Rodrigo Fernandez-Gonzalez

Myosin II dynamics are regulated by tension in intercalating cells.

Axis elongation in Drosophila occurs through polarized cell rearrangements driven by actomyosin contractility. Myosin II promotes neighbor exchange through the contraction of single cell boundaries, while the contraction of myosin II structures spanning multiple pairs of cells leads to rosette formation. Here we show that multicellular actomyosin cables form at a higher frequency than expected by chance, indicating that cable assembly is an active process. Multicellular cables are sites of increased mechanical tension as measured by laser ablation. Fluorescence recovery after photobleaching experiments show that myosin II is stabilized at the cortex in regions of increased tension. Myosin II is recruited in response to an ectopic force and relieving tension leads to a rapid loss of myosin, indicating that tension is necessary and sufficient for cortical myosin localization. These results demonstrate that myosin II dynamics are regulated by tension in a positive feedback loop that leads to multicellular actomyosin cable formation and efficient tissue elongation.

Integration of contractile forces during tissue invagination

Transcription factor Twist promotes cell junctions to link individual cells into a contractile network responsible for the apical constriction pulses during epithelial morphogenesis.

3D reconstruction of histological sections: Application to mammary gland tissue

In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high‐content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer‐based system integrates image acquisition, annotation, registration, and three‐dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high‐resolution molecular data. Microsc. Res. Tech. 73:1019–1029, 2010.

Rho-Kinase Directs Bazooka/Par-3 Planar Polarity during Drosophila Axis Elongation

Cell rearrangements shape the Drosophila embryo via spatially regulated changes in cell shape and adhesion. We show that Bazooka/Par-3 (Baz) is required for the planar polarized distribution of myosin II and adherens junction proteins and polarized intercalary behavior is disrupted in baz mutants. The myosin II activator Rho-kinase is asymmetrically enriched at the anterior and posterior borders of intercalating cells in a pattern complementary to Baz. Loss of Rho-kinase results in expansion of the Baz domain, and activated Rho-kinase is sufficient to exclude Baz from the cortex. The planar polarized distribution of Baz requires its C-terminal domain. Rho-kinase can phosphorylate this domain and inhibit its interaction with phosphoinositide membrane lipids, suggesting a mechanism by which Rho-kinase could regulate Baz association with the cell cortex. These results demonstrate that Rho-kinase plays an instructive role in planar polarity by targeting Baz/Par-3 and myosin II to complementary cortical domains.

Oscillatory behaviors and hierarchical assembly of contractile structures in intercalating cells

Fluctuations in the size of the apical cell surface have been associated with apical constriction and tissue invagination. However, it is currently not known if apical oscillatory behaviors are a unique property of constricting cells or if they constitute a universal feature of the force balance between cells in multicellular tissues. Here, we set out to determine whether oscillatory cell behaviors occur in parallel with cell intercalation during the morphogenetic process of axis elongation in the Drosophila embryo. We applied multi-color, time-lapse imaging of living embryos and SIESTA, an integrated tool for automated and semi-automated cell segmentation, tracking, and analysis of image sequences. Using SIESTA, we identified cycles of contraction and expansion of the apical surface in intercalating cells and characterized them at the molecular, cellular, and tissue scales. We demonstrate that apical oscillations are anisotropic, and this anisotropy depends on the presence of intact cell-cell junctions and spatial cues provided by the anterior-posterior patterning system. Oscillatory cell behaviors during axis elongation are associated with the hierarchical assembly and disassembly of contractile actomyosin structures at the medial cortex of the cell, with actin localization preceding myosin II and with the localization of both proteins preceding changes in cell shape. We discuss models to explain how the architecture of cytoskeletal networks regulates their contractile behavior and the mechanisms that give rise to oscillatory cell behaviors in intercalating cells.

Wounded cells drive rapid epidermal repair in the early Drosophila embryo.

In early Drosophila embryos, wound closure is driven by the apical constriction of the wounded cells, which assemble actomyosin networks that lose actin and myosin as they contract. In late embryos, wound closure is mediated by the cells around the wound, which assemble an actomyosin purse string that contracts with constant actomyosin levels.

Cell Mechanics and Feedback Regulation of Actomyosin Networks

Mechanical signals shape the organization and dynamics of contractile networks in cells during morphogenesis. Actomyosin contractility is the major force-generating machinery that shapes cells and tissues during morphogenesis. New evidence from Drosophila demonstrates that these forces are spatially organized by a combination of biochemical and mechanical signals that provide dynamic feedback in a complex cellular environment.

Local mechanical forces promote polarized junctional assembly and axis elongation in Drosophila

Axis elongation is a conserved process in which the head-to-tail or anterior-posterior (AP) axis of an embryo extends. In Drosophila, cellular rearrangements drive axis elongation. Cells exchange neighbours by converging into transient multicellular vertices which resolve through the assembly of new cell interfaces parallel to the AP axis. We found that new interfaces elongate in pulses correlated with periodic contractions of the surrounding cells. Inhibiting actomyosin contractility globally, or specifically in the cells around multicellular vertices, disrupted the rate and directionality of new interface assembly. Laser ablation indicated that new interfaces sustained greater tension than non-elongating ones. We developed a method to apply ectopic tension and found that increasing AP tension locally increased the elongation rate of new edges by more than twofold. Increasing dorsal-ventral tension resulted in vertex resolution perpendicular to the AP direction. We propose that local, periodic contractile forces polarize vertex resolution to drive Drosophila axis elongation. DOI: http://dx.doi.org/10.7554/eLife.10757.001

A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior. Summary: A high-throughput in vitro system allowing the induction of peri-gastrulation-like fates in geometrically confined hPSC colonies reveals that a two-step process underlies the observed self-organization and subsequent fate acquisition.

Automated multidimensional image analysis reveals a role for Abl in embryonic wound repair

The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. We introduce MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. We validate MEDUSA by quantifying wound closure in Drosophila embryos, and we show that the results of our automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. We demonstrate that MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, we find that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. We demonstrate that Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein β-catenin at the wound margin during embryonic wound repair. Finally, we discuss different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin.