Gut Development: A squash and a squeeze

Advanced imaging techniques reveal details of the interactions between the two layers of the embryonic midgut that influence its ultimate shape.
  1. Danelle Devenport  Is a corresponding author
  1. Department of Molecular Biology, Princeton University, United States

The gastrointestinal tract of most animal species is far longer than the body in which it is housed. The human gut, for example, is approximately 20 feet long and must fold, loop and twist to fit inside the body (Helander and Fändriks, 2014). Remarkably, contortions of the gut tube are highly stereotyped and species-specific, indicating that the formation of the folds is genetically controlled (Savin et al., 2011). However, it remains unclear how the instructions encoded within the genome lead to such precise and reproducible changes of shape.

To get to the bottom of this, developmental biologists seek to describe the movements of individual cells and connect these to a change in the shape of the whole organ. This remains a challenge, especially for internal organs, which develop deep within embryos and whose shape is determined by the interactions between multiple layers of tissue. Recent advances in live imaging using 3D light-sheet microscopy have allowed biologists to visualize morphological change on the surface of whole embryos, but internal organs like the gut have remained largely out of reach (Wan et al., 2019).

Now, in eLife, Sebastian Streichan of the University of California Santa Barbara and colleagues – including Noah Mitchell as first author – report a new method that combines deep-tissue light-sheet microscopy with a framework to analyze shape changes between tissue layers in the gut of fruit flies (Mitchell et al., 2022).

The midgut of fruit flies begins as a simple tube consisting of an inner epithelial layer ensheathed by smooth muscle. The gut tube then constricts at three precise positions, which subdivides the tube into four chambers as it changes shape and gets longer (Figure 1). To visualize this folding and elongation, Mitchell et al. expressed fluorescent markers selectively in cells of the midgut and used genetically modified, transparent embryos to reduce light scatter. Using confocal multiview light-sheet microscopy, the researchers generated time-lapse movies of full, 3D volumes of the developing midgut (de Medeiros et al., 2015). By measuring the geometry of whole organs, they found that the length of the gut tube triples during folding, while maintaining a near constant volume. This occurs in the absence of cell divisions, suggesting that changes in the shape of cells may be responsible for the elongation.

Shaping of the developing midgut of fruit flies.

Top: Automatic segmentation tools enable layer-specific imaging of the muscle (yellow ) and endoderm (blue) to generate a 3D shape. Bottom: The midgut initially consists of muscle cells (yellow) and a layer of endodermal cells (blue), which interact to mold the gut into shape. The gut tube constricts at three precise positions, which subdivide it into four chambers before it starts to coil.

To find out how the behavior of individual cells drives the constriction and elongation of the gut, Mitchell et al. developed an image analysis package aptly named TubULAR. This programme combines machine learning and computer vision techniques that link cell movements to changes in the shape of the whole organ. In many epithelia, cell intercalations – a process during which neighboring cells switch places – drive tissue convergence and extension (Paré and Zallen, 2020; Sutherland et al., 2020). In the gut tube, however, constriction and elongation correlated with patterned changes in the epithelial cell shape (Figure 2A and B).

Changes in the shape of endodermal cells are linked to a change in the shape of the whole organ.

(A) Top: Layer-specific imaging of the developing gut (early stages to the left, more developed ones to the right). Endodermal cells are initially elongated along the circumferential direction, but they change their shape during organ folding. (B) Three-dimensional representation of cells near the anterior fold. The aspect ratio of the endodermal cells (a/b, where a and b are the lengths of the cells in the circumferential and longitudinal directions) changes from greater than two to about one. (C) Hox genes regulate calcium signaling, which mediates muscle contraction (yellow cells), thus linking hox genes to organ shape through tissue mechanics. The resulting muscle contractions are mechanically coupled to the endoderm (blue), which places strain on the tissue and ultimately influences the shape of the organ.

Image credit: Adapted from Mitchell et al., 2022 (CC BY 4.0).

Modeling the gut epithelium as an incompressible material, they found that localized changes in the shape of cells in the gut folds accounted entirely for both folding and extending of the organ. In other words, gut constrictions simultaneously converge the tissue circumferentially and extend the tissue longitudinally. Mitchell et al. term this new morphological mechanism “convergent extension via constriction”.

Based on prior work, the researchers hypothesized that localized muscle contractions by the outer layer of the gut could provide the force necessary for the gut to constrict (Bilder and Scott, 1995; Wolfstetter et al., 2009). To test this idea, they employed optogenetic tools to either inhibit or stimulate muscle contractions at specific positions along the gut tube. Strikingly, they found that localized muscle contractions were both necessary and sufficient for gut constriction. Both gain or loss of muscle constrictions led to defects in the shapes of the underlying epithelial cells and in the folding of the organ.

These data provide a clear and convincing example of one tissue layer exerting both mechanical force and morphological change onto another layer. But if gut contortions are ultimately genetically encoded, what molecular information drives muscle contractions at precise positions? The homeotic (hox) transcription factors Antp and Ubx are known regulators of organ shape and are expressed at various positions along the muscle layer (Tremml and Bienz, 1989). Antp mutants lack the anterior gut fold, while Ubx mutants lack the central fold, suggesting these patterned transcription factors could promote contractions in local muscle cells. Using high-speed calcium imaging as a proxy for muscle contraction, Mitchell et al. found that calcium pulses in the muscle layer concentrated at the positions of all three folds (Figure 2C). Moreover, localized calcium pulses were lost in Antp mutants.

The results of this study demonstrate that regional hox gene expression promotes calcium signaling and muscle contractions at precise positions in the developing gut. Further, they show how mechanical coupling between layers of tissue both folds and extends the tissue into stereotyped contortions. These findings add to the growing body of research emphasizing the importance of smooth muscle as a sculptor of epithelial organs, such as the vertebrate gut and mammalian lung (Shyer et al., 2013; Huycke et al., 2019; Jaslove and Nelson, 2018). The advances in deep-tissue imaging and image analysis open new possibilities for in toto imaging of a vast variety of internal organs. Moreover, they provide a framework for evaluating how adjacent tissue layers may mechanically interact.


    1. Jaslove JM
    2. Nelson CM
    (2018) Smooth muscle: a stiff sculptor of epithelial shapes
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 373:20170318.

Article and author information

Author details

  1. Danelle Devenport

    Danelle Devenport is in the Department of Molecular Biology, Princeton University, United States

    For correspondence
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5464-259X

Publication history

  1. Version of Record published: June 30, 2022 (version 1)


© 2022, Devenport

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.


  • 504
    Page views
  • 75
  • 0

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Danelle Devenport
Gut Development: A squash and a squeeze
eLife 11:e80416.

Further reading

    1. Developmental Biology
    2. Genetics and Genomics
    Divya Khattar et al.
    Research Article

    The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+ progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+ progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.

    1. Developmental Biology
    Qiyan Mao et al.
    Research Article Updated

    Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.