Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes
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Evaluation Summary:
This manuscript by Yu et al. captures the transcriptional heterogeneity of mouse POMC neurons across hypothalamic development. This study unifies multiple other observations about the role for other neuron al cell types that express POMC transiently during development. The paper is an important resource understanding of the diversity of POMC neuron classes and their relationship to other cell types in the arcuate nucleus.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use single-cell RNA-sequencing (scRNA-seq) to define the transcriptomes characterizing Pomc -expressing cells in the developing hypothalamus and translating ribosome affinity purification with RNA-sequencing (TRAP-seq) to analyze the subsequent translatomes of mature POMC neurons. Our data showed that Pomc -expressing neurons give rise to multiple developmental pathways expressing different levels of Pomc and unique combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts, represents the canonical arcuate POMC neurons. Additional cell clusters expressing medium or low levels of Pomc mature into different neuronal phenotypes featured by distinct sets of transcription factors, neuropeptides, processing enzymes, cell surface, and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to mature postnatally.
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Author response
Reviewer #2 (Public Review):
This work addresses the developmental origins of functionally distinct neuronal populations in the arcuate nucleus of the hypothalamus (ARH). During gestation, immature Pomc-expressing neurons differentiate into at least 3 subpopulations of mature POMC neurons, as well as non-POMC neuronal sub-types (eg., AgRP and KNDy neurons). The authors set out to address the issue of whether these diverse populations arise from a common progenitor or from multiple, molecularly distinct progenitor populations.
They performed single cell RNA-seq on Pomc-expressing neurons (FACS-purified on the basis of expression of a Pomc-driven reporter transgene) across embryonic and early postnatal stages (E11.5 to P12). They also compared these transcriptional profiles to translational profiles of Pomc-expressing …
Author response
Reviewer #2 (Public Review):
This work addresses the developmental origins of functionally distinct neuronal populations in the arcuate nucleus of the hypothalamus (ARH). During gestation, immature Pomc-expressing neurons differentiate into at least 3 subpopulations of mature POMC neurons, as well as non-POMC neuronal sub-types (eg., AgRP and KNDy neurons). The authors set out to address the issue of whether these diverse populations arise from a common progenitor or from multiple, molecularly distinct progenitor populations.
They performed single cell RNA-seq on Pomc-expressing neurons (FACS-purified on the basis of expression of a Pomc-driven reporter transgene) across embryonic and early postnatal stages (E11.5 to P12). They also compared these transcriptional profiles to translational profiles of Pomc-expressing neurons at P5 and P12 generated with the TRAP-Seq approach. Clustering and developmental trajectory analyses confirm reports by other groups that immature Pomc-expressing neurons give rise to non-POMC cell fates (including AgRP/NPY and KNDy neurons) and that terminal differentiation of POMC neurons is achieved after P12. While the data generated here will be a useful resource for the field, there are weaknesses in the analyses used to support the central claim that POMC neurons arise from heterogenous progenitor populations.
Strengths
• This is an interesting topic that would be of interest to scientists studying neural circuits regulating energy balance. • The expression databases provide a valuable resource for the community. They can be mined to identify genes that can be used as markers and as the foundation for functional studies. They can also inform efforts to generate and stage specific ARH cell types using induced pluripotent stem cell technology.
Weaknesses
• My main concerns stem from the fact that all Pomc-expressing neurons in the developing ARH are considered as a single category of "progenitors" in these analyses. While they meet this strict definition because they do not express markers of terminally differentiated POMC neurons, the failure to distinguish between early and late progenitors limits the conclusions that can be drawn.
We agree with the reviewer’s concern and in this revised version of the manuscript we have avoided the use of the terms “progenitors” or “precursors” while focusing on the embryonic and postnatal ages of the collected hypothalamic neurons.
o The earliest stage analyzed here is E11.5, which represents the peak of POMC neuronal differentiation. To capture the precursors of these neurons (1.Pomchigh/Prdm12 at E11.5), it is necessary to perform transcriptomic analyses at earlier stages.
This is answered in the Essential Revision 1.
o EBFs have been shown to regulate neuronal differentiation and migration out of ventricular layer into mantle layer. It is critical to determine whether EBF-expressing neurons in the ARH similarly represent an "early" progenitor stage that follows cell cycle exit migration out of the ventricular zone and precedes the expression of transcription factors that specify a particular cell fate. If so, EBF-expressing neurons in 2.Pomc-med-Ebf1 could represent progenitors of 1.Pomc-hi-Prdm12 neurons. In support of this idea, the transcriptome of 2.Pomc-med-Ebf1 subcluster 1 neurons map onto the two major subpopulations of POMC neurons (Supplemental Figure 15).
This matter has also been answered in the Essential Revision 1.
• Because key terminal markers of POMC neurons are not expressed at P12 (i.e. Ttr, Anxa2), it is hard to precisely map progenitor populations onto neuronal subpopulations in the adult.
We agree with the reviewer’s concern and have decided to remove this level of comparison from the revised manuscript. Therefore, the paragraphs from lines 453 to 497 in the Results section of the original manuscript are no longer present. Similarly, we have deleted from the Discussion the paragraphs referring to the comparison of the two transcriptome data sets.
• While the data support the idea that there are several molecularly distinct subpopulations of POMC progenitors, these analyses do not provide clear answers to the following key questions: 1) Do AgRP/NPY and KNDy neurons arise from molecularly distinct populations of Pomc-expressing progenitors? 2) At what point in the developmental trajectory are molecularly distinct subpopulations of POMC neurons specified?
One of the most intriguing findings of this study is that most Pomc-expressing neuronal clusters are already present in E11.5 embryos, showing their distinctive feature genes and characteristic transcript level from this early time point. For example, cells from cluster 4.Pomclow/Otp, which give rise to Npy/Agrp neurons, express the feature gene, Otp already at E11.5. At this time point cells from cluster 5.Pomclow/Tac2, which give rise to the KNDy neurons, express similar feature genes as cluster 1, except that the level of *Pomc *transcripts in cluster 5 is much lower than in cluster 1. In addition, cells from cluster 5 do never express Otp. Thus, cluster 4 and 5 do not share their repertoire of feature transcripts at E11.5 suggesting that they arise from molecularly distinct populations of Pomc-expressing neurons.
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Evaluation Summary:
This manuscript by Yu et al. captures the transcriptional heterogeneity of mouse POMC neurons across hypothalamic development. This study unifies multiple other observations about the role for other neuron al cell types that express POMC transiently during development. The paper is an important resource understanding of the diversity of POMC neuron classes and their relationship to other cell types in the arcuate nucleus.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The biggest criticism is that the data is descriptive. The authors spend a great deal of time stating the genes in each cluster, but they don't make many inferences as to the biological function of these diverse populations. For example, do these subpopulations express unique axon guidance molecules or receptors that point to the developmental and functional heterogeneity in these genetic clusters.
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Reviewer #2 (Public Review):
This work addresses the developmental origins of functionally distinct neuronal populations in the arcuate nucleus of the hypothalamus (ARH). During gestation, immature Pomc-expressing neurons differentiate into at least 3 subpopulations of mature POMC neurons, as well as non-POMC neuronal sub-types (eg., AgRP and KNDy neurons). The authors set out to address the issue of whether these diverse populations arise from a common progenitor or from multiple, molecularly distinct progenitor populations.
They performed single cell RNA-seq on Pomc-expressing neurons (FACS-purified on the basis of expression of a Pomc-driven reporter transgene) across embryonic and early postnatal stages (E11.5 to P12). They also compared these transcriptional profiles to translational profiles of Pomc-expressing neurons at P5 and …
Reviewer #2 (Public Review):
This work addresses the developmental origins of functionally distinct neuronal populations in the arcuate nucleus of the hypothalamus (ARH). During gestation, immature Pomc-expressing neurons differentiate into at least 3 subpopulations of mature POMC neurons, as well as non-POMC neuronal sub-types (eg., AgRP and KNDy neurons). The authors set out to address the issue of whether these diverse populations arise from a common progenitor or from multiple, molecularly distinct progenitor populations.
They performed single cell RNA-seq on Pomc-expressing neurons (FACS-purified on the basis of expression of a Pomc-driven reporter transgene) across embryonic and early postnatal stages (E11.5 to P12). They also compared these transcriptional profiles to translational profiles of Pomc-expressing neurons at P5 and P12 generated with the TRAP-Seq approach. Clustering and developmental trajectory analyses confirm reports by other groups that immature Pomc-expressing neurons give rise to non-POMC cell fates (including AgRP/NPY and KNDy neurons) and that terminal differentiation of POMC neurons is achieved after P12. While the data generated here will be a useful resource for the field, there are weaknesses in the analyses used to support the central claim that POMC neurons arise from heterogenous progenitor populations.
Strengths
• This is an interesting topic that would be of interest to scientists studying neural circuits regulating energy balance.• The expression databases provide a valuable resource for the community. They can be mined to identify genes that can be used as markers and as the foundation for functional studies. They can also inform efforts to generate and stage specific ARH cell types using induced pluripotent stem cell technology.
Weaknesses
• My main concerns stem from the fact that all Pomc-expressing neurons in the developing ARH are considered as a single category of "progenitors" in these analyses. While they meet this strict definition because they do not express markers of terminally differentiated POMC neurons, the failure to distinguish between early and late progenitors limits the conclusions that can be drawn.
o The earliest stage analyzed here is E11.5, which represents the peak of POMC neuronal differentiation. To capture the precursors of these neurons (1.Pomc-hi/Prdm12 at E11.5), it is necessary to perform transcriptomic analyses at earlier stages.
o EBFs have been shown to regulate neuronal differentiation and migration out of ventricular layer into mantle layer. It is critical to determine whether EBF-expressing neurons in the ARH similarly represent an "early" progenitor stage that follows cell cycle exit migration out of the ventricular zone and precedes the expression of transcription factors that specify a particular cell fate. If so, EBF-expressing neurons in 2.Pomc-med-Ebf1 could represent progenitors of 1.Pomc-hi-Prdm12 neurons. In support of this idea, the transcriptome of 2.Pomc-med-Ebf1 subcluster 1 neurons map onto the two major subpopulations of POMC neurons (Supplemental Figure 15).• Because key terminal markers of POMC neurons are not expressed at P12 (i.e. Ttr, Anxa2), it is hard to precisely map progenitor populations onto neuronal subpopulations in the adult.
• While the data support the idea that there are several molecularly distinct subpopulations of POMC progenitors, these analyses do not provide clear answers to the following key questions: 1) Do AgRP/NPY and KNDy neurons arise from molecularly distinct populations of Pomc-expressing progenitors? 2) At what point in the developmental trajectory are molecularly distinct subpopulations of POMC neurons specified?
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Reviewer #3 (Public Review):
This study reports the transcriptional program of develop of multiple subtypes of POMC neurons, which are important for multiple physiological and behavioral functions, including appetite.
The paper uses cutting edge scRNA-Seq methods and analysis to reveal the develop of these neurons. This work is likely to be important for research studying the physiology of the arcuate nucleus and those interested in defining the underlying neuronal components based on their transcriptional similarity and differences.
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