Correct regionalization of a tissue primordium is essential for coordinated morphogenesis

  1. Yara E Sánchez-Corrales
  2. Guy B Blanchard
  3. Katja Röper

  • Curated by eLife

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    Evaluation Summary:

    Sanchez et al investigate how morphogenetic movements driving epithelial tube formation are patterned to occur with the correct spatiotemporal dynamics, a fundamental question in developmental biology. By correlating dynamic patterns of transcription factor expression with rigorous, quantitative analyses of cell behaviors across the salivary gland primordium, their results suggest Hkb and Fkh transcription factor patterning induces switches in cell behaviors at fixed positions to promote continued morphogenesis of the tubular structure. This mechanism is likely to be more generally important for the development of complex tubular organs.

    (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 #3 agreed to share their name with the authors.)

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Abstract

During organ development, tubular organs often form from flat epithelial primordia. In the placodes of the forming tubes of the salivary glands in the Drosophila embryo, we previously identified spatially defined cell behaviors of cell wedging, tilting, and cell intercalation that are key to the initial stages of tube formation. Here, we address what the requirements are that ensure the continuous formation of a narrow symmetrical tube from an initially asymmetrical primordium whilst overall tissue geometry is constantly changing. We are using live-imaging and quantitative methods to compare wild-type placodes and mutants that either show disrupted cell behaviors or an initial symmetrical placode organization, with both resulting in severe impairment of the invagination. We find that early transcriptional patterning of key morphogenetic transcription factors drives the selective activation of downstream morphogenetic modules, such as GPCR signaling that activates apical-medial actomyosin activity to drive cell wedging at the future asymmetrically placed invagination point. Over time, transcription of key factors expands across the rest of the placode and cells switch their behavior from predominantly intercalating to predominantly apically constricting as their position approaches the invagination pit. Misplacement or enlargement of the initial invagination pit leads to early problems in cell behaviors that eventually result in a defective organ shape. Our work illustrates that the dynamic patterning of the expression of transcription factors and downstream morphogenetic effectors ensures positionally fixed areas of cell behavior with regards to the invagination point. This patterning in combination with the asymmetric geometrical setup ensures functional organ formation.

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  1. Version published to 10.7554/elife.72369 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    Sanchez et al investigate how the complex morphogenetic movements that drive epithelial tube formation are patterned to occur with the correct spatiotemporal dynamics by upstream transcription factor expression, a fundamental question in developmental biology. In prior work, the authors defined the cell behaviors that drive tube formation in the Drosophila salivary gland, demonstrating that localized apical constriction induces epithelial bending to form a pit and circumferential cell intercalations narrow the primordium. In this study, they show that as more peripheral cells flow into the deepening pit they switch behaviors to constrict apically, promoting continued morphogenesis of the structure. These behaviors, as well as correlated patterns of myosin pathway activation, require transcription factors Fkh and Hkb suggesting the expression pattern of these TFs may drive the dynamic changes in cell behavior. Using endogenously tagged protein reporters of Fkh and Hkb, they show the two TFs display dynamic expression patterns that initiate roughly where apical constriction will predominate and spread outward to cells that will later constrict into the pit. Fog appears to be a key downstream target of Fkh and Hkb, and interfering with the radial pattern Fog expression disrupts tube formation. Strengths of the study include the high quality, quantitative morphometric analysis in both time and space, the use of endogenously tagged reporters of Fkh and Hkb together with time-lapse analysis, and the multiscale nature of the study encompassing and connecting upstream patterning events to intermediate regulators of cell shape to downstream cell and tissue-level behaviors. A minor weakness of the study in its current form is a lack of cell tracking data that connect cell identity with the stated changes in behavior, something that should be straightforward to address.

    These data are inherent in our quantitative analysis, but were not explicitly shown.

    Reviewer #2 (Public Review):

    The authors analyze the cellular dynamics responsible for the formation of a tubular structure, taking as a model the formation of salivary gland in Drosophila embryo, which is initiated by the asymmetric invagination of cells from a circular placode. They observed a regionalized cellular behavior, dependent on Hkb and Fkh dynamic expression in the placode. Both these transcription factors are required to ensure the correct expression of Fog, which drives localized apical constriction and ensures correct morphogenesis of the salivary gland.

    Strengths: This is a detailed analysis of cellular dynamics during salivary gland morphogenesis. This study highlights the regionalized behavior of cells from the presumptive gland (or placode) with a region close to the invagination pit where Hbk and Fkh drive Fog expression, leading to medio-apical myosin accumulation and apical constriction and a more distant region where cells mostly intercalate, a process driven by a junctional Myosin polarity. Although mainly descriptive, these data are precise and convincing. The conclusions fit with the observations.

    Weaknesses: Although this work is interesting, it raises a lot of unanswered questions. How is the timing of apical constriction in the placode controlled?

    The question about the timing of apical constriction and how it evolves across the placode is exactly what we are trying to address in our study. As we show and discuss, it is the patterned and dynamic expression of the transcription of Fkh and Hkb, starting at the future pit position, that initiates the apical constriction (via downstream expression and action of Fog,) and then part of the later maintenance and continuation of apical constriction as cells move into a position in proximity to the pit is through the continued expansion of the Fkh expression.

    What is responsible for the delay in apical constriction observed in Hkb mutant?

    We explain this in the discussion of the manuscript (line 570 onwards): We conclude from our data that the initial constriction at the eccentric position where the pit forms depends on both Fkh and Hkb. In the hkb-/- mutant the central cells in the placode manage to constrict in the absence of Hkb likely only require Fkh activity to initiate Fog expression and function (and hence constriction). These cells therefore undergo their normal apical constriction once Fkh expression has reached the central position (as Fkh expression is unaffected in the hkb-/- mutant as we show). As they now are the first cells to constrict and invaginate, the hkb-/- mutants show a central and delayed constriction and invagination.

    How does apical constriction propagate? How is the switch between apical contraction and intercalating domains regulated?

    Once apical constriction is initiated at the pit position in the dorsal-posterior corner due to the action of both Hkb and Fkh (and downstream Fog), the constriction spreads across the placode, as we describe and analyse, mostly due to expanding expressing of Fkh, driving the expansion of Fog expression in its wake. The cell intercalation behaviour we observe and describe here and in (Sanchez-Corrales et al., 2018) leads to a convergence and extension of the tissue that feeds more cells into a position near the pit. Once Fkh expression has reached cells now in a closer position to the pit they also start to apically constrict.

  3. eLife

    Evaluation Summary:

    Sanchez et al investigate how morphogenetic movements driving epithelial tube formation are patterned to occur with the correct spatiotemporal dynamics, a fundamental question in developmental biology. By correlating dynamic patterns of transcription factor expression with rigorous, quantitative analyses of cell behaviors across the salivary gland primordium, their results suggest Hkb and Fkh transcription factor patterning induces switches in cell behaviors at fixed positions to promote continued morphogenesis of the tubular structure. This mechanism is likely to be more generally important for the development of complex tubular organs.

    (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 #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Sanchez et al investigate how the complex morphogenetic movements that drive epithelial tube formation are patterned to occur with the correct spatiotemporal dynamics by upstream transcription factor expression, a fundamental question in developmental biology. In prior work, the authors defined the cell behaviors that drive tube formation in the Drosophila salivary gland, demonstrating that localized apical constriction induces epithelial bending to form a pit and circumferential cell intercalations narrow the primordium. In this study, they show that as more peripheral cells flow into the deepening pit they switch behaviors to constrict apically, promoting continued morphogenesis of the structure. These behaviors, as well as correlated patterns of myosin pathway activation, require transcription factors Fkh and Hkb suggesting the expression pattern of these TFs may drive the dynamic changes in cell behavior. Using endogenously tagged protein reporters of Fkh and Hkb, they show the two TFs display dynamic expression patterns that initiate roughly where apical constriction will predominate and spread outward to cells that will later constrict into the pit. Fog appears to be a key downstream target of Fkh and Hkb, and interfering with the radial pattern Fog expression disrupts tube formation. Strengths of the study include the high quality, quantitative morphometric analysis in both time and space, the use of endogenously tagged reporters of Fkh and Hkb together with time-lapse analysis, and the multiscale nature of the study encompassing and connecting upstream patterning events to intermediate regulators of cell shape to downstream cell and tissue-level behaviors. A minor weakness of the study in its current form is a lack of cell tracking data that connect cell identity with the stated changes in behavior, something that should be straightforward to address. A limitation of the study is whether the timing of events described here are consistent their model - the dynamics of TF expression patterns precede the corresponding Fog/myosin patterns and morphogenetic changes by ~30-60 minutes. An analysis of Fog transcriptional dynamics could fill this gap.

  5. eLife

    Reviewer #2 (Public Review):

    The authors analyze the cellular dynamics responsible for the formation of a tubular structure, taking as a model the formation of salivary gland in Drosophila embryo, which is initiated by the asymmetric invagination of cells from a circular placode. They observed a regionalized cellular behavior, dependent on Hkb and Fkh dynamic expression in the placode. Both these transcription factors are required to ensure the correct expression of Fog, which drives localized apical constriction and ensures correct morphogenesis of the salivary gland.

    Strengths:

    This is a detailed analysis of cellular dynamics during salivary gland morphogenesis. This study highlights the regionalized behavior of cells from the presumptive gland (or placode) with a region close to the invagination pit where Hbk and Fkh drive Fog expression, leading to medio-apical myosin accumulation and apical constriction and a more distant region where cells mostly intercalate, a process driven by a junctional Myosin polarity. Although mainly descriptive, these data are precise and convincing. The conclusions fit with the observations.

    Weaknesses:

    Although this work is interesting, it raises a lot of unanswered questions. How is the timing of apical constriction in the placode controlled? What is responsible for the delay in apical constriction observed in Hkb mutant? How does apical constriction propagate? How is the switch between apical contraction and intercalating domains regulated?

  6. eLife

    Reviewer #3 (Public Review):

    In the manuscript by Sanchez-Corrales, Blanchard, and Röper, the authors examine how the Drosophila salivary glands form from a primordium where the specifying transcription factors are expressed asymmetrically. Previous recent studies have shown the roles of apical constriction and cell intercalation to salivary gland formation (Sanchez-Corrales et al., 2018; Chung et al., 2017). In this study, the authors characterize the pattern of these cell behaviors across the primordium and correlate these with the expression of transcription factors, Hkb and Fkh. The authors found that cells in the dorsal-posterior of the primordium apically constrict and invaginate and that the position of this behavior is controlled by Hkb. The authors conclude that the expression of a GPCR ligand, Fog, is patterned by the transcription factors Hkb and Fkh, which leads to primordium regionalization.

    The finding of this transcription factor patterning and switches in cell behaviors at fixed positions in the primordium is likely to be more generally important for the development of complex tubular organs. The authors' conclusions are mostly well supported by the data, which is rigorously quantified. One limitation is that the conclusion that the pattern of Hkb expression regulates this cell behavior is based on analyzing a loss-of-function mutant in hkb, rather than altering its expression pattern.

  7. Version published to 10.1101/2020.08.29.273219v1 on bioRxiv