Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons
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Abstract
GABAergic neurons of the hypothalamus regulate many innate behaviors, but little is known about the mechanisms that control their development. We previously identified hypothalamic neurons that express the LIM homeodomain transcription factor Lhx6, a master regulator of cortical interneuron development, as sleep-promoting. In contrast to telencephalic interneurons, hypothalamic Lhx6 neurons do not undergo long-distance tangential migration and do not express cortical interneuronal markers such as Pvalb . Here, we show that Lhx6 is necessary for the survival of hypothalamic neurons. Dlx1/2 , Nkx2-2 , and Nkx2-1 are each required for specification of spatially distinct subsets of hypothalamic Lhx6 neurons, and that Nkx2-2+/Lhx6+ neurons of the zona incerta are responsive to sleep pressure. We further identify multiple neuropeptides that are enriched in spatially segregated subsets of hypothalamic Lhx6 neurons, and that are distinct from those seen in cortical neurons. These findings identify common and divergent molecular mechanisms by which Lhx6 controls the development of GABAergic neurons in the hypothalamus.
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##Author Response
###Reviewer #1
This paper investigates the role of Lhx6 and other transcription factors in the development of GABAergic neurons in the hypothalamus. The authors report that a small fraction of hypothalamic GABAergic neurons express Lhx6 and further depend on this expression for their survival. Dlx1/2, Nkx1-1 and Nkx2-2 define 5 subpopulations and at least three of these populations depend on these TFs to maintain Lhx6 expression. A strength of the paper is the multimodal analysis and the fact that descriptive assays like RNAseq and ATACseq are followed up with specific knockouts of candidate transcription factors. However, the relationships between the developmental populations identified and adult subtypes of hypothalamic neurons remain unclear. Although the results will surely interest those already interested in …
##Author Response
###Reviewer #1
This paper investigates the role of Lhx6 and other transcription factors in the development of GABAergic neurons in the hypothalamus. The authors report that a small fraction of hypothalamic GABAergic neurons express Lhx6 and further depend on this expression for their survival. Dlx1/2, Nkx1-1 and Nkx2-2 define 5 subpopulations and at least three of these populations depend on these TFs to maintain Lhx6 expression. A strength of the paper is the multimodal analysis and the fact that descriptive assays like RNAseq and ATACseq are followed up with specific knockouts of candidate transcription factors. However, the relationships between the developmental populations identified and adult subtypes of hypothalamic neurons remain unclear. Although the results will surely interest those already interested in hypothalamic development, it is not clear that broader developmental or functional principles have been identified. The authors make much of the fact that the identified populations do not resemble forebrain interneurons defined by Lhx6 expression, but it is not clear why this should have been expected. Many developmental transcription factors are utilized both across diverse brain regions and across tissues outside of the brain. Perhaps the emphasis of this point could be tempered.
We thank the Reviewer for his/her comments, although we respectfully but strongly disagree with the statement that “it is not clear that broader developmental or functional principles have been identified”. This manuscript aims to provide a broad overview, and by no means exhaustive, an overview of the molecular mechanisms controlling the development of hypothalamic neurons that express Lhx6. Although these neurons comprise only approximately 2% of all hypothalamic GABAergic neurons, they are highly heterogeneous at the molecular level. Using traditional methods such as histology and more recent methods such as scRNA-Seq, we have not found a selective marker of hypothalamic Lhx6+ neurons other than Lhx6 itself. However, we have found multiple spatially distinct domains in hypothalamic Lhx6+ neurons that express specific sets of transcription factors such as Dlx1/2, Nkx2-1, and Nkx2-2, as we and others have previously observed in developing hypothalamic nuclei.
In addition, a subpopulation of these neurons later gives rise to a subset of Lhx6+ neurons of the zona incerta, which have been previously shown by us to promote sleep. Unlike all previously described sleep-promoting neurons, Lhx6+ zona incerta neurons are only one of few neuronal subtypes that can regulate both REM and NREM, which likely reflects molecular and functional heterogeneity among these neurons.
Our thus manuscript speaks to both broader developmental principles by demonstrating the molecular heterogeneity of hypothalamic Lhx6+ cells that arises through the action of diverse transcriptional networks, and broader functional principles by identifying developmental networks that potentially control the specification, differentiation, and survival of sleep-promoting neurons.
We believe that there are several compelling reasons for including a direct comparison of hypothalamic and cortical Lhx6 neurons, both of which arise from different regions of the forebrain (or secondary prosencephalon, if using the prosomere model). First, the role of Lhx6 in development of telencephalic interneurons is extensively studied, with 72 publications ((Pubmed: Lhx6 AND development AND (cortex OR telencephalon OR interneuron), accessed 7/27/20), and virtually all our understanding of how Lhx6 controls neuronal development has been acquired from this work. It is thus critically important that we directly connect our findings to a prior understanding of the mechanism of action of Lhx6.
Second, current work in the field of developmental neuroscience in general, is heavily focused on studying telencephalic development. It is very much an open question, however, whether telencephalic structures are themselves particularly good models for studying the development of physiologically vital brain regions, such as the hypothalamus. By identifying many key differences in the function of this extensively studied gene between Lhx6+ MGE-derived neural precursors and hypothalamic Lhx6+ neurons, we establish some important caveats in generalizing studies of telencephalic development even to nearby forebrain structures.
Nonetheless, we certainly agree with the Reviewer that the organization and clarity of the manuscript can be substantially improved. To this end, we have revised the manuscript carefully to improve clarity, focusing on its key findings.
The presentation of the manuscript could be improved by clarifying the relationships between embryonic and more mature structure within the hypothalamus. For example, It is extremely hard to follow the evidence split across figures 5, S6 and S7 for parsing the cell groups by TF expression.
We have revised the manuscript carefully to improve clarity. We have moved scRNA-Seq analysis of postnatal Lhx6-expressing neurons as Fig 3, and embryonic Lhx6-expressing neurons as Fig. 4, to improve the overall flow of the manuscript.
The ATAC seems to be used only to bolster the impression that the populations identified by gene expression are different. The description of footprinting seems to imply an effort to analyze binding sites for specific factors (e.g. to identify targets of the TFs studied), but the statistical approach employed and even the conclusions reached are not fully spelled out. As such, this part of the study is underdeveloped or not well enough described.
Specific details of the ATAC-Seq analysis are extensively described in the Method section, with each bioinformatics package (and package version) listed and, when non-default parameters were used, parameters clearly stated. However, we have added details of the statistical approaches used for data analysis to the revised manuscript.
There is little use in conducting ATAC-Seq analysis without a matched RNA-Seq dataset, as changes in peaks (open chromatin regions) do not necessarily correlate with changes in gene expression levels. By integrating ATAC-Seq data with differential gene expression obtained using RNA-Seq, we have been able to identify changes in motif accessibility and candidate transcription factor footprinting that to identify changes in gene regulatory networks that control Lhx6 expression in both hypothalamus and cortex. We have revised the manuscript to make this clearer, and better explain the findings of this part of the study.
###Reviewer #2:
Kim and colleagues used a combination of state-of-art sequencing and mouse genetic tools to study the mechanisms that control the development of a subset of GABAergic neurons in the developing hypothalamus.
While neurodevelopment of GABAergic neurons has been extensively studied in the developing telencephalon, little is known about their counterparts in the developing hypothalamus. The authors focused their work on a specific subset of GABAergic neurons that express the LIM homeodomain factor Lhx6. Lhx6 is a master regulator of GABAergic neuron differentiation, specification, and migration in cortical interneurons. In contrast, Lhx6-expressing neurons make up only 2-3% of GABAergic neurons in the hypothalamus. The authors' previous work demonstrated that these neurons play a critical role in sleep homeostasis. Therefore, understanding how these neurons are formed and maintained is of great importance.
The authors show that hypothalamic Lhx6 is necessary for neuronal differentiation and survival. Furthermore, by profiling and comparing multiple RNA-seq, scRNA-seq, and ATAC-seq datasets, they were able to identify three transcription factors Nkx2.1, Nkx2.2, and Dlx1/2 that each delineates non-overlapping subdomains of Lhx6 neurons and are necessary for Lhx6 expression in the hypothalamus. Finally, the authors demonstrate that mature Lhx6 neurons manifest extensive molecular heterogeneity that is distinct from their counterparts in the telencephalon.
We thank the Reviewer for his/her comments, and for appreciating the key findings of the manuscript.
The work presented is of high quality and is a technological tour de force. The scope and depth of the study are unparalleled among similar studies of hypothalamic neurodevelopment. That said I only have a couple of minor suggestions.
- In Figure S2, the number of tomato+ cells appear to be reduced, but not eliminated. Do the authors think that Lhx6 is necessary for the survival of all Lhx6 neurons, or just a subset? The use of the floxed Bax allele is clever, but is there evidence directly supporting increased cell death? Can the authors completely rule out the possibility of the mismigration of cell bodies after the postnatal deletion of Lhx6?
We appreciate the Reviewer for his/her comments. We conclude that Lhx6 is necessary for the survival of all Lhx6 neurons due to the lack of read-through transcription in Lhx6-CreER/CreER mice (Fig 2), and the rescue of Lhx6-deficient mice that is seen using conditional Bax mutants (Fig. 2). The fact that numbers of cells labeled with Lhx6-CreER are rescued by the deletion of this key positive regulator of apoptosis strongly implies that Lhx6-deficient neurons simply die. Finally, we observe very few Lhx6-expressing hypothalamic neurons that undergo even short-range tangential migration (Fig. 1), and observe no evidence for an increase in these cells in the analysis described in Fig. 2.
The fact that postnatal loss of function of Lhx6 leads to a more modest cell loss than the constitutive mutant may simply reflect a reduced overall requirement for Lhx6 in regulating neuronal survival in the postnatal hypothalamus or may indicate that the survival of a specific subset of Lhx6+ neurons is no longer Lhx6-dependent at this age. We cannot currently distinguish between these alternatives, and state this fact in the text.
- In Figure 4, the authors acknowledged that the ectopic gene expression in Lhx6CreER/lox; Baxlox/lox mice could be due to the loss of function of Bax. If so, would Lhx6CreER/+; Baxlox/lox mice be a better control in this experiment?
We initially thought of using Lhx6-CreER/+;Baxlox/lox as a control since our phenotype could be due to loss of Bax itself, but not due changes in cell survival. However, we observed the same rescue phenotype in initial experiments using Lhx6-CreER/Bak-null (#006329), which strengthened our initial hypothesis. We now discuss potential limitations that may result from the fact that RNA-Seq data from Lhx6CreER/+;Baxlox/lox mice is not included in this study.
###Reviewer #3:
Kim et al. aimed to characterize the similarities and differences between the development and molecular identity of telencephalic versus hypothalamic (HT) Lhx6+ GABAergic neurons. By analyzing a diverse repertoire of transgenic mice at different developmental stages and through the use of fate mapping, bulk and single cell sequencing approaches, ISH and immunostaining, the authors descriptively compare transcriptional networks and upstream regulators of LHX6. They found essential differences between LHX6-dependent networks and those in telencephalic neurons and suggest a role of LHX6 in survival instead of migration regulation HT neurons. Moreover, spatially distinct LHX6+ HT cell clusters were identified and transcriptionally profiled.
- Only 1-2% of the GABAergic neurons express LHX6, and the cells expressing LHX6 in the HT were identified to be very diverse. Apart from a putative role for LHX6 in promoting the survival of HT neurons, which in my opinion is not analyzed convincingly, nothing functional was revealed. For this, I do not judge the potential significance and influence of the findings as broad or fundamental.
We respectfully but strongly disagree with this conclusion, most of which have already been described at length in our response to Reviewer #1. In brief, hypothalamic Lhx6+ neurons are key regulators of sleep initiation and maintenance, and nothing is known about their development. In much the same way that studies of the development of Lhx6+ cortical interneurons potentially help inform our understanding of neurodevelopmental disorders such as autism, so too may an understanding of the development of hypothalamic Lhx6+ neurons improve our understanding of sleep disorders and their treatment. In this study, we characterize the fate of hypothalamic Lhx6+ neurons, identify transcriptional regulatory networks that control their patterning and survival, and characterize their molecular heterogeneity in the postnatal period. We identify the homeodomain factor Nkx2.2 as a key regulator of both regional patterning of hypothalamic Lhx6 neurons, but also as a marker of a substantial subset of Lhx6+ ZI neurons that are activated by sleep pressure. This represents the groundwork needed for a basic understanding of the development of this physiologically important cell type, and forms the basis of more detailed future studies.
Unless the Reviewer simply believes that studies of hypothalamic development are inherently uninteresting and of little significance, these comments simply do not seem to reflect a careful reading of the manuscript, and come across as vague and unconstructive. In future reviews, we urge the Reviewer to be more specific, and to offer concrete and constructive comments, to support sweeping statements of this sort.
- The manuscript could be better focused, and more coherent. The authors jump between different aspects of the story. First, the authors address a potential role of LHX6 in survival regulation in HT interneurons, and try to identify potential LHX6 target genes mediating this effect. The latter was neither analyzed convincingly nor validated. Then the authors switch to the comparative analysis of transcriptional networks in cortical versus hypothalamic LHX6+ interneurons, and the identification of different clusters of LHX6+ HT cells. Next, potential upstream regulators of LHX6 in HT neurons were addressed by fate mapping studies. Then, the authors again switch focus, and analyzed distinct anatomical regions covered by Lhx6+ neurons by single cell RNA seq and investigated an instructive role of Nkx2-1, Nkx2-2 and Dlx1/2 in the establishment of these hypothalamic regions.
Subheadings in the result section might be very useful. However, the focus of this study requires clarification and also respective consideration in the introduction.
As stated in our response to Reviewer #1, we have sought to conduct a broad characterization of the development and diversity of hypothalamic Lhx6+ neurons, a subset of which are important regulators of sleep. While we cover multiple aspects of this question, we strongly disagree that the manuscript “lacks focus”. However, we do agree that organization and clarity could be improved. To this end, we have incorporated subheadings into the Results section, and clearly outlined the experiments conducted, and the reasons why each were conducted.
- The authors use a variety of different reporter and loss of function mouse models and jump between developmental stages for analysis. Apart from being confusing, the experimental/analytical pipeline is not sufficiently rigorous with respect to age and genetic background. E.g. to analyze target genes of LHX6 through which the effect on cell survival could be mediated, the authors compared expression profiles from P10 Lhx6CreER/+;Ai9 neurons with hypothalamic and cortical Lhx6-GFP positive and negative cells from P8 mice. Hypothalamic enriched genes were then compared to single-cell RNA-Sequencing (scRNA-Seq) datasets of E15.5 and P8 hypothalamic Lhx6-expressing neurons. Transcriptional profiles tremendously change with progressing development, and different mouse lines were used, which were not all time-matched. This might have caused Lhx6-independent variation, which likely masks relevant genes. This could be an explanation why so few LHX6 target genes were identified through which LHX6 putatively acts on neuronal survival.
This is another instance where the Reviewer seems to have failed to appreciate the rationale for the work presented here. We have modified the text to make this clearer. In summary, while it is certainly true that gene expression patterns are dynamic during development, cells of common origin and/or function also typically show core patterns of gene expression that are expressed across multiple stages of development. Our findings suggest that constitutive loss of function seen in Lhx6CreER/Lhx6CreER mice leads to a complete loss of hypothalamic Lhx6+ cells (Fig. 2), while the postnatal loss of function leads to a partial loss of Lhx6+ cells (Fig. 2). This suggests that Lhx6 may control the expression of similar target genes in both embryonic and postnatal hypothalamus to promote neuronal survival. In addition, since Lhx6 clearly is not required for survival of telencephalic neurons, we predict that Lhx6 will regulate the expression of specific sets of genes in both embryonic and postnatal hypothalamus, but not telencephalon, which promotes neuronal survival.
In Figure 4, we therefore identify candidates for these prosurvival genes both by comparing gene expression profiles between embryonic (E15) and postnatal (P8) hypothalamic and cortical Lhx6+ cells and also by directly comparing the gene expression profile of P10 control Lhx6-CreER;Ai9 and Lhx6-deficient but viable Lhx6CreER/Lhx6lox;Baxlox/lox;Ai9 mice. These were analyzed at P10 rather than P8 because of the need to ensure efficient disruption of the conditional alleles of Lhx6 and Bax, and induction of sufficient levels of tdTom to allow for efficient cell isolation, following daily 4-OHT administration between P1 and P5. While this might lead to the failure to identify whatever the small number of Lhx6-regulated genes that are differentially expressed between P8 and P10, we believe that this will identify the great majority of Lhx6-dependent genes that promote neuronal survival. Any readers who wish to delve further into this dataset, and identify additional genes we may have missed in this initial screen, can do so using the data in Table S1.
We are frankly puzzled by the Reviewer’s statement that we “identified so few Lhx6 target genes”, when we clearly state in Figure S2 that over 2,000 differentially expressed genes were observed between control and Lhx6/Bax-deficient hypothalamic neurons. A major reason why data was incorporated from the E15 and P8 datasets was to better select strong candidate regulators of neuronal survival from this very long list of genes.
- The proposed survival regulatory function of LHX6 in HT interneurons represents the main functional finding of this study, which however was not analyzed in great detail. Likewise, the analysis of LHX6 target genes that mediate the survival regulating function was not very successful, identifying only the ERBB4 receptor and other genes related to the neurotrophic neuregulin pathway. Of note, the authors proposed a clear difference of LHX6-associated transcriptional networks and LHX6 function in telencephalic versus HT neurons (migration versus survival). However, THE identified target gene of LHX6 suggested to regulate survival in HT neurons was Erbb4. Erbb4 is likewise expressed in telencephalic neurons, here being involved in migration regulation. Studies that confirm Erbb4 function in survival regulation in HT neurons are lacking. By applying a more coherent analysis, comparing transcriptional profiles of Lhx6 KO and WT cells of the same age, better candidates might be identified. For this, the time window of the LHX6-dependent survival regulation needs to be identified.
This is exactly the point we were trying to make here. Lhx6 is strongly expressed in a large subset of progenitors and precursors of GABAergic neurons in the telencephalon, and in a much smaller subset of GABAergic neuronal precursors in has different functions between telencephalic and hypothalamic populations, yet is strongly expressed in both populations.
Quoting Reviewer #1 “Many developmental transcription factors are utilized both across diverse brain regions and across tissues outside of the brain”. Errb4 has been shown to regulate tangential migration in cortical interneurons but has been shown to promote neuronal survival in other cell types. Since hypothalamic Lhx6+ neurons do not undergo long-range tangential migration, we therefore conclude that the function of Errb4 in hypothalamic Lhx6+ neurons is likely related to promoting survival, rather than controlling migration. It is certainly possible, however, that Erbb4 could also contribute to the regulation of short-range tangential migration of Lhx6-expressing neuronal precursors, such as the likely migration of Nkx2.2-expressing cells from the hinge to the ZI. We have revised the text to make this point clearer. We certainly believe that further functional studies of these genes are worthwhile and compelling, but are also beyond the scope of this study.
- With respect to the survival analysis, the analysis of Lhx6CreER/lox;Baxlox/lox;Ai9 mice although elegant, should be supplemented with other data, eg caspase and/or TUNEL labeling to support this main conclusion.
Both TUNEL and Caspase-3 staining is detectable for only a relatively brief period during apoptosis, and neither are highly sensitive tools for detecting neuronal death. We were unable to observe changes in staining with either marker between P5 and P10 following the postnatal loss of function of Lhx6 (Fig. 2). This is now mentioned in the text. The use of Bax mutants in this analysis, in which apoptosis altogether, was done with the aim of maximizing our ability to detect Lhx6-dependent regulation of neuronal survival.
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###Reviewer #3:
Kim et al. aimed to characterize the similarities and differences between the development and molecular identity of telencephalic versus hypothalamic (HT) Lhx6+ GABAergic neurons. By analyzing a diverse repertoire of transgenic mice at different developmental stages and through the use of fate mapping, bulk and single cell sequencing approaches, ISH and immunostaining, the authors descriptively compare transcriptional networks and upstream regulators of LHX6. They found essential differences between LHX6-dependent networks and those in telencephalic neurons and suggest a role of LHX6 in survival instead of migration regulation HT neurons. Moreover, spatially distinct LHX6+ HT cell clusters were identified and transcriptionally profiled.
Only 1-2% of the GABAergic neurons express LHX6, and the cells expressing LHX6 in the …
###Reviewer #3:
Kim et al. aimed to characterize the similarities and differences between the development and molecular identity of telencephalic versus hypothalamic (HT) Lhx6+ GABAergic neurons. By analyzing a diverse repertoire of transgenic mice at different developmental stages and through the use of fate mapping, bulk and single cell sequencing approaches, ISH and immunostaining, the authors descriptively compare transcriptional networks and upstream regulators of LHX6. They found essential differences between LHX6-dependent networks and those in telencephalic neurons and suggest a role of LHX6 in survival instead of migration regulation HT neurons. Moreover, spatially distinct LHX6+ HT cell clusters were identified and transcriptionally profiled.
Only 1-2% of the GABAergic neurons express LHX6, and the cells expressing LHX6 in the HT were identified to be very diverse. Apart from a putative role for LHX6 in promoting the survival of HT neurons, which in my opinion is not analyzed convincingly, nothing functional was revealed. For this, I do not judge the potential significance and influence of the findings as broad or fundamental.
The manuscript could be better focused, and more coherent. The authors jump between different aspects of the story. First, the authors address a potential role of LHX6 in survival regulation in HT interneurons, and try to identify potential LHX6 target genes mediating this effect. The latter was neither analyzed convincingly nor validated. Then the authors switch to the comparative analysis of transcriptional networks in cortical versus hypothalamic LHX6+ interneurons, and the identification of different clusters of LHX6+ HT cells. Next, potential upstream regulators of LHX6 in HT neurons were addressed by fate mapping studies. Then, the authors again switch focus, and analyzed distinct anatomical regions covered by Lhx6+ neurons by single cell RNA seq and investigated an instructive role of Nkx2-1, Nkx2-2 and Dlx1/2 in the establishment of these hypothalamic regions.
Subheadings in the result section might be very useful. However, the focus of this study requires clarification and also respective consideration in the introduction.
The authors use a variety of different reporter and loss of function mouse models and jump between developmental stages for analysis. Apart from being confusing, the experimental/analytical pipeline is not sufficiently rigorous with respect to age and genetic background. E.g. to analyze target genes of LHX6 through which the effect on cell survival could be mediated, the authors compared expression profiles from P10 Lhx6CreER/+;Ai9 neurons with hypothalamic and cortical Lhx6-GFP positive and negative cells from P8 mice. Hypothalamic enriched genes were then compared to single-cell RNA-Sequencing (scRNA-Seq) datasets of E15.5 and P8 hypothalamic Lhx6-expressing neurons. Transcriptional profiles tremendously change with progressing development, and different mouse lines were used, which were not all time-matched. This might have caused Lhx6-independent variation, which likely masks relevant genes. This could be an explanation why so few LHX6 target genes were identified through which LHX6 putatively acts on neuronal survival.
The proposed survival regulatory function of LHX6 in HT interneurons represents the main functional finding of this study, which however was not analyzed in great detail. Likewise, the analysis of LHX6 target genes that mediate the survival regulating function was not very successful, identifying only the ERBB4 receptor and other genes related to the neurotrophic neuregulin pathway. Of note, the authors proposed a clear difference of LHX6-associated transcriptional networks and LHX6 function in telencephalic versus HT neurons (migration versus survival). However, THE identified target gene of LHX6 suggested to regulate survival in HT neurons was Erbb4. Erbb4 is likewise expressed in telencephalic neurons, here being involved in migration regulation. Studies that confirm Erbb4 function in survival regulation in HT neurons are lacking. By applying a more coherent analysis, comparing transcriptional profiles of Lhx6 KO and WT cells of the same age, better candidates might be identified. For this, the time window of the LHX6-dependent survival regulation needs to be identified.
With respect to the survival analysis, the analysis of Lhx6CreER/lox;Baxlox/lox;Ai9 mice although elegant, should be supplemented with other data, eg caspase and/or TUNEL labeling to support this main conclusion.
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###Reviewer #2:
Kim and colleagues used a combination of state-of-art sequencing and mouse genetic tools to study the mechanisms that control the development of a subset of GABAergic neurons in the developing hypothalamus.
While neurodevelopment of GABAergic neurons has been extensively studied in the developing telencephalon, little is known about their counterparts in the developing hypothalamus. The authors focused their work on a specific subset of GABAergic neurons that express the LIM homeodomain factor Lhx6. Lhx6 is a master regulator of GABAergic neuron differentiation, specification, and migration in cortical interneurons. In contrast, Lhx6-expressing neurons make up only 2-3% of GABAergic neurons in the hypothalamus. The authors' previous work demonstrated that these neurons play a critical role in sleep homeostasis. …
###Reviewer #2:
Kim and colleagues used a combination of state-of-art sequencing and mouse genetic tools to study the mechanisms that control the development of a subset of GABAergic neurons in the developing hypothalamus.
While neurodevelopment of GABAergic neurons has been extensively studied in the developing telencephalon, little is known about their counterparts in the developing hypothalamus. The authors focused their work on a specific subset of GABAergic neurons that express the LIM homeodomain factor Lhx6. Lhx6 is a master regulator of GABAergic neuron differentiation, specification, and migration in cortical interneurons. In contrast, Lhx6-expressing neurons make up only 2-3% of GABAergic neurons in the hypothalamus. The authors' previous work demonstrated that these neurons play a critical role in sleep homeostasis. Therefore, understanding how these neurons are formed and maintained is of great importance.
The authors show that hypothalamic Lhx6 is necessary for neuronal differentiation and survival. Furthermore, by profiling and comparing multiple RNA-seq, scRNA-seq, and ATAC-seq datasets, they were able to identify three transcription factors Nkx2.1, Nkx2.2, and Dlx1/2 that each delineates non-overlapping subdomains of Lhx6 neurons and are necessary for Lhx6 expression in the hypothalamus. Finally, the authors demonstrate that mature Lhx6 neurons manifest extensive molecular heterogeneity that is distinct from their counterparts in the telencephalon.
The work presented is of high quality and is a technological tour de force. The scope and depth of the study are unparalleled among similar studies of hypothalamic neurodevelopment. That said I only have a couple of minor suggestions.
In Figure S2, the number of tomato+ cells appear to be reduced, but not eliminated. Do the authors think that Lhx6 is necessary for the survival of all Lhx6 neurons, or just a subset? The use of the floxed Bax allele is clever, but is there evidence directly supporting increased cell death? Can the authors completely rule out the possibility of the mismigration of cell bodies after the postnatal deletion of Lhx6?
In Figure 4, the authors acknowledged that the ectopic gene expression in Lhx6CreER/lox; Baxlox/lox mice could be due to the loss of function of Bax. If so, would Lhx6CreER/+; Baxlox/lox mice be a better control in this experiment?
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###Reviewer #1:
This paper investigates the role of Lhx6 and other transcription factors in the development of GABAergic neurons in the hypothalamus. The authors report that a small fraction of hypothalamic GABAergic neurons express Lhx6 and further depend on this expression for their survival. Dlx1/2, Nkx1-1 and Nkx2-2 define 5 subpopulations and at least three of these populations depend on these TFs to maintain Lhx6 expression. A strength of the paper is the multimodal analysis and the fact that descriptive assays like RNAseq and ATACseq are followed up with specific knockouts of candidate transcription factors. However, the relationships between the developmental populations identified and adult subtypes of hypothalamic neurons remain unclear. Although the results will surely interest those already interested in hypothalamic …
###Reviewer #1:
This paper investigates the role of Lhx6 and other transcription factors in the development of GABAergic neurons in the hypothalamus. The authors report that a small fraction of hypothalamic GABAergic neurons express Lhx6 and further depend on this expression for their survival. Dlx1/2, Nkx1-1 and Nkx2-2 define 5 subpopulations and at least three of these populations depend on these TFs to maintain Lhx6 expression. A strength of the paper is the multimodal analysis and the fact that descriptive assays like RNAseq and ATACseq are followed up with specific knockouts of candidate transcription factors. However, the relationships between the developmental populations identified and adult subtypes of hypothalamic neurons remain unclear. Although the results will surely interest those already interested in hypothalamic development, it is not clear that broader developmental or functional principles have been identified. The authors make much of the fact that the identified populations do not resemble forebrain interneurons defined by Lhx6 expression, but it is not clear why this should have been expected. Many developmental transcription factors are utilized both across diverse brain regions and across tissues outside of the brain. Perhaps the emphasis of this point could be tempered.
The presentation of the manuscript could be improved by clarifying the relationships between embryonic and more mature structure within the hypothalamus. For example, It is extremely hard to follow the evidence split across figures 5, S6 and S7 for parsing the cell groups by TF expression.
The ATAC seems to be used only to bolster the impression that the populations identified by gene expression are different. The description of footprinting seems to imply an effort to analyze binding sites for specific factors (e.g. to identify targets of the TFs studied), but the statistical approach employed and even the conclusions reached are not fully spelled out. As such, this part of the study is underdeveloped or not well enough described.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
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