectively, suggesting that these two clusters may also represent different sets of regionally specified B cells with distinct embryonic origins. These regions become blurred by cells intermixing in the formation of the wedge region in the postnatal V-SVZ making it difficult to confirm their origin based on expression patterns. In addition to pallial and dorsal subpallial markers, this wedge region likely also includes what has been termed the ventral pallium (Puelles et al. 2016), which is characterized in the embryo by the expression of Dbx1. Unfortunately, our scRNA-Seq analysis did not detect this marker. Further lineage tracing experiments will help determine the precise embryonic origin and nature of the dorsal V-SVZ, including the wedge region.”

   1. The percentages of dividing cells based on gene expression is given for some clusters of cells but not others. It might be useful to have a chart showing the percentages of cells in cycle (ki67 expression) for each cluster. This might be especially useful in characterizing some fo the differences between various subclusters of B, A and C cells. On page 9 it is suggested that the heterogeneity amongst C cell clusters was driven by cell cycle genes. However, it is possible to remove the cell cycle genes from the data analysis to see if this then produces clearer dorsal versus ventral positional identities. This may be an important issue as the dorsal versus ventral positional identity genes appear to be expressed more in less dividing A and B cells, than in the more dividing C cells. This leads to a potentially alternative conclusion - that dorsal/ventral regional identity genes are primarily expressed in the non-dividing post mitotic cells in their resident dorsal or ventral region, and not in precursor cells in the lineage.This could be easiy tested by removing the cell cycle genes from the analysis of highly dividing clusters to see if they then break down into doral versus ventral clusters.

   We now provide a table indicating the fraction of proliferating cells (defined as in S phase or G2-M phase) for all scRNA-Seq clusters.

   Concerning whether dorsal and ventral identities are maintained during the period of proliferation we have analyzed our data looking at dorsal and ventral signature levels over pseudotime (Figure 6-Supplement 1F). Here we do not observe a reduction in either dorsal or ventral score at the proliferative cell stages (pseudotime ~0.75, Figure 2L). This is in contrast to gene signatures that show clear up- or down-regulation over pseudotime, such as Gfap, Egfr & Dcx (Figure 2M). To understand how cell clustering is affected in the absence of proliferative gene influence, and whether clearer dorsal/ventral signatures are observed in proliferating cells, we are performing additional analyses using our scRNA-Seq dataset that is clustered after cell-cycle gene regression.

   References Cited:

   Chaker, Zayna, Paolo Codega, and Fiona Doetsch. 2016. “A Mosaic World: Puzzles Revealed by Adult Neural Stem Cell Heterogeneity.” Wiley Interdisciplinary Reviews. Developmental Biology 5 (6): 640–58.

   Delgado, Ryan N., Benjamin Mansky, Sajad Hamid Ahanger, Changqing Lu, Rebecca E. Andersen, Yali Dou, Arturo Alvarez-Buylla, and Daniel A. Lim. 2020. “Maintenance of Neural Stem Cell Positional Identity by.” Science 368 (6486): 48–53.

   Fiorelli, Roberto, Kasum Azim, Bruno Fischer, and Olivier Raineteau. 2015. “Adding a Spatial Dimension to Postnatal Ventricular-Subventricular Zone Neurogenesis.” Development 142 (12): 2109–20.

   Fuentealba, Luis C., Santiago B. Rompani, Jose I. Parraguez, Kirsten Obernier, Ricardo Romero, Constance L. Cepko, and Arturo Alvarez-Buylla. 2015. “Embryonic Origin of Postnatal Neural Stem Cells.” Cell 161 (7): 1644–55.

   Furutachi, Shohei, Hiroaki Miya, Tomoyuki Watanabe, Hiroki Kawai, Norihiko Yamasaki, Yujin Harada, Itaru Imayoshi, et al. 2015. “Slowly Dividing Neural Progenitors Are an Embryonic Origin of Adult Neural Stem Cells.” Nature Neuroscience 18 (5): 657–65.

   Gorski, Jessica A., Tiffany Talley, Mengsheng Qiu, Luis Puelles, John L. R. Rubenstein, and Kevin R. Jones. 2002. “Cortical Excitatory Neurons and Glia, but Not GABAergic Neurons, Are Produced in the Emx1-Expressing Lineage.” The Journal of Neuroscience: The Official Journal of the Society for Neuroscience 22 (15): 6309–14.

   Kazanis, Ilias, Kimberley A. Evans, Evangelia Andreopoulou, Christina Dimitriou, Christos Koutsakis, Ragnhildur Thora Karadottir, and Robin J. M. Franklin. 2017. “Subependymal Zone-Derived Oligodendroblasts Respond to Focal Demyelination but Fail to Generate Myelin in Young and Aged Mice.” Stem Cell Reports 8 (3): 685–700.

   Kooy, D. van der, and S. Weiss. 2000. “Why Stem Cells?” Science 287 (5457): 1439–41.

   Merkle, Florian T., Luis C. Fuentealba, Timothy A. Sanders, Lorenza Magno, Nicoletta Kessaris, and Arturo Alvarez-Buylla. 2014. “Adult Neural Stem Cells in Distinct Microdomains Generate Previously Unknown Interneuron Types.” Nature Neuroscience 17 (2): 207–14.

   Merkle, Florian T., Zaman Mirzadeh, and Arturo Alvarez-Buylla. 2007. “Mosaic Organization of Neural Stem Cells in the Adult Brain.” Science 317 (5836): 381–84.

   Morshead, C. M., B. A. Reynolds, C. G. Craig, M. W. McBurney, W. A. Staines, D. Morassutti, S. Weiss, and D. van der Kooy. 1994. “Neural Stem Cells in the Adult Mammalian Forebrain: A Relatively Quiescent Subpopulation of Subependymal Cells.” Neuron 13 (5): 1071–82.

   Ponti, Giovanna, Kirsten Obernier, Cristina Guinto, Lingu Jose, Luca Bonfanti, and Arturo Alvarez-Buylla. 2013. “Cell Cycle and Lineage Progression of Neural Progenitors in the Ventricular-Subventricular Zones of Adult Mice.” Proceedings of the National Academy of Sciences of the United States of America 110 (11): E1045–54.

   Puelles, Luis, Loreta Medina, Ugo Borello, Isabel Legaz, Anne Teissier, Alessandra Pierani, and John L. R. Rubenstein. 2016. “Radial Derivatives of the Mouse Ventral Pallium Traced with Dbx1-LacZ Reporters.” Journal of Chemical Neuroanatomy 75 (Pt A): 2–19.

   Reeve, Rachel L., Samantha Z. Yammine, Cindi M. Morshead, and Derek van der Kooy. 2017. “Quiescent Oct4 Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein NSCs in the Adult Mouse Brain.” Stem Cells 35 (9): 2071–82.

   Reynolds, B. A., and S. Weiss. 1992. “Generation of Neurons and Astrocytes from Isolated Cells of the Adult Mammalian Central Nervous System.” Science 255 (5052): 1707–10.

   Tsai, Hui-Hsin, Huiliang Li, Luis C. Fuentealba, Anna V. Molofsky, Raquel Taveira-Marques, Helin Zhuang, April Tenney, et al. 2012. “Regional Astrocyte Allocation Regulates CNS Synaptogenesis and Repair.” Science 337 (6092): 358–62.

   Xie, Xuanhua P., Dan R. Laks, Daochun Sun, Asaf Poran, Ashley M. Laughney, Zilai Wang, Jessica Sam, et al. 2020. “High Resolution Mouse Subventricular Zone Stem Cell Niche Transcriptome Reveals Features of Lineage, Anatomy, and Aging.”Cold Spring Harbor Laboratory. https://doi.org/10.1101/2020.07.27.223602.

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3. 

   eLife

   Apr 7, 2021

   Reviewer #2 (Public Review):

   The paper is well written, and the data are well analyzed and presented. My concerns centre on terminology and alternative explanations of some of the data, which the authors might deal with in the introduction or discussion.

   1. I am slightly confused about some of the data shown in Figure 1. If B cells are defined as GFAP expressing cells, then why do only 25% of the B cells in the plot in Figure 1C express GFAP? I may be missing something here, as other readers may as well. Similarly in the same panel, only 25% of astrocytes seem to be expressing GFAP or GFP driven by a GFAP promotor.

   2. The authors term the germinal zone of the adult mouse brain - the ventricular-subventricular zone. They should discuss the evidence that the adult germinal zone is made up of cells from both the ventricular zone and the sub …

   Reviewer #2 (Public Review):

   The paper is well written, and the data are well analyzed and presented. My concerns centre on terminology and alternative explanations of some of the data, which the authors might deal with in the introduction or discussion.

   1. I am slightly confused about some of the data shown in Figure 1. If B cells are defined as GFAP expressing cells, then why do only 25% of the B cells in the plot in Figure 1C express GFAP? I may be missing something here, as other readers may as well. Similarly in the same panel, only 25% of astrocytes seem to be expressing GFAP or GFP driven by a GFAP promotor.

   2. The authors term the germinal zone of the adult mouse brain - the ventricular-subventricular zone. They should discuss the evidence that the adult germinal zone is made up of cells from both the ventricular zone and the sub ventricular zone in the late embryo, where those zones are described clearly on the basis of morphology. Many of the early embryonic neural stem cells are present in the ventricular zone before the sub ventricular zone has developed and continue to be present into the adult. If there is not clear mouse evidence that the progeny of embryonic sub ventricular cells are present in the adult germinal zone independent of embryonic ventricular zone progeny, then the authors might consider calling the zone - the adult ventricular zone, or alternatively terming the neurogenic area around the lateral ventricle the adult germinal zone or by a more straightforward descriptive term - the adult subependymal zone or the adult periventricular zone. Also, I think the first word in line 6 on page 3 should be neural rather than neuronal.

   3. The authors refer to their molecularly described B cells as stem cells. Certainly, their lab and others have shown that adult olfactory bulb neurons are the progeny of those B cells, however the classic definition of stem cells (in the blood or intestine for example) require demonstration that single stem cells can make all of the differentiated cells in that tissue. Is their evidence that a single adult B1 cell can make astrocytes, neurons and oligodendrocytes? Indeed, what percentage of the single adult B cells characterized here on the bases of RNA expression can be shown to be multipoint for both macroglial and neuron lineages in vivo or in vitro? Perhaps progenitor or precursor cells might be a better term for a B cells that appears to give rise to neurons primarily.

   4. This may be more than a semantic issue, as the rare clonal neurophere forming neural stem cells that do make all three neural cell types in vitro, and also maintain their AP and DV positional identity through clonal passaging in vitro (Hitoshi et al, 2002). However, Emx1 expressing cortical neural stem cells can be lineage traced as they migrate from the embryonic cortical germinal zone to the striata germinal zone in the perinatal period (Willaime-Morawek et al, 2006). Surprisingly, in their new striatal home the Emx1 lineage cortical neural stem cells will turn down Emx1 expression and turn up Dlx2 striatal germinal zone expression. The switch in positional identities of clonal neural stem cells can be seen also in vitro when the stem cells are co-cultured with an excess of cells from a different region and then regrown as clonal neural stem cells. This may suggested that Emx1 expressing neural stem cells (the clonal neurosphere forming cells), may switch their positional identities in vivo as they migrate into the striatal germinal zone, but the downstream neuron producing precursor B cells studied in this paper may maintain their Emx1 expression into the adult germinal zone. This raises an interesting issue concerning which cells in the neural stem cell lineage can be regionally re-specified.

   5. The authors nicely show dorsal versus ventral germinal zone lineages are marked by some of the same positional genes from B cells to A cells, suggesting complete dorsal versus ventral neurogenic lineages giving rise to different types of olfactory bulb neurons. Indeed, they nicely test this idea with dissection of the dorsal versus ventral germinal zones, followed by nuclear RNA sequencing. However, they don't discuss the broader issues concerning the embryological origins of the dorsal versus ventral germinal zones. Emx1 is one of the genes the authors use to mark dorsal lineages. The authors reference papers (Young et al, 2007; Willaime-Morawek et al, 2006;2008) that use Emx1 lineage tracing to show that certain classes of olfactory bulb neurons originate from embryonic cortical neural stem cells that migrate perinatally from the cortical germinal zone into the dorsal subcortical germinal zone. Could cortical versus subcortical embryonic origins of the dorsal versus ventral adult germinal zone explain the origin of different sets of adult olfactory bulb neurons? Further, the authors report that one of the GO terms for their dorsal lineages in cortical regionalization.

   6. The percentages of dividing cells based on gene expression is given for some clusters of cells but not others. It might be useful to have a chart showing the percentages of cells in cycle (ki67 expression) for each cluster. This might be especially useful in characterizing some fo the differences between various subclusters of B, A and C cells. On page 9 it is suggested that the heterogeneity amongst C cell clusters was driven by cell cycle genes. However, it is possible to remove the cell cycle genes from the data analysis to see if this then produces clearer dorsal versus ventral positional identities. This may be an important issue as the dorsal versus ventral positional identity genes appear to be expressed more in less dividing A and B cells, than in the more dividing C cells. This leads to a potentially alternative conclusion - that dorsal/ventral regional identity genes are primarily expressed in the non-dividing post mitotic cells in their resident dorsal or ventral region, and not in precursor cells in the lineage.This could be easiy tested by removing the cell cycle genes from the analysis of highly dividing clusters to see if they then break down into doral versus ventral clusters.

   Read the original source
4. 

   eLife

   Apr 7, 2021

   Reviewer #1 (Public Review):

   Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the manuscript is well …

   Reviewer #1 (Public Review):

   Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse V-SVZ. The authors generated two datasets: one which was whole cell RNA-seq of whole V-SVZ and one which consisted of nuclear RNA-seq of V-SVZ microdissected into anterior-posterior and dorsal-ventral quadrants. The authors first identified distinct subtypes of B cells and showed that these B cell subtypes correspond to dorsal and ventral identities. Then, they identified distinct subtypes of A cells and classified them into dorsal and ventral identities. Finally, the authors identified a handful of genes that they conclude constitute a conserved molecular signature for dorsal or ventral lineages. The text of the manuscript is well written and clear, and the figures are organized and polished. The datasets generated in this manuscript will be a great resource for the field of adult neurogenesis. However, the arguments and supporting data used to assign dorsal/ventral identities to B cells and A cells could be strengthened, and more rigorous data analysis could result in new biological insights into stem and progenitor cell heterogeneity in the V-SVZ.

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5. 

   eLife

   Apr 7, 2021

   Evaluation Summary:

   Redmond et al. use single-cell and single-nucleus RNA-sequencing to reveal the molecular heterogeneity that underlies regional differences in neural stem cells in the adult mouse. Prior work had separate subventricular stem cells as type A and B. By generating bulk and single cell transcriptome sequence data, the authors identified a distinct subtype of both A and B cells that differentiate into dorsal and ventral identities. They also identify a set of genes that constitute a conserved molecular signature of these cell types.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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6. 

   Version published to 10.1101/2021.02.10.430525v2 on bioRxiv

   Feb 16, 2021
7. 

   Version published to 10.1101/2021.02.10.430525v1 on bioRxiv

   Feb 10, 2021