A neuroepithelial wave of BMP signalling drives anteroposterior specification of the tuberal hypothalamus
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eLife assessment
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick embryo. It thus presents a useful tool for scientists working in this particular subfield. However, the manuscript is incomplete as it is impossible for the reader to follow the conclusions made by the authors because the presentation of the data is not streamlined and the text is difficult to follow, even for experts.
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Abstract
The tuberal hypothalamus controls life-supporting homeostatic processes, but despite its fundamental role, the cells and signalling pathways that specify this unique region of the central nervous system in embryogenesis are poorly characterised. Here, we combine experimental and bioinformatic approaches in the embryonic chick to show that the tuberal hypothalamus is progressively generated from hypothalamic floor plate-like cells. Fate-mapping studies show that a stream of tuberal progenitors develops in the anterior-ventral neural tube as a wave of neuroepithelial-derived BMP signalling sweeps from anterior to posterior through the hypothalamic floor plate. As later-specified posterior tuberal progenitors are generated, early specified anterior tuberal progenitors become progressively more distant from these BMP signals and differentiate into tuberal neurogenic cells. Gain- and loss-of-function experiments in vivo and ex vivo show that BMP signalling initiates tuberal progenitor specification, but must be eliminated for these to progress to anterior neurogenic progenitors. scRNA-Seq profiling shows that tuberal progenitors that are specified after the major period of anterior tuberal specification begin to upregulate genes that characterise radial glial cells. This study provides an integrated account of the development of the tuberal hypothalamus.
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Author Response
Reviewer #1 (Public Review):
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick by carefully analyzing the expression patterns of a plethora of proteins involved and perturbation of BMP signaling.
Strengths:
This manuscript presents the results of an in-depth analysis aimed to unravel the expression of a variety of transcription factors, and the role of signaling molecules, in particular BMP, SHH and Notch, and, and the role of BMP for the development of the tubular hypothalamus. For this, the authors applied a variety of methods, including in-situ RNA hybridizations to chick embryos, fate mapping, explant cultures, and loss and gain-functions studies in embryos, complemented by carefully mining previously performed scRNA-Seq data. From the data …
Author Response
Reviewer #1 (Public Review):
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick by carefully analyzing the expression patterns of a plethora of proteins involved and perturbation of BMP signaling.
Strengths:
This manuscript presents the results of an in-depth analysis aimed to unravel the expression of a variety of transcription factors, and the role of signaling molecules, in particular BMP, SHH and Notch, and, and the role of BMP for the development of the tubular hypothalamus. For this, the authors applied a variety of methods, including in-situ RNA hybridizations to chick embryos, fate mapping, explant cultures, and loss and gain-functions studies in embryos, complemented by carefully mining previously performed scRNA-Seq data. From the data they derive a model, which explains the dynamic changes of expression of signaling molecules and transcription factors from anterior to posterior during chick development. In addition, they show that fate specification and growth occur concomitantly. Overall, the data provide a plethora of information on expression patterns and consequences of BMP signaling perturbation, which will be valuable for scientists interested in the events taking place during the development of the chick tubular hypothalamus.
We thank the reviewer for recognising the value of this study for development of the chick tuberal hypothalamus.
Weaknesses:
The plethora of data presented makes it very difficult for a reader, who is not familiar with this system, to follow the major conclusions from each of the panels. This difficulty is enhanced by the lack of a concise, simple and focused summary at the end of most chapters, which, from my point of view, still contains too many details. Similarly, the discussion too often refers to details presented in the figures of the Results section, rather than giving a broader and focused summary and pointing out to novel conclusions.
We have extensively revised the manuscript, to ensure that it is easier to follow and is less detailed. We have tightened and shortened the Introduction, without losing content or context. We have revised the narrative in the Results section, to reflect revisions to figures (detailed below and in response to Reviewer 2 comments), cut back on detail, and summarised each section. We have streamlined the Discussion, so that the broader points and novel conclusions are more prominent.
Revisions to figures are as follows:
Several main Figures and associated Supplementary Figures have been rearranged so that the text and figures are easier to follow. The rearrangements mean that the reader can follow critical conceptual points without having to jump from main to supplementary figures. Key rearrangements have been made between Figure 1 and Figure 1-figure supplement 1; Figure 2 and Figure 2-figure supplement 1; Figure 2 and Figure 2-figure supplement 2; Figure 6 and Figure 6 supplement 1.
Throughout the manuscript, we have added new images/replaced previous images in cases where key points were not coming across clearly (see Reviewer 2 comments). New data is shown in Figures 1F, G, T-T”; Figures 2G-P’; Figure 2-figure supplement 1 (panels A and E); Figure 2-figure supplement 2 (panels B, E-G; Q-T).
Throughout the manuscript we have improved the schematics, making it easier to follow key domains and, separately, gene expression patterns
Finally, in light of the comment on the plethora of data, detail and the overall difficulty in following the manuscript, we have removed in situ data that was not needed for our central arguments (previous panels 1F-J and 1R-T).
I also suggest that the authors check the Materials and Methods section, which does not always contain the information required. For example, in the chapter on "Chicken HCR": I guess they used the HCR IHC kit from Molecular Instruments? What kind of "modification" of the Molecular Instruments protocol did they introduce?
We have revised the Material and Methods section as required. We followed the Molecular Instrument Protocol HCRv3-Chicken, but included a methanol dehydration step, which we have now added.
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eLife assessment
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick embryo. It thus presents a useful tool for scientists working in this particular subfield. However, the manuscript is incomplete as it is impossible for the reader to follow the conclusions made by the authors because the presentation of the data is not streamlined and the text is difficult to follow, even for experts.
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Reviewer #1 (Public Review):
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick by carefully analyzing the expression patterns of a plethora of proteins involved and perturbation of BMP signaling.
Strengths:
This manuscript presents the results of an in-depth analysis aimed to unravel the expression of a variety of transcription factors, and the role of signaling molecules, in particular BMP, SHH and Notch, and, and the role of BMP for the development of the tubular hypothalamus. For this, the authors applied a variety of methods, including in-situ RNA hybridizations to chick embryos, fate mapping, explant cultures, and loss and gain-functions studies in embryos, complemented by carefully mining previously performed scRNA-Seq data. From the data they derive a model, which …Reviewer #1 (Public Review):
The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick by carefully analyzing the expression patterns of a plethora of proteins involved and perturbation of BMP signaling.
Strengths:
This manuscript presents the results of an in-depth analysis aimed to unravel the expression of a variety of transcription factors, and the role of signaling molecules, in particular BMP, SHH and Notch, and, and the role of BMP for the development of the tubular hypothalamus. For this, the authors applied a variety of methods, including in-situ RNA hybridizations to chick embryos, fate mapping, explant cultures, and loss and gain-functions studies in embryos, complemented by carefully mining previously performed scRNA-Seq data. From the data they derive a model, which explains the dynamic changes of expression of signaling molecules and transcription factors from anterior to posterior during chick development. In addition, they show that fate specification and growth occur concomitantly.
Overall, the data provide a plethora of information on expression patterns and consequences of BMP signaling perturbation, which will be valuable for scientists interested in the events taking place during the development of the chick tubular hypothalamus.Weaknesses:
The plethora of data presented makes it very difficult for a reader, who is not familiar with this system, to follow the major conclusions from each of the panels. This difficulty is enhanced by the lack of a concise, simple and focused summary at the end of most chapters, which, from my point of view, still contains too many details. Similarly, the discussion too often refers to details presented in the figures of the Results section, rather than giving a broader and focused summary and pointing out to novel conclusions.I also suggest that the authors check the Materials and Methods section, which does not always contain the information required. For example, in the chapter on "Chicken HCR": I guess they used the HCR IHC kit from Molecular Instruments? What kind of "modification" of the Molecular Instruments protocol did they introduce?
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Reviewer #2 (Public Review):
Chinnaiya et al. integrated recent scRNA transcriptomics with high-resolution multiplexing in situ hybridization, fate mapping and tissue explants to unravel the spatiotemporal development of early chick tuberal hypothalamus. They show that a wave of BMP signaling passes through anterior and posterior regions sequentially. Interestingly, they showed that neuroepithelial-intrinsic BMPs drive and maintain tuberal hypothalamus late development. Using bioinformatical and in situ profiling, the authors indicated the potential of the tuberal progenitors transferring into radial glia-like cells.
This is a remarkable piece of work and I commend the authors for their bold endeavor to decipher the complex developmental of the tuberal hypothalamus.
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