Dörthe Jülich

Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling.

Extracellular matrixes (ECMs) coat and subdivide animal tissues, but it is unclear how ECM formation is restricted to tissue surfaces and specific cell interfaces. During zebrafish somite morphogenesis, segmental assembly of an ECM composed of Fibronectin (FN) depends on the FN receptor Integrinα 5β1. Using in vivo imaging and genetic mosaics, our studies suggest that incipient Itgα5 clustering along the nascent border precedes matrix formation and is independent of FN binding. Integrin clustering can be initiated by Eph/Ephrin signaling, with Ephrin reverse signaling being sufficient for clustering. Prior to activation, Itgα5 expressed on adjacent cells reciprocally and non-cell-autonomously inhibits spontaneous Integrin clustering and assembly of an ECM. Surface derepression of this inhibition provides a self-organizing mechanism for the formation and maintenance of ECM along the tissue surface. Within the tissue, interplay between Eph/Ephrin signaling, ligand-independent Integrin clustering and reciprocal Integrin inhibition restricts de novo ECM production to somite boundaries.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1-/-. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.

Cell-Fibronectin Interactions Propel Vertebrate Trunk Elongation via Tissue Mechanics

During embryonic development and tissue homeostasis, cells produce and remodel the extracellular matrix (ECM). The ECM maintains tissue integrity and can serve as a substrate for cell migration. Integrin α5 (Itgα5) and αV (ItgαV) are the α subunits of the integrins most responsible for both cell adhesion to the ECM protein fibronectin (FN) and FN matrix fibrillogenesis. We perform a systems-level analysis of cell motion in the zebrafish tail bud during trunk elongation in the presence and absence of normal cell-FN interactions. Itgα5 and ItgαV have well-described roles in cell migration in vitro. However, we find that concomitant loss of itgα5 and itgαV leads to a trunk elongation defect without substantive alteration of cell migration. Tissue-specific transgenic rescue experiments suggest that the FN matrix on the surface of the paraxial mesoderm is required for body elongation via its role in defining tissue mechanics and intertissue adhesion.

Essential roles of fibronectin in the development of the left-right embryonic body plan.

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left-right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left-right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left-right axis of asymmetry in vertebrates.

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

Segmental Assembly of Fibronectin Matrix Requires rap1b and integrin α5

Background: During segmentation of the zebrafish embryo, inside‐out signaling activates Integrin α5, which is necessary for somite border morphogenesis. The direct activator of Integrin α5 during this process is unknown. One candidate is Rap1b, a small monomeric GTPase implicated in Integrin activation in the immune system. Results: Knockdown of rap1b, or overexpression of a dominant negative rap1b, causes a mild axis elongation defect in zebrafish. However, disruption of rap1b function in integrin α5−/− mutants results in a strong reduction in Fibronectin (FN) matrix assembly in the paraxial mesoderm and a failure in somite border morphogenesis along the entire anterior‐posterior axis. Somite patterning appears unaffected, as her1 oscillations are maintained in single and double morphants/mutants, but somite polarity is gradually lost in itgα5−/−; rap1b MO embryos. Conclusions: In itgα5−/− mutants, rap1b is required for proper somite border morphogenesis in zebrafish. The loss of somite borders is not a result of aberrant segmental patterning. Rather, somite boundary formation initiates but is not completed, due to the failure to assemble FN matrix along the nascent boundary. We propose a model in which Rap1b activates Integrin/Fibronectin receptors as part of an “inside‐out” signaling pathway that promotes Integrin binding to FN, FN matrix assembly, and subsequent stabilization of morphological somite boundaries. Developmental Dynamics 242:122–131, 2013.

A Sawtooth Pattern of Cadherin 2 Stability Mechanically Regulates Somite Morphogenesis

Differential cadherin (Cdh) expression is a classical mechanism for in vitro cell sorting. Studies have explored the roles of differential Cdh levels in cell aggregates and during vertebrate gastrulation, but the role of differential Cdh activity in forming in vivo tissue boundaries and boundary extracellular matrix (ECM) is unclear. Here, we examine the interactions between cell-cell and cell-ECM adhesion during somitogenesis, the formation of the segmented embryonic precursors of the vertebral column and musculature. We identify a sawtooth pattern of stable Cdh2 adhesions in which there is a posterior-to-anterior gradient of stable Cdh2 within each somite, while there is a step-like drop in stable Cdh2 along the somite boundary. Moreover, we find that the posterior somite boundary cells with high levels of stable Cdh2 have the most columnar morphology. Cdh2 is required for maximal cell aspect ratio and thus full epithelialization of the posterior somite. Loss-of-function analysis also indicates that Cdh2 acts with the fibronectin (FN) receptor integrin α5 (Itgα5) to promote somite boundary formation. Using genetic mosaics, we demonstrate that differential Cdh2 levels are sufficient to induce boundary formation, Itgα5 activation, and FN matrix assembly in the paraxial mesoderm. Elevated cytoskeletal contractility is sufficient to replace differential Cdh2 levels in genetic mosaics, suggesting that Cdh2 promotes ECM assembly by increasing cytoskeletal and tissue stiffness along the posterior somite boundary. Throughout somitogenesis, Cdh2 promotes ECM assembly along tissue boundaries and inhibits ECM assembly in the tissue mesenchyme.