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A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.

ABSTRACT: Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a nonmuscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and nonmuscle myosin II activity decreased integrin ?1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis.

SUBMITTER: Daley WP

PROVIDER: S-EPMC3647453 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.

Daley William P WP Kohn Joshua M JM Larsen Melinda M

Developmental dynamics : an official publication of the American Association of Anatomists 20110901 9

Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a nonmuscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and nonmuscle myosin II activity decreased in ...[more]

PMID: 22016182

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Project description:Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.

Project description:This study considers the problem of describing and predicting cleft formation during the early stages of branching morphogenesis in mouse submandibular salivary glands (SMG) under the influence of varied concentrations of epidermal growth factors (EGF). Given a time-lapse video of a growing SMG, first we build a descriptive model that captures the underlying biological process and quantifies the ground truth. Tissue-scale (global) and morphological features related to regions of interest (local features) are used to characterize the biological ground truth. Second, we devise a predictive growth model that simulates EGF-modulated branching morphogenesis using a dynamic graph algorithm, which is driven by biological parameters such as EGF concentration, mitosis rate, and cleft progression rate. Given the initial configuration of the SMG, the evolution of the dynamic graph predicts the cleft formation, while maintaining the local structural characteristics of the SMG. We determined that higher EGF concentrations cause the formation of higher number of buds and comparatively shallow cleft depths. Third, we compared the prediction accuracy of our model to the Glazier-Graner-Hogeweg (GGH) model, an on-lattice Monte-Carlo simulation model, under a specific energy function parameter set that allows new rounds of de novo cleft formation. The results demonstrate that the dynamic graph model yields comparable simulations of gland growth to that of the GGH model with a significantly lower computational complexity. Fourth, we enhanced this model to predict the SMG morphology for an EGF concentration without the assistance of a ground truth time-lapse biological video data; this is a substantial benefit of our model over other similar models that are guided and terminated by information regarding the final SMG morphology. Hence, our model is suitable for testing the impact of different biological parameters involved with the process of branching morphogenesis in silico, while reducing the requirement of in vivo experiments.

Project description:Prostatic branching morphogenesis is an intricate event requiring precise temporal and spatial integration of numerous hormonal and growth factor-regulated inputs, yet relatively little is known about the downstream signaling pathways that orchestrate this process. In this study, we use a novel mesenchyme-free embryonic prostate culture system, newly available mTOR inhibitors and a conditional PTEN loss-of-function model to investigate the role of the interconnected PI3K and mTOR signaling pathways in prostatic organogenesis. We demonstrate that PI3K levels and PI3K/mTOR activity are robustly induced by androgen during murine prostatic development and that PI3K/mTOR signaling is necessary for prostatic epithelial bud invasion of surrounding mesenchyme. To elucidate the cellular mechanism by which PI3K/mTOR signaling regulates prostatic branching, we show that PI3K/mTOR inhibition does not significantly alter epithelial proliferation or apoptosis, but rather decreases the efficiency and speed with which the developing prostatic epithelial cells migrate. Using mTOR kinase inhibitors to tease out the independent effects of mTOR signaling downstream of PI3K, we find that simultaneous inhibition of mTORC1 and mTORC2 activity attenuates prostatic branching and is sufficient to phenocopy combined PI3K/mTOR inhibition. Surprisingly, however, mTORC1 inhibition alone has the reverse effect, increasing the number and length of prostatic branches. Finally, simultaneous activation of PI3K and downstream mTORC1/C2 via epithelial PTEN loss-of-function also results in decreased budding reversible by mTORC1 inhibition, suggesting that the effect of mTORC1 on branching is not primarily mediated by negative feedback on PI3K/mTORC2 signaling. Taken together, our data point to an important role for PI3K/mTOR signaling in prostatic epithelial invasion and migration and implicates the balance of PI3K and downstream mTORC1/C2 activity as a critical regulator of prostatic epithelial morphogenesis.

Dataset Information

A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.

Publications

A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis.

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