Chromatin regulator Kdm6b is required for the establishment and maintenance of neural stem cells in mouse hippocampus

Reviewer #1 (Public Review):

Summary:

The authors have previously studied the function of the lysine demethylase Kdm6b as a positive regulator of neurogenesis from subventricular zone neural precursors. Here they knockout Kdm6b in progenitors of the dentate gyrus and show convincingly that deletion causes precocious differentiation of these stem cells. These data are valuable and show that Kdm6b can have very different functions in distinct populations of neuronal progenitors.

Strengths:

Kdm6b has repeatedly been implicated as a positive regulator of differentiation in the cellular transitions where it has been studied before. By contrast, here the authors show convincingly that it is required for maintenance of the stem cell state in the hippocampus, and that Kdm6b deletion is associated with premature stem cell differentiation and a small dentate gyrus in the adult hippocampus. Inducible deletion of Kdm6b in adult hippocampal stem cells confirms the precocious differentiation and loss of this population in the absence of Kdm6b even when induced at this later age.

Weaknesses:

This is a surprising finding in light of many other papers that are well-cited by the authors, including their own studies of SVZ progenitors where Kdm6b promotes neuronal differentiation. However, the weakness of the study is that the authors shed very little light on why the effects of Kdm6b would be so different (in fact, largely opposite) in the two stem cell populations they have studied.

Reviewer #2 (Public Review):

Summary:

Gil & Lim et al. applied mouse genetic models to study the roles of chromatin regulator KDM6B in regulating the development of the hippocampal dentate gyrus (DG), as well as the establishment and maintenance of DG NSCs. KDM6B is expressed in postnatal DGs. Importantly, conditional knockout of Kdm2b in embryonic DG progenitors leads to a significantly smaller DG with loss of DG NSCs. Hippocampal-dependent behaviors are defective in Kdm6b-cKO mice. Deletion of Kdm6b results in precocious neuronal differentiation and loss of the NSC population in both postnatal and adult DGs. Single-cell RNA-seq reveals disrupted stem cell maintenance gene signature in Kdm6b-deleted NSCs. Moreover, CUT&RUN studies showed that Kdm6b deletion increases H3K27me3 levels at a few NSC maintenance genes.

Strengths:

The conclusions of this paper are mostly well supported by data. The discussion is thorough.

Weaknesses:

I concur with the two reviewing editors who noted that the paper lacks insights into how KDM6B regulates the expression of NSC genes in DG precursors. Additionally, the authors did not provide evidence regarding whether the function of KDM6B is enzymatically dependent.

The Kdm6b-cKO brain exhibited apparently smaller DGs, indicating compromised neurogenesis. While the authors observed an increased number of IPCs in the E17.5 DGs (Figure 4B-4C) and an increased number of BrdU+TBR2+PROX1+ cells in the P0.5 DGs (Figure 5B-5C), it is perplexing why this does not lead to an increased number of PROX1+ DG neurons? Further investigation into the cellular mechanisms underlying these events would enhance the understanding of Kdm6b's role in neurogenesis.

Many data were not of sufficient quality and should be improved.

Reviewer #3 (Public Review):

Gil et al provide novel evidence that the chromatin regulator KDM6B is important for establishing and maintaining the neural stem cell (NSC) pool within the dentate gyrus in development and adulthood. They show compelling evidence that loss of KDM6B promotes precocious neuronal differentiation, resulting in a failure to establish and maintain the dentate gyrus NSC pool. The strongest evidence they provide is their immunohistochemistry analysis, in which they observed precocious expression of later differentiation markers from cells marked by BrdU. However, given that KDM6B is ubiquitously expressed, it is difficult to ascertain if their dysregulation is due to a direct loss of KDM6B within NSCs or caused by dysregulation of other glial cells impacted by KDM6B loss through the hGFAP-Cre. Characterization of mature glia would strengthen the work.

They additionally provide evidence of precocious differentiation through scRNA-seq by highlighting key genes that are dysregulated with KDM6B loss. It appears the clustering analysis into cell types was done with WT and KDM6b-depleted cells together. The evidence for precocious differentiation would be greatly strengthened if they instead determined cell-type specific clusters using their WT samples and then observed if fewer cells are characterized as NSCs and more cells align to later developmental stage clusters with KDM6B depletion.

Gil et al propose that KDM6B loss leads to hippocampus-specific impairments in learning and memory. While KDM6B-depleted mice do show a significant decrease in freezing time in contextual fear conditioning, Figure 2 Supplement 1 shows KDM6B-depleted mice are hyperactive compared to WT in the open field test. Thus, the reduction in freezing could be due to hyperactivity. Plotting freezing time in short bins throughout the duration of the test can help clarify this. It would be additionally helpful to plot the training baseline and the test on the same graph and compare their freezing from baseline to clarify if they completely fail to freeze or show a reduction in freezing compared to the wild-type.

Author response:

We thank the reviewers for their positive evaluation and constructive comments. In our revision, we will aim to improve the analysis of our existing data and perform new experiments to address questions raised by the reviewers.

Reviewer 1 found it interesting that Kdm6b-deletion in hippocampal dentate gyrus (DG) neural stem cells causes precocious neuronal differentiation, whereas in contrast, Kdm6b is required for the maturation of neural progenitors in the ventricular-subventricular zone (V-SVZ). In the submitted manuscript, we did not provide much insight into the differences in Kdm6b function in these two neural stem cell populations. We plan on performing new experiments and expanding on our prior V-SVZ studies in a way that allows a direct comparison to the analyses of the DG. We hope that the addition of this data will shed light on why Kdm6b-deletion produces such different phenotypes in postnatal neural stem cells of the mouse brain.

Reviewer 2 noted that our submitted manuscript lacked insight into how KDM6B regulates gene expression. In particular, this reviewer asked whether the function of KDM6B is mediated by its enzymatic activity. The CUT&RUN experiment in our manuscript revealed an increase in H3K27me3 levels at select neural maintenance genes in the DG of Kdm6b-deleted mice. However, we agree that this data is insufficient to assess the significance of KDM6B-mediated H3K27me3 demethylation in regulating the NSC transcriptome. To address this point, we are performing experiments that can directly test this mechanistic model of KDM6B function and answer the question of whether the H3K27me3 demethylase activity of KDM6B is required for its ability to activate transcription. Reviewer 2 also had a specific question about the cell types observed in the developing hippocampus after Kdm6b-deletion, and we believe that additional analyses will provide clarity to the overall phenotype. More generally, we will aim to improve data quality and visualization.

Reviewer 3 raised the concern that because Kdm6b is not exclusively expressed in neural stem cells, the phenotype of precocious neuronal differentiation in mice with Kdm6b-deletion driven by the hGFAP-Cre transgene may be indirect, such as through changes in mature glial populations. We will study the mature glia, as suggested by the reviewer. We will also more thoroughly describe how our experiments targeting Kdm6b-deletion to adult neural stem cells with the tamoxifen-inducible Nestin-CreER provide evidence for the precocious neuronal differentiation phenotype being cell autonomous, at least in adult mice. Reviewer 3 also had helpful suggestions for analyzing our single-cell RNA-seq data and behavioral studies, and we will address these comments in the revision.

Again, we thank the editors and reviewers for their time and consideration. We believe that our manuscript will be greatly improved through this review process and hope to construct a stronger understanding of the role of KDM6B in DG neurogenesis.