Integrin α5β1 is necessary for regulation of radial migration of cortical neurons during mouse brain development

During cerebral cortex development, post-mitotic neurons interact with radial glial fibers and the extracellular environment to migrate away from the ventricular region and form a correct laminar structure. Integrin receptors are major mediators of cell–cell and cell–extracellular matrix interactions. Several integrin heterodimers are present during formation of the cortical layers. The α5β1 receptor is expressed in the neural progenitors of the ventricular zone during cerebral cortex formation. Using in utero electroporation to introduce short hairpin RNAs in the brain at embryonic day 15.5, we were able to inhibit acutely the expression of α5 integrin in the developing cortex. The knockdown of α5 integrin expression level in neural precursors resulted in an inhibition of radial migration, without perturbing the glial scaffold. Moreover, the same inhibitory effect on neuronal migration was observed after electroporation of a Cre recombinase expression plasmid into the neural progenitors of conditional knockout mice for α5 integrin. In both types of experiments, the electroporated cells expressing reduced levels of α5 integrin accumulated in the premigratory region with an abnormal morphology. At postnatal day 2, ectopic neurons were observed in cortical layer V, while a deficit of neurons was observed in cortical layer II–IV. We show that these neurons do not express a layer V-specific marker, suggesting that they have not undergone premature differentiation. Overall, these results indicate that α5β1 integrin functions in the regulation of neural morphology and migration during cortical development, playing a role in cortical lamination.National Institutes of Health (U.S.) (NHLBI PO1HL66105)National Institutes of Health (U.S.) (NIGMS GM064346)Australian Research CouncilInstitut national de physique nucléaire et de physique des particulesUniversité de StrasbourgLigue nationale contre le cancer (France)Association française contre les myopathie

Laminin α5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through β1 integrin signaling

AbstractLaminin α chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin α5 in mouse submandibular glands (SMGs). Lama5−/− SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs α5 and α1 are present in epithelial clefts but as branching begins α5 expression increases while α1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-β1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that β1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3−/−:Itga6−/− SMGs have a similar phenotype to Lama5−/−. Our findings suggest that laminin α5 controls SMG epithelial morphogenesis through β1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling

Cell adhesion: parallels between vertebrate and invertebrate focal adhesions

International audienceRecent studies highlight the striking similarity between vertebrate focal adhesion plaques and Caenorhabditis elegans muscle adhesion structures and position LIM domain proteins as central players at focal adhesions

Essential role of alpha 6 integrins in cortical and retinal lamination

International audienceExtracellular matrix (ECM) is believed to play important roles in many aspects of nervous system development [1]. The laminins are ECM glycoproteins expressed in neural tissues and are potent stimulators of neurite outgrowth in vitro [1-3]. Genetic approaches using Drosophila and Caenorhabditis elegans have demonstrated a role for laminin and a laminin receptor in vivo in axon pathfinding and fasciculation, respectively [4,5]. In higher organisms, however, the role of laminins in the development of the nervous system is poorly understood. Integrins alpha 6 beta 1 and alpha 6 beta 4 are major laminin receptors. A role for the alpha 6 integrin in neurulation has been reported in amphibians [6]. We previously described mice lacking integrin alpha 6; these mice died at birth with severe skin blistering [7]. Detailed analyses of integrin alpha 6-/- mice reported here revealed abnormalities in the laminar organization of the developing cerebral cortex and retina. Ectopic neuroblastic outgrowths were found on the brain surface and in the vitreous body in the eye. Alterations of laminin deposition were found in mutant brains. Thus, this study provides evidence for an essential role of integrin-laminin interactions in the proper development of the nervous system. These observations are particularly significant given the recent report that human patients suffering from epidermolysis bullosa can carry mutations in ITGA6, the gene encoding the alpha 6 integrin chain [8,9]

Expression of fascin-1, the gene encoding the actin-bundling protein fascin-1, during mouse embryogenesis

Fascin-1 is an actin-bundling protein that contributes to the architecture and function of cell protrusions and microfilaments in cell adhesion, interactions and motility. Fascin-1 has been studied in cultured cells and by biophysical methods, but little is known about its distribution and functions in vertebrate development. As a first step to understanding the role of fascin-1 in embryogenesis, we have characterised the expression pattern of fascin-1 by in situ hybridisation on whole-mount and sectioned mouse embryos from embryonic day (E)8.0-E16.5. Fascin-1 was widely expressed throughout the embryo and the developing nervous system and mesenchymal tissues represented major sites of expression. Intense signals were observed in different regions of the brain, in the spinal cord and retina, and the cranial and dorsal root ganglia (DRG) appeared strongly positive. This neural expression remained strong throughout development. Fascin-1 was also present in the developing somites. High expression was detected in branchial arches and limb bud mesenchyme. At later stages, fascin-1 was expressed in different muscles of the face, skeletal muscles of the body, and in smooth muscle layers of several organs. Limb tendons appeared strongly positive. There was weak expression in heart ventricles. These results show that fascin-1 is principally expressed in neural and mesenchymal derivatives during embryonic development

An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse

International audienceHemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6β4 integrin recycling. We find that α6β4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6β4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover

Basement membrane attachment is dispensable for radial glial cell fate and for proliferation, but affects positioning of neuronal subtypes.

International audienceRadial glial cells have been shown to act as neuronal precursors in the developing cortex and to maintain their radial processes attached to the basement membrane (BM) during cell division. Here, we examined a potential role of direct signalling from the BM to radial glial cells in three mouse mutants where radial glia attachment to the BM is disrupted. This is the case if the nidogen-binding site of the laminin gamma1 chain is mutated, in the absence of alpha6 integrin or of perlecan, an essential BM component. Surprisingly, cortical radial glial cells lacking contact to the BM were not affected in their proliferation, interkinetic nuclear migration, orientation of cell division and neurogenesis. Only a small subset of precursors was located ectopically within the cortical parenchyma. Notably, however, neuronal subtype composition was severely disturbed at late developmental stages (E18) in the cortex of the laminin gamma1III4-/- mice. Thus, although BM attachment seems dispensable for precursor cells, an intact BM is required for adequate neuronal composition of the cerebral cortex