Functional definition of the Drosophila airway progenitor field through overlapping compensatory regulators
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eLife Assessment
In this important manuscript, Matsuda and colleagues present a model describing the regulation of tracheal tubulogenesis in Drosophila melanogaster embryos. The authors support this model using convincing approaches that combine novel experimental results with previously published work from their group. While some conclusions are consistent with earlier studies, the present manuscript introduces distinct molecular markers not previously reported, which reinforce the authors' prior findings. In addition, the manuscript analyses, using experimental strategies, the requirement of the Dpp and EGFR signalling pathways for the maintenance of trachealess (trh), one of the key transcription factors governing tracheal development.
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Abstract
Tubular organs present a common solution to fluid transport in multicellular organisms. They often arise by an initial bulging of flat epithelial progenitor cells, which then undergo branching morphogenesis. Here, we present 3 cooperative programs fully defining the Drosophila airway progenitor field and their roles in early morphogenesis linking the radial pattern of the 2-dimensional (2D) field to the proximo-distally patterning of the 3D tubes. We previously showed that extrinsic Hedgehog (Hh) and intrinsic POU-Homeobox TF Ventral-veinless (Vvl)/Drifter/U-turn dominantly drive the transcriptional program toward the distal airway cell identity at the expense of a proximal program specified by the GATA TF grain (grn). Both programs require the basic-HLH-POU TF trachealess (trh) (Matsuda et. al, 2015). Whereas trh is not essential for primordia invagination, we show that in hh vvl double mutants, the oval-shaped primordia frequently remain at the 2D plane, retaining trh expression in a grn dependent manner. Therefore, hh and vvl are the principal regulators of progenitor invagination independent of trh. Each of the 3 regulators, Trh, Vvl and Grn fulfills only complementary or compensatory functions in transcription and morphogenesis but their combinations functionally define the airway progenitor field. We further provide a comprehensive description for allocating the airway progenitors on the body coordinates, involving dorsal Decapentaplegic/BMP signaling along the dorso-ventral axis and subsequent radial EGFR signaling along the proximo-distal axis. The presence of 3 complementary, regulatory programs in early gene expression and morphogenesis of the simple Drosophila airways may reflect the vital needs for respiration, and their influence on the evolution of various strategies in tubular organ development.
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Author response:
Reviewer #1
- The results showing that hh and vvl drive tracheal invaginaton independently of trh are reported in Figure 5 of (Matsuda et al. 2015 eLife 4:e09646).Reviewer #2
Many images primarily show lateral views of whole embryos, which can make it difficult to fully assess some phenotypes; higher-magnification or sectional views would enhance clarity. There are also some minor inconsistencies in the description of invagination phenotypes, particularly regarding whether all trh+ cells remain in a 2D plane versus indications of partial invagination in hh vvl double mutants blocking apoptosis, which would benefit from further clarification.
The data in our previous eLife publication (DOI: 10.7554/eLife.09646)1 were mostly projection views. Therefore, it is hard to conclude if the airway progenitors of hh vvl …
Author response:
Reviewer #1
- The results showing that hh and vvl drive tracheal invaginaton independently of trh are reported in Figure 5 of (Matsuda et al. 2015 eLife 4:e09646).Reviewer #2
Many images primarily show lateral views of whole embryos, which can make it difficult to fully assess some phenotypes; higher-magnification or sectional views would enhance clarity. There are also some minor inconsistencies in the description of invagination phenotypes, particularly regarding whether all trh+ cells remain in a 2D plane versus indications of partial invagination in hh vvl double mutants blocking apoptosis, which would benefit from further clarification.
The data in our previous eLife publication (DOI: 10.7554/eLife.09646)1 were mostly projection views. Therefore, it is hard to conclude if the airway progenitors of hh vvl double mutants failed to invaginate or they invaginated to form sacs. We will provide magnified views of the progenitor invagination in hh vvl double mutants and describe the degrees of their invagination phenotypes.
Reviewer #1
The results showing dpp requirement for trh maintenance are partially reported in Figure 6 of (Matsuda 2015 eLife 4:e09646).
Reviewer #2
Finally, some statements in the abstract, especially regarding the role of grn, are not directly supported by data in this study and could be better aligned with the scope of the presented results.
trh-lacZ (1-eve-1) has been used as the earliest and the strongest enhancer trap line to mark the airway primordia and the airway progenitors. Perdurance of beta-galactocidase proteins makes it difficult to conclude if the marker signals result from the active transcriptional state of the trh locus. In our previous eLife publication we showed that Trh proteins and _trh_transcripts are not detectable in H99 grn hh vvl quadruple mutants and in grn hh vvl triple mutants (Figure 5H and Figure 5-figure supplement 2A of DOI: 10.7554/eLife.09646, respectively)1, although trh-LacZ signals are detected in grn hh vvl triple mutants.
Similarly, although we previously showed trh-LacZ expression in dpp mutant combinations, Figure 2 in the current manuscript, shows that even strong trh-LacZ signals do not always correlate with trh transcripts in dpp mutants. Therefore, in the current manuscript we included the data of dpp-driven positive regulation of trh transcripts at later stages since they have not been shown before.
Assessments and advices of the Editors and the Reviewers are indispensable for improving the manuscript. We will address all the Reviewers comments (Weakness of Public review, major and minor issues of Recommendations for the authors) both experimentally and in the text.
Sincerely yours,
Christos Samakovlis on behalf of all authors
- (1) Matsuda, R., Hosono, C., Samakovlis, C. & Saigo, K. Multipotent versus differentiated cell fate selection in the developing Drosophila airways. eLife 4 (2015).
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eLife Assessment
In this important manuscript, Matsuda and colleagues present a model describing the regulation of tracheal tubulogenesis in Drosophila melanogaster embryos. The authors support this model using convincing approaches that combine novel experimental results with previously published work from their group. While some conclusions are consistent with earlier studies, the present manuscript introduces distinct molecular markers not previously reported, which reinforce the authors' prior findings. In addition, the manuscript analyses, using experimental strategies, the requirement of the Dpp and EGFR signalling pathways for the maintenance of trachealess (trh), one of the key transcription factors governing tracheal development.
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Reviewer #1 (Public review):
Summary:
In this manuscript, Matsuda and collaborators present a model of how tracheal tubulogenesis is controlled in Drosophila embryos. Some of the results backing the model are new, but others are based on information already published by the authors. However, the results in this manuscript present different molecular markers not published before, which agree with previous conclusions. The manuscript also analyses the requirement of the dpp and EGFR signalling pathways for trachealess (trh) maintenance, one of the main tracheal transcription factors.
Strengths:
The two most interesting novel points of the manuscript are:
(1) Its contribution to the analysis of how the dpp and EGFR pathways contribute to the maintenance of trh expression.
(2) The experimental evidence showing that mechanical invagination …
Reviewer #1 (Public review):
Summary:
In this manuscript, Matsuda and collaborators present a model of how tracheal tubulogenesis is controlled in Drosophila embryos. Some of the results backing the model are new, but others are based on information already published by the authors. However, the results in this manuscript present different molecular markers not published before, which agree with previous conclusions. The manuscript also analyses the requirement of the dpp and EGFR signalling pathways for trachealess (trh) maintenance, one of the main tracheal transcription factors.
Strengths:
The two most interesting novel points of the manuscript are:
(1) Its contribution to the analysis of how the dpp and EGFR pathways contribute to the maintenance of trh expression.
(2) The experimental evidence showing that mechanical invagination is not a requirement for trh maintenance in the tracheal cells, an intriguing hypothesis previously suggested by (Kondo Hayashi 2019 eLife 8:e45145) that can now be discarded by the data presented in this work.
Weaknesses:
Because of the mixture of new and already published data, this manuscript can be considered as a review/experimental paper.
Already known data:
- The results showing that hh and vvl drive tracheal invaginaton independently of trh are reported in Figure 5 of (Matsuda et al. 2015 eLife 4:e09646).
- The results showing dpp requirement for trh maintenance are partially reported in Figure 6 of (Matsuda 2015 eLife 4:e09646). -
Reviewer #2 (Public review):
Summary:
Matsuda et al. investigate the regulatory mechanisms controlling gene expression and morphogenesis in the Drosophila embryonic trachea. Building on previous findings that tracheal invagination can occur independently of trh, they identify extrinsic hh and intrinsic vvl as key regulators that cooperatively promote this process. The study also integrates major signaling pathways (Dpp/BMP and EGFR) in defining tracheal cell identity and demonstrates that Ras activation can upregulate trh. Overall, the work supports a model in which multiple transcription factors and signaling inputs coordinate airway progenitor specification.
Strengths:
This study uses genetic analysis of various mutants to dissect regulatory relationships underlying tracheal development. While the uncoupling of tracheal invagination …
Reviewer #2 (Public review):
Summary:
Matsuda et al. investigate the regulatory mechanisms controlling gene expression and morphogenesis in the Drosophila embryonic trachea. Building on previous findings that tracheal invagination can occur independently of trh, they identify extrinsic hh and intrinsic vvl as key regulators that cooperatively promote this process. The study also integrates major signaling pathways (Dpp/BMP and EGFR) in defining tracheal cell identity and demonstrates that Ras activation can upregulate trh. Overall, the work supports a model in which multiple transcription factors and signaling inputs coordinate airway progenitor specification.
Strengths:
This study uses genetic analysis of various mutants to dissect regulatory relationships underlying tracheal development. While the uncoupling of tracheal invagination from trh function has been previously recognized, this work advances the field by identifying hh and vvl as key regulators of invagination independent of trh. The study also integrates multiple signaling pathways, such as Dpp/BMP and EGFR, into a coherent framework for tracheal cell specification. In addition, the demonstration that Ras activation can upregulate trh provides a clear mechanistic link between RTK signaling and transcriptional regulation. Overall, the work offers important and broadly relevant insights into how gene expression and morphogenesis are coordinated during development.
Weaknesses:
Data presentation and clarity of interpretation could be improved. Many images primarily show lateral views of whole embryos, which can make it difficult to fully assess some phenotypes; higher-magnification or sectional views would enhance clarity. There are also some minor inconsistencies in the description of invagination phenotypes, particularly regarding whether all trh+ cells remain in a 2D plane versus indications of partial invagination in hh vvl double mutants blocking apoptosis, which would benefit from further clarification. Finally, some statements in the abstract, especially regarding the role of grn, are not directly supported by data in this study and could be better aligned with the scope of the presented results.
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