A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The transcription factor Bcl11b/Ctip2 promotes hippocampal progenitor proliferation and neural differentiation in a non-cell autonomous manner by regulating the expression of the cell adhesion molecule Desmoplakin. Forebrain-specific ablation causes defective spatial learning and memory.

1. The microarray profiling experiment is performed from dentate gyrus tissue from control and mutant animals at P14. However the phenotypic changes affect a variety of cell types in the dentate gyrus in mutants and WT, including progenitors, immature and mature granule neurons. Also, there may be non-cell autonomous effects on cellular populations not examined, such as astrocytes, endothelial cells, and neural stem cell populations. Thus, caution should be taken to interpret changes in gene expression corresponding to mutant and WT dentate gyurs.
2. Along these lines, which cell populations express Desmoplakin? In Fig 6A-D, co-localization with cell-type specific markers should be performed along with Desmoplakin. Since the authors postulate that Desmoplakin is a direct transcriptional target of Bcl11b, does overexpression of Desmoplakin rescue, at least in part, the Bcl11b mutant phenotype? This could be explored, perhaps more easily, in the context of in vitro co-cultures with neural progenitors and mature hippocampal neurons?

Referee #2
Simon et al., investigated the role of Bcl11b in hippocampal development. Using a deletion strategy, they found that this gene is important for survival and maturation of hippocampal neurons and that the mutant mice display smaller hippocampi, impaired circuitry and reduced learning and memory. Transcriptome analyses and chromatin immunoprecipitation identify Dsp as a primary target of Bcl11b and, in fact, Dsp mutants display very similar phenotypes than Bcl11b mutants. The authors also report a reduced proliferation and abundance of stem/progenitor cells. Since Bcl11b is not expressed in neural stem/progenitors, the authors conclude that Bcl11b deletion, in addition to an impaired maturation of newborn neurons, also leads to cell-extrinsic signalling within the niche that depletes stem/progenitor cells.
The authors have identified and characterized a novel step in the differentiation and function of an important neurogenic niche. Their manuscript is well organized and written and the data are of exceptional quality. The conclusions about neuronal maturation and the involvement of Dsp as a downstream target of Bcl11b seem totally compelling to me.
Yet, I find some claim on stem/progenitor cells rather weak. The main reason for my criticism is that all quantifications (BrdU, Sox2, Tbr2 etc.) are given as average of cells / per section. As such, since the mutant hippocampi are smaller, a reduction in, say, BrdU may not necessarily imply a reduced proliferation of progenitors since progenitors could have died together with neurons. I could not find any evidence that the number of cells in the two sets of mice is being considered for normalization. If claims about proliferation of progenitors need to be made, then BrdU should be measured in cells expressing stem cell markers. Along these lines, I do not agree that the number of Sox2+ cells is reduced ( Fig 3J). The sum of Sox2+ cells in the GCL and SGZ is essentially identical in mutant and control (ca. 140 per section) and the fact that the cells are distributed differently between the two layers might just be a reflection of the fact that the whole hippocampus is disorganized, as the authors say: "missing sharp borders". All cell counts in the manuscript should take into account differences in tissue volume/area. Minor comments: I am rather puzzled by the differences in the Bcl11b and Dsp knock-outs with regard to apoptosis and behaviour. Perhaps the increase in apoptosis in Bcl11b at P14 is one of the earliest phenotypes of this mutant. Since Dsp is downstream of Bcl11b, it can be that apoptosis in Dsp knock out will become apparent later, say, at p21 (and that the apparent 'decrease' in apoptosis may again be due to lack of normalization of cell numbers). For the behavioural tests, what is the genetic background of the two mutants and what are their respective controls?

Referee #3
Simon et al show in their paper "A dual phase-specific function of Bcl11b/Ctip2 in hippocampal Neurogenesis" that the zinc-finger transcription factor Bcl11b is expressed in postmitotic dentate gyrus granule cells and that conditional deletion of Bcl11b during embryonic forebrain development disrupts proper development of the dentate formation. Furthermore, they show that Bcl11b appears to directly regulate the expression of desmoplakin and that genetic deletion of desmoplakin phenocopies at least partially the effects of Bcl11b deletion. They go on to show that Bcl11b deletion impairs certain forms of hippocampus-dependent learning and memory. Given these data they come to the following main conclusions: 1) Bcl11b indirectly controls progenitor proliferation in the developing DG, 2) Bcl11b is involved in granule cell differentiation and their functional integration. The data presented are convincing and the experimental design is straight-forward. Given that the transcriptional codes regulating dentate neurogenesis, persisting throughout life, remain poorly understood the data presented are of general interest. However, the manuscript has in its current form several shortcomings that should be addressed.
Simon and colleagues used Emx1Cre to delete Bcl11b early during development. Even though they did not observe substantial defects in the initial stages of dentate development the architecture of the granule cell layer and dentate formation are severely affected at early and later postnatal stages. Thus, a major concern is that Bcl11b is important for the layering and structural integrity of the dentate but that basically all other effects are secondary (e.g., altered proliferation, impaired differentiation). The authors speculate about a feedback mechanism depending on Bcl11b to regulate proliferation but there is no evidence for that. In fact, the proper niche appears to be fundamentally important for postnatal dentate neurogenesis. Thus, disruption of the niche may cause basically all phenotypes, somewhat independent of more direct mechanisms. We understand that this general concern is difficult to address but we feel that the authors need additional approaches to justify their conclusions. In fact the same holds true for the differentiation deficits. It is intriguing that the cells fail to express Calbindin but for instance it remains unclear if the stage of DCX expression is prolonged or if the cells eventually die (at which stage do they die?). Additional experiments with more mosaic (or sparse) deletion of Bcl11b in newborn cells would partially address this concern (what happens to newborn cells in an intact "surrounding" when Bcl11b is deleted?). Furthermore, the addition of in vitro experiments would at least partially allow to test for indirect effects of Bcl11b deletion on progenitor proliferation (using co-culture systems) and direct effects on granule cell differentiation.
Summarized, we think that this is a very solid and extensive study. However, we feel that more experiments are required to understand if Bcl11b deletion truly exerts a feedback mechanism on progenitor proliferation and if there is a cell-autonomous effect on granule cell differentiation.

Referee #1
This manuscript describes the feedback role of the transcription factor Bcl11b/Ctip2 during hippocampal neurogenesis. These findings extend the exciting and growing body of work that embryonic and postnatal neurons express molecules that regulate the onset of progenitor and neuroblast proliferation and differentiation. Bcl11b is specifically expressed in postmitotic dentate gyris granule neurons. Loss of Bcl11b at the onset of dentate development in Emx1-Cre; floxed Bcll11b mice leads to not only a decrease in granule neurons, but a reduction in the proliferation of neural progenitors, suggesting that Bcl111b plays both cell autonomous and non-cell autonomous roles on granule cell differerentiation. Surprisingly, the fraction of DCX+ and NeuroD+ immature neuroblasts/neurons is increased in Bcl111b cKO mice. The Bcl11b mutant phenotype is robust and the images are beautiful. It is very interesting that there is a negative feedback from the mature granule neurons to the proliferation of progenitor cells, however the mechanism by which Bcl11b mediates its actions is still somewhat unclear and could be further elaborated upon to strengthen the conclusions of this work.
> We added several additional experiments to the revised ms to further characterize the mechanisms of Bcl11b function and its downstream effector Desmoplakin in dentate gyrus development.
Together, these further support the dual role of Bcl11b in progenitor cell proliferation and neuronal differentiation of the dentate gyrus; they now unambiguously demonstrate that the role of Bcl11b in progenitor proliferation is caused by indirect mechanism. > We agree with reviewer 1, the tissue specimens we used for microarray analysis contain different cell populations, and the microarray expression data might as well reflect (indirect) changes in gene expression within non-neuronal (glia, endothelia, etc.) cells, which may in turn contribute to the Bcl11b mutant phenotype. To further explore this, we analyzed the expression of additional markers in the Bcl11b mutant dentate gyrus (new supplementary Figure S6). We found no obvious changes in glial cells, Cajal-Retzius cells, or the vascular pattern as determined by GFAP-and Reelin-and Pecam expression, respectively, and the overall architecture of the dentate gyrus based on the expression of these molecular markers was unchanged. Fig 6A- > We performed co-expression analyses using antibodies against Desmoplakin (Dsp), as well as markers for postmitotic granule neurons (Bcl11b) and dentate progenitor cells (Sox2; new Figure  6E-H). We found that high levels of Dsp protein expression are restricted to Bcl11b positive postmitotic granule cell neurons. Only faint Dsp expression was detected in cells located in the SGZ with part of them co-expressing Sox2.

Along these lines, which cell populations express Desmoplakin? In
> Using ex utero electroporation followed by organotypic slice culture of the dentate gyrus, we introduced Dsp cDNA into Bcl11b mutant dentate granule neurons (new Figure 8). Mosaic reexpression of Dsp in part of the cells completely rescues the proliferation deficits observed in Bcl11b mutants. This suggests that Dsp is an important downstream effector of Bcl11b in the control of dentate progenitor proliferation. We further observed that increased BrdU incorporation was not confined to GFP/Dsp expressing cells, providing additional support for an indirect feedback function of Bcl11b/Dsp in the control of progenitor proliferation. This is in good agreement with our expression data (new Figure 6E-H). Due to the limited numbers of mice available, and the short culture periods used for the proliferation studies, we did not analyze whether re-expression of Dsp rescues differentiation deficits.

reduction in, say, BrdU may not necessarily imply a reduced proliferation of progenitors since progenitors could have died together with neurons. I could not find any evidence that the number of cells in the two sets of mice is being considered for normalization. If claims about proliferation of progenitors need to be made, then BrdU should be measured in cells expressing stem cell markers. Along these lines, I do not agree that the number of Sox2+ cells is reduced (Fig 3J). The sum of Sox2+ cells in the GCL and SGZ is essentially identical in mutant and control (ca. 140 per section) and the fact that the cells are distributed differently between the two layers might just be a reflection of the fact that the whole hippocampus is disorganized, as the authors say: "missing sharp borders". All cell counts in the manuscript should take into account differences in tissue volume/area.
> Although we observed granule cell dispersion in the Bcl11b mutant dentate gyrus resulting in less sharp borders of the granule cell band, it was still possible to identify the SGZ, i.e. the accumulation of BrdU incorporating cells was confined to a narrow zone beneath the actual granule cell layer. Furthermore, we used markers that identify progenitors (Sox2, Tbr2). To clarify this, we included in the revised manuscript triple immuno-stainings (BrdU/Sox2/NeuN) visualizing the SGZ in controls and Bcl11b mutants (revised Figure 3I-J).
> These experiments show that the total numbers of Sox2+ cells are unchanged in the mutant dentate gyrus, but their distribution is changed, with a significant shift from the SGZ to the GCL (new Figure 3). This raises the question whether the mislocated Sox2+ cells have changed their proliferation capacities. To explore this, we determined the numbers and distributions of Sox2+, BrdU+, and Sox2/BrdU or Tbr2/BrdU double positive cells (as suggested by reviewer 2) in the SGZ, the GCL, and the entire dentate gyrus of controls and Bcl11b mutants. Compared to controls, we observe a significant reduction of BrdU-, Sox2-, Sox2/BrdU-positive cells in the SGZ. This suggests that dentate progenitor cells are depleted from the SGZ. While numbers of Sox2+ cells in the GCL are significantly increased, numbers of BrdU positive cells are unchanged. Thus, the mislocated mutant progenitors of the GCL have partially lost their proliferation capacity.
> Similar changes in proliferation and distribution of Sox2+ cells are also observed when we mutate Bcl11b using NexCre, which restricts the mutation to postmitotic granule neurons but spares progenitor cells (Goebbels et al., Genesis 44:611-621 (2006); Seuntjens et al., Nature Neuroscience 12:1369-1376). Both mutants display very similar phenotypes (new Figure 3C, K-P, new supplementary Figure S5). Figure 3C) were increased in the GCL but not significantly changed in the SGZ. This argues against the hypothesis that Bcl11b mutant progenitors died together with neurons. Clearly, apoptotic loss of cells contributes to the overall change observed in the Bcl11b mutant dentate gyrus but it mostly affects postmitotic neurons in the GCL. We speculate that these neurons die due to impaired differentiation and integration into the hippocampal circuitry.

> Numbers of apoptotic cells observed in Bcl11b mutants (new
> Mosaic re-expression of Dsp rescues the proliferation deficits in Bcl11b mutant dentate gyrus, providing further experimental support for an indirect feedback control of progenitor proliferation by Bcl11b and its downstream effector Dsp.
> The overall volume of the Bcl11b mutant dentate gyrus is smaller as compared to controls. However, this does not necessarily reflect reduced neuron numbers. The volume/sectional area of the dentate gyrus is very much influenced by the molecular layer and the length, branching behavior and connectivity of dendrites therein. Thus, even if numbers of dentate granule neurons are unchanged, the overall volume of the dentate gyrus can be significantly smaller due to, for example, hypoplastic dendrites. We observe changes in the molecular layer and in dendrites of Bcl11b mutants, and this is most likely part of the impaired differentiation of mutant granule neurons. Moreover, we observed that normalization of cell counts for reduced organ volume would have led to the inappropriate overestimation of certain aspects of the Bcl11b mutant phenotype, as for example the piling-up of immature neurons.
> Please see also response to reviewer 1.

Minor comments: I am rather puzzled by the differences in the Bcl11b and Dsp knock-outs with regard to apoptosis and behavior. Perhaps the increase in apoptosis in Bcl11b at P14 is one of the earliest phenotypes of this mutant. Since Dsp is downstream of Bcl11b, it can be that apoptosis in Dsp knock out will become apparent later, say, at p21 (and that the apparent 'decrease' in apoptosis may again be due to lack of normalization of cell numbers). For the behavioural tests, what is the genetic background of the two mutants and what are their respective controls?
> We analyzed apoptosis in Dsp mutants also on later developmental stages. At P30 (additional data are included in the revised ms) apoptosis in the Dsp mutant dentate gyrus was unchanged. Thus, the increased apoptosis observed in Bcl11b mutants is Dsp independent. Bcl11b controls several aspects of dentate gyrus development: progenitor cell proliferation through an indirect feedback mechanism; correct differentiation and functional integration of postmitotic granule cell neurons. It is likely that Bcl11b has more than just a single critical target in granule cell neurons. We identified Dsp to be one target, and in good agreement, deletion of Dsp in the hippocampus recapitulates part of the phenotype (i.e. progenitor proliferation and granule cell differentiation) of Bcl11b mutants. One important difference in the phenotypes of both mutants is that mossy fiber projections are severely altered in Bcl11b but not in Dsp mutant mice. Correct mossy fiber projections are critical for the formation of trisynaptic circuitry of the hippocampus, and thus may well account for the behavioral differences between both mutants. We did not intend to describe Dsp as the only "critical" downstream effector of Bcl11b in hippocampal development. Simon and colleagues used Emx1Cre to delete Bcl11b early during development. Even though they did not observe substantial defects in the initial stages of dentate development the architecture of the granule cell layer and dentate formation are severely affected at early and later postnatal stages. Thus, a major concern is that Bcl11b is important for the layering and structural integrity of the dentate but that basically all other effects are secondary (e.g., altered proliferation, impaired differentiation). The authors speculate about a feedback mechanism depending on Bcl11b to regulate proliferation but there is no evidence for that. In fact, the proper niche appears to be fundamentally important for postnatal dentate neurogenesis. Thus, disruption of the niche may cause basically all phenotypes, somewhat independent of more direct mechanisms. We understand that this general concern is difficult to address but we feel that the authors need additional approaches to justify their conclusions. > We addressed both issues by several additional experiments. We analyzed the progenitor compartment in Bcl11b mutants in detail and used a NexCre line for mutation, thereby restricting the deletion of Bcl11b to the postmitotic neuronal compartment (new Figure 3). In addition, using ex utero electroporation followed by organotypic slice culture of the dentate gyrus, we show that reexpression of Desmoplakin in Bcl11b in a fraction of the mutant granule cells suffices to normalize the proliferation deficits observed in Bcl11b mutant animals (new Figure 8). Finally, using ex utero electroporation of GFP-Cre followed by slice culture, we generated a mosaic deletion of Bcl11b in the dentate gyrus. By this approach we addressed the question whether Bcl11b is required cellautonomously for neuronal differentiation. We now show that impaired granule cell differentiation, as determined by the piling-up of immature, NeuroD positive cells, is not restricted to Bcl11b mutant cells (new Figure 4). Thus, deletion of Bcl11b in a fraction of the dentate neurons suffices to disrupt neuronal differentiation in surrounding tissue still expressing Bcl11b. This suggests that the effect of Bcl11b on neuronal differentiation is, in part, caused by indirect mechanisms. An indirect role of Bcl11b in granule cell differentiation is in good agreement with recently published work on the functions of Bcl11b in keratinocytes, and it is well established that several transcription factors expressed in the developing dentate gyrus act indirectly on neurons through paracrine signals (for example Sox2/Shh) In the revised ms we discuss our findings in light of the recent literature.
> Please see also our detailed response to reviewers 1 and 2 on these issues. Thank you for submitting your revised manuscript to the EMBO Journal. Your revision has now been seen by the three referees. As you can see below, the referees appreciate the introduced changes and they support publication in the EMBO Journal.
I am therefore very pleased to proceed with the acceptance of the paper for publication here. You will receive the formal acceptance letter shortly.
Thank you for submitting your interesting manuscript to the EMBO Journal.
Yours sincerely

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