Visceral organ morphogenesis via calcium-patterned muscle constrictions
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Evaluation Summary:
This study employs cutting-edge, multiview light-sheet microscopy and advanced image analysis to investigate how the mechanical interplay of two adjacent tissue layers shapes a developing organ. The finding that genetically-patterned calcium pulses induce local muscle contractions that constrict and fold the adjacent endoderm offers a novel mechanism by which genetically encoded patterning information shapes organs across tissue layers.
(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
Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. Here, we trace the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from subcellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, we find a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calcium pulses trigger muscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. Our analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer – revealing post-translational mechanisms that govern shape change.
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Evaluation Summary:
This study employs cutting-edge, multiview light-sheet microscopy and advanced image analysis to investigate how the mechanical interplay of two adjacent tissue layers shapes a developing organ. The finding that genetically-patterned calcium pulses induce local muscle contractions that constrict and fold the adjacent endoderm offers a novel mechanism by which genetically encoded patterning information shapes organs across tissue layers.
(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):
Mitchell et al. investigated the gut morphogenesis in Drosophila. Light-sheet microscopy was used to obtain detailed 3D live images of the folding process. Authors developed a new computational framework called 'TubULAR', which enabled them to process 3D images to extract the detailed shape of the gut as well as to image endoderm cells on the surface of the gut. Using this novel framework, the authors demonstrated that endoderm cells change their aspect ratio during morphogenesis while maintaining their area approximately constant. Furthermore, the authors demonstrated that the endoderm and muscle cells move in unison during morphogenesis. By combining the results of the experiments with the knockout of hox genes, optogenetically controlled induction or inhibition of muscle contractions, and modulation of …
Reviewer #1 (Public Review):
Mitchell et al. investigated the gut morphogenesis in Drosophila. Light-sheet microscopy was used to obtain detailed 3D live images of the folding process. Authors developed a new computational framework called 'TubULAR', which enabled them to process 3D images to extract the detailed shape of the gut as well as to image endoderm cells on the surface of the gut. Using this novel framework, the authors demonstrated that endoderm cells change their aspect ratio during morphogenesis while maintaining their area approximately constant. Furthermore, the authors demonstrated that the endoderm and muscle cells move in unison during morphogenesis. By combining the results of the experiments with the knockout of hox genes, optogenetically controlled induction or inhibition of muscle contractions, and modulation of the calcium signaling pathway, the authors were able to uncover the molecular mechanism that regulates the folding of the gut. In particular, hox genes regulate calcium signaling, which induces muscle contractions that deform the tightly connected endoderm.
This is an excellent manuscript that significantly advanced our understanding of gut morphogenesis in Drosophila. Furthermore, the novel computational framework 'TubULAR' for the analysis of 3D images will be a great resource for the community.
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Reviewer #2 (Public Review):
Using in toto, multiview light-sheet imaging of the Drosophila midgut, the authors investigate how the endoderm and surrounding muscle layer interact to fold and elongate the developing organ. They find that the movements of both layers closely coincide and that cell flows and shape changes in the endodermal closely correlate with midgut constrictions. Optogenetic inhibition or stimulation of muscle contractility impairs folding patterns, consistent with localized contractility being the driving force for gut constriction and organ shape change. Using a GCAMP6 calcium sensor, they find that Hox-dependent calcium pulses spatially correlate and are required for local muscle contraction and midgut folding. This study represents a substantial advance in whole-organ imaging, between layers, in a deep internal …
Reviewer #2 (Public Review):
Using in toto, multiview light-sheet imaging of the Drosophila midgut, the authors investigate how the endoderm and surrounding muscle layer interact to fold and elongate the developing organ. They find that the movements of both layers closely coincide and that cell flows and shape changes in the endodermal closely correlate with midgut constrictions. Optogenetic inhibition or stimulation of muscle contractility impairs folding patterns, consistent with localized contractility being the driving force for gut constriction and organ shape change. Using a GCAMP6 calcium sensor, they find that Hox-dependent calcium pulses spatially correlate and are required for local muscle contraction and midgut folding. This study represents a substantial advance in whole-organ imaging, between layers, in a deep internal organ. The image processing and analysis pipeline represent another important technical advance. The data are simply beautiful and presented in a clear and convincing way. Multiple parallel approaches are used to test the hypothesis that patterned midgut contractions generate whole organ shape changes in the developing midgut, and thus, the conclusions are well supported by the data. The main weakness of the paper in its current form is the overly-concise nature of the main text. Descriptions of the approach and results would need to be elaborated. Some terms are not well defined in the main text, particularly in the sections accompanying Figure 2, making it difficult to follow some of the logic and conclusions.
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Reviewer #3 (Public Review):
This manuscript addresses important questions in developmental biology: how Hox codes are transformed into organ shapes and how different tissue layers mechanically interact during morphogenesis. The authors set up a deep tissue imaging system that allowed them to simultaneously track the endoderm and overlaying muscles during gut folding in Drosophila embryos. By combining the imaging system with optogenetic manipulation of muscle constriction, the authors showed that Hox genes are required to trigger Ca++ spikes and induce muscle constriction specifically in the future-fold region, which results in changes in endodermal cell shape. The authors' claims were supported by data. However, the quantification of endodermal cell behaviors, especially with respect to the link between cell- and tissue-scale …
Reviewer #3 (Public Review):
This manuscript addresses important questions in developmental biology: how Hox codes are transformed into organ shapes and how different tissue layers mechanically interact during morphogenesis. The authors set up a deep tissue imaging system that allowed them to simultaneously track the endoderm and overlaying muscles during gut folding in Drosophila embryos. By combining the imaging system with optogenetic manipulation of muscle constriction, the authors showed that Hox genes are required to trigger Ca++ spikes and induce muscle constriction specifically in the future-fold region, which results in changes in endodermal cell shape. The authors' claims were supported by data. However, the quantification of endodermal cell behaviors, especially with respect to the link between cell- and tissue-scale deformations would benefit from comparisons of folded and unfolded regions that could be generated from existing data.
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