Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development
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Evaluation Summary:
The formation of the cardiopulmonary circuit is a vital terrestrial adaptation, and precise mechanisms defining how the heart and lung co-develop would be interesting to a broad developmental biology audience. In this manuscript, Rankin et al. propose a nuanced model that bridges previous observations regarding the role of Tbx5 and retinoic acid in forming the cardiopulmonary circuit. This manuscript nicely utilizes forward and reverse genetic approaches in Xenopus model system to rigorously study to describe a Tbx5-Aldh1a2- Shh pathway that leads to reciprocal mesoderm-endoderm interactions and co-induction of segmental heart and lung identities.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.
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Author Response:
Reviewer #1:
In this manuscript by Rankin et al., the authors proposes a model of reciprocal mesoderm-endoderm interactions involving Tbx5 activation of retinoic acid (RA) production in the posterior second heart field (pSHF) that activates endoderm expression of patterning ligands such as Shh, which feeds back to activate the pSHF to coordinate cardiopulmonary development. This is a nice model that bridges previous work from the same authors, which had shown that Tbx5 can alter Shh expression in a non-cell autonomous manner (Steimle et al, 2018), along with prior experiments showing that RA induces Shh expression (Rankin et al, 2016). As such, the novelty here lies in the mechanistic portion of the paper that describes how Tbx5 induces Aldh1a2, a gene responsible for RA production in the pSHF, along with the …
Author Response:
Reviewer #1:
In this manuscript by Rankin et al., the authors proposes a model of reciprocal mesoderm-endoderm interactions involving Tbx5 activation of retinoic acid (RA) production in the posterior second heart field (pSHF) that activates endoderm expression of patterning ligands such as Shh, which feeds back to activate the pSHF to coordinate cardiopulmonary development. This is a nice model that bridges previous work from the same authors, which had shown that Tbx5 can alter Shh expression in a non-cell autonomous manner (Steimle et al, 2018), along with prior experiments showing that RA induces Shh expression (Rankin et al, 2016). As such, the novelty here lies in the mechanistic portion of the paper that describes how Tbx5 induces Aldh1a2, a gene responsible for RA production in the pSHF, along with the interactions of Tbx5 with putative enhancers of Aldh1a2. The use of the xenopus model system allows the authors to perform elegant epistasis experiments using morpholinos and Crispr/CAS9 excisions in the whole embryos, which nicely illustrates the role of Tbx5 in inducing Ald1a2 expression and the role of Tbx5 in downstream pathways described. The experiments are well-presented and follows a clear logic, and they are mostly supportive of the experimental models presented at the end of the manuscript. However, a potential weakness of this manuscript is the reliance on pharmacologic methods of modifying RA pathways rather than using a genetic/RNA targeting approach, which would confer more specificity related to the functional importance of the Tbx5 transcriptional target described (in this case, Aldh1a2). Furthermore, more precise colocalization of Tbx5 and Aldh1a2 within the developing cardiopulmonary tissues is important, with some clarification as to why there appears to be broad Aldh1a2 expression independent of Tbx5.
We thank the reviewer for appreciating the mechanistic novelty and for the fair constructive criticisms. We have addressed your concerns with additional data and revisions to the text, which we agree have greatly improved the study.
Reviewer #2:
In this manuscript Rankin et al. combined mouse and frog genetic models to study the gene regulatory network orchestrated by the transcription factor Tbx5. The authors demonstrated that Tbx5 regulates expression of Aldh1a2 which catalyzes the production of retinoid acid (RA) in the lateral plate mesenchyme, thereby activating RA signaling which then signals to the foregut endoderm and induces Shh expression there. In turn Shh activates Hedgehog signaling in the mesenchyme where it works with Tbx5 to promote expression of Wnt2/2b. Wnt2/2b then initiates lung specification from the anterior foregut endoderm. Biochemistry assays were used to assess howTbx5 and RA regulate the transcription of Aldh1a2 and Shh, respectively. Two evolutionarily conserved enhancers were identified for the regulation of the transcription of Aldh1a2 and Shh. The authors in the end suggested that their work provides knowledge basis for better understanding the pathogenesis of the human birth defects DiGeorge Syndrome and Holt-Oram Syndrome. The findings help to fill the knowledge gap, connecting several observations made by previous studies. Moving forward, Tbx5 and other Tbx genes (e.g. Tbx4) continue to be expressed in the developing lungs. Whether a similar regulatory axis is present to modulate lung epithelial and mesenchymal development remains to be explored.
We thanks the reviewer for appreciating the value of the work and for the helpful suggestions.
Reviewer #3:
Previously the Zorn lab has published that retinoic acid-hedgehog signaling is a key step in lung specification. (Rankin et al, Cell Rep 2016, 66-78.) Previously, molecular networks have been proposed for the early heart/lung differentiation. Examples include: (Xie, L.et al (2012). Dev. Cell 23 (2), 280-291; Steimle, J. D., et al. (2018). Proc. Natl. Acad. Sci. USA 115 (45), E10615-E10624. Peng T, et al, (2013) Nature 500(7464):589-92). Although many pieces of these signaling and transcription factor activities have been described, this manuscript demonstrates additional information. The strengths are the use of an in vivo system to tease out the transcriptional elements regulated by Tbx5 and RA. The authors perform sufficient experiments to support their claims although some of these readouts are qualitative rather than quantitative. The authors include relevant controls where possible. The authors were also rigorous by providing a time window for when Tbx5 control of Raldh2 occurs. One weakness is that the manuscript is difficult to follow for individuals who are not familiar with past published data in these networks. Another weakness is that some of the data is drawn from whole mount images and bulk sequencing which could lead to overstatements. A third weakness is that the manuscript does not have a clear focus. Its main concept is filling in the gaps for some of the gene transcription networks that have been described previously. An additional weakness is that almost all of the gene manipulations are global either by morpholino or chemical treatment (inhibition and activation). Finally, it is unclear what the outcomes of the signaling disruptions are in the embryos. We see a snapshot of gene expression but not how this affects organ development in the long run.
We thank the reviewer for appreciating the value of elucidating the molecular mechanisms by which Tbx-RA interactions regulate cardiopulmonary development- we agree that this is the main value of our study. We also appreciate the reviewer’s perspective that we did not describe the work as clearly as we could, particularly for a non-expert, and that we may have overstated some of the conclusions. We have tried to modify the text to address these issues and have tempered our claims. In addition, we added new experiments to complement the inhibitor studies and test more rigorously our hypothesis, all of which support our model. We also provide additional data and a better description of previous publications on the final anatomical outcome of Tbx5-RA deficiency.
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Evaluation Summary:
The formation of the cardiopulmonary circuit is a vital terrestrial adaptation, and precise mechanisms defining how the heart and lung co-develop would be interesting to a broad developmental biology audience. In this manuscript, Rankin et al. propose a nuanced model that bridges previous observations regarding the role of Tbx5 and retinoic acid in forming the cardiopulmonary circuit. This manuscript nicely utilizes forward and reverse genetic approaches in Xenopus model system to rigorously study to describe a Tbx5-Aldh1a2- Shh pathway that leads to reciprocal mesoderm-endoderm interactions and co-induction of segmental heart and lung identities.
(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 …
Evaluation Summary:
The formation of the cardiopulmonary circuit is a vital terrestrial adaptation, and precise mechanisms defining how the heart and lung co-develop would be interesting to a broad developmental biology audience. In this manuscript, Rankin et al. propose a nuanced model that bridges previous observations regarding the role of Tbx5 and retinoic acid in forming the cardiopulmonary circuit. This manuscript nicely utilizes forward and reverse genetic approaches in Xenopus model system to rigorously study to describe a Tbx5-Aldh1a2- Shh pathway that leads to reciprocal mesoderm-endoderm interactions and co-induction of segmental heart and lung identities.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript by Rankin et al., the authors proposes a model of reciprocal mesoderm-endoderm interactions involving Tbx5 activation of retinoic acid (RA) production in the posterior second heart field (pSHF) that activates endoderm expression of patterning ligands such as Shh, which feeds back to activate the pSHF to coordinate cardiopulmonary development. This is a nice model that bridges previous work from the same authors, which had shown that Tbx5 can alter Shh expression in a non-cell autonomous manner (Steimle et al, 2018), along with prior experiments showing that RA induces Shh expression (Rankin et al, 2016). As such, the novelty here lies in the mechanistic portion of the paper that describes how Tbx5 induces Aldh1a2, a gene responsible for RA production in the pSHF, along with the …
Reviewer #1 (Public Review):
In this manuscript by Rankin et al., the authors proposes a model of reciprocal mesoderm-endoderm interactions involving Tbx5 activation of retinoic acid (RA) production in the posterior second heart field (pSHF) that activates endoderm expression of patterning ligands such as Shh, which feeds back to activate the pSHF to coordinate cardiopulmonary development. This is a nice model that bridges previous work from the same authors, which had shown that Tbx5 can alter Shh expression in a non-cell autonomous manner (Steimle et al, 2018), along with prior experiments showing that RA induces Shh expression (Rankin et al, 2016). As such, the novelty here lies in the mechanistic portion of the paper that describes how Tbx5 induces Aldh1a2, a gene responsible for RA production in the pSHF, along with the interactions of Tbx5 with putative enhancers of Aldh1a2. The use of the xenopus model system allows the authors to perform elegant epistasis experiments using morpholinos and Crispr/CAS9 excisions in the whole embryos, which nicely illustrates the role of Tbx5 in inducing Ald1a2 expression and the role of Tbx5 in downstream pathways described. The experiments are well-presented and follows a clear logic, and they are mostly supportive of the experimental models presented at the end of the manuscript. However, a potential weakness of this manuscript is the reliance on pharmacologic methods of modifying RA pathways rather than using a genetic/RNA targeting approach, which would confer more specificity related to the functional importance of the Tbx5 transcriptional target described (in this case, Aldh1a2). Furthermore, more precise colocalization of Tbx5 and Aldh1a2 within the developing cardiopulmonary tissues is important, with some clarification as to why there appears to be broad Aldh1a2 expression independent of Tbx5.
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Reviewer #2 (Public Review):
In this manuscript Rankin et al. combined mouse and frog genetic models to study the gene regulatory network orchestrated by the transcription factor Tbx5. The authors demonstrated that Tbx5 regulates expression of Aldh1a2 which catalyzes the production of retinoid acid (RA) in the lateral plate mesenchyme, thereby activating RA signaling which then signals to the foregut endoderm and induces Shh expression there. In turn Shh activates Hedgehog signaling in the mesenchyme where it works with Tbx5 to promote expression of Wnt2/2b. Wnt2/2b then initiates lung specification from the anterior foregut endoderm. Biochemistry assays were used to assess howTbx5 and RA regulate the transcription of Aldh1a2 and Shh, respectively. Two evolutionarily conserved enhancers were identified for the regulation of the …
Reviewer #2 (Public Review):
In this manuscript Rankin et al. combined mouse and frog genetic models to study the gene regulatory network orchestrated by the transcription factor Tbx5. The authors demonstrated that Tbx5 regulates expression of Aldh1a2 which catalyzes the production of retinoid acid (RA) in the lateral plate mesenchyme, thereby activating RA signaling which then signals to the foregut endoderm and induces Shh expression there. In turn Shh activates Hedgehog signaling in the mesenchyme where it works with Tbx5 to promote expression of Wnt2/2b. Wnt2/2b then initiates lung specification from the anterior foregut endoderm. Biochemistry assays were used to assess howTbx5 and RA regulate the transcription of Aldh1a2 and Shh, respectively. Two evolutionarily conserved enhancers were identified for the regulation of the transcription of Aldh1a2 and Shh. The authors in the end suggested that their work provides knowledge basis for better understanding the pathogenesis of the human birth defects DiGeorge Syndrome and Holt-Oram Syndrome. The findings help to fill the knowledge gap, connecting several observations made by previous studies. Moving forward, Tbx5 and other Tbx genes (e.g. Tbx4) continue to be expressed in the developing lungs. Whether a similar regulatory axis is present to modulate lung epithelial and mesenchymal development remains to be explored.
-
Reviewer #3 (Public Review):
Previously the Zorn lab has published that retinoic acid-hedgehog signaling is a key step in lung specification. (Rankin et al, Cell Rep 2016, 66-78.) Previously, molecular networks have been proposed for the early heart/lung differentiation. Examples include: (Xie, L.et al (2012). Dev. Cell 23 (2), 280-291; Steimle, J. D., et al. (2018). Proc. Natl. Acad. Sci. USA 115 (45), E10615-E10624. Peng T, et al, (2013) Nature 500(7464):589-92). Although many pieces of these signaling and transcription factor activities have been described, this manuscript demonstrates additional information. The strengths are the use of an in vivo system to tease out the transcriptional elements regulated by Tbx5 and RA. The authors perform sufficient experiments to support their claims although some of these readouts are …
Reviewer #3 (Public Review):
Previously the Zorn lab has published that retinoic acid-hedgehog signaling is a key step in lung specification. (Rankin et al, Cell Rep 2016, 66-78.) Previously, molecular networks have been proposed for the early heart/lung differentiation. Examples include: (Xie, L.et al (2012). Dev. Cell 23 (2), 280-291; Steimle, J. D., et al. (2018). Proc. Natl. Acad. Sci. USA 115 (45), E10615-E10624. Peng T, et al, (2013) Nature 500(7464):589-92). Although many pieces of these signaling and transcription factor activities have been described, this manuscript demonstrates additional information. The strengths are the use of an in vivo system to tease out the transcriptional elements regulated by Tbx5 and RA. The authors perform sufficient experiments to support their claims although some of these readouts are qualitative rather than quantitative. The authors include relevant controls where possible. The authors were also rigorous by providing a time window for when Tbx5 control of Raldh2 occurs. One weakness is that the manuscript is difficult to follow for individuals who are not familiar with past published data in these networks. Another weakness is that some of the data is drawn from whole mount images and bulk sequencing which could lead to overstatements. A third weakness is that the manuscript does not have a clear focus. Its main concept is filling in the gaps for some of the gene transcription networks that have been described previously. An additional weakness is that almost all of the gene manipulations are global either by morpholino or chemical treatment (inhibition and activation). Finally, it is unclear what the outcomes of the signaling disruptions are in the embryos. We see a snapshot of gene expression but not how this affects organ development in the long run.
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