Cell-autonomous integrin control of Wnt and Notch signalling during somitogenesis

Abstract

SUMMARYIntegrins act at signalling crossroads, and their interactions with other signal transduction pathways are key to the regulation of normal and pathological cell cytoarchitecture and behaviour. Here, we describe a signalling cascade that acts during the formation of the defining segmental features of the vertebrate body -the somites -in which 1-integrin activity regulates epithelialisation by controlling downstream Wnt and Notch activity crucial for somite border formation. Using in vivo transcriptional inhibition in the developing chick embryo, we show that 1-integrin in the anterior presomitic mesoderm activates canonical Wnt signalling in a cell-autonomous, 'outside-inside' manner. Signalling is mediated by integrin-linked kinase (ILK), leading to modulation of glycogen synthase kinase 3 (GSK3) phosphorylation, and activates Notch signalling in the anterior presomitic mesoderm. The two signalling pathways then cooperate to promote somite formation via cMESO1/Mesp2. Our results show that 1-integrin can regulate cell shape and tissue morphogenesis indirectly, by regulation of downstream signalling cascades. further define an outside-inside signalling pathway in which 1-integrin signals cell-autonomously via ILK to activate Wnt signalling in the anterior PSM. Wnt signalling then activates Notch signalling, and these target pathways act in combination to drive somite boundary formation. These results define a regulatory cascade by which 1-integrin regulates cell and tissue architecture in vivo. KEY WORDS MATERIALS AND METHODS EggsFertilised chicken eggs (Henry Stewart, Louth, UK) were incubated at 37°C and were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1951). ElectroporationElectroporation conditions were based on those described previously (Momose et al., 1999). In summary, DNA plasmids were diluted to 1-2 mg/ml in PBS containing 2% sucrose and mixed with Fast Green for visualisation of the injection site. For electroporations at HH9, DNA was injected in ovo over the anterior primitive streak. Electrodes were placed at either side of the embryo and five 50-msecond pulses of 35V were applied. For electroporations in the node and primitive streak at HH4/5, the negative and positive electrodes were placed above and below the embryo, respectively, and five 50-msecond pulses of 10V were applied. Embryo cultureChick embryos were cultured in L15 medium supplemented with 15% foetal calf serum in a roller incubator at 37°C. For pharmacological inhibition of signalling pathways, embryos were cultured for 6 hours in 10-100 mM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), 1 mg/ml recombinant mouse Dkk1 (R&D Systems), 5 mM LiCl, or 200 mM CKI-7 (Sigma-Aldrich). In situ hybridisation, immunohistochemistry and western blotWhole-mount in situ hybridisation was carried out as previously described (Henrique et al., 1995). Immunohistochemistry was performed using standard methods with mouse anti-chick fibronectin (B3/D6 and VA1; supernatant 1/5, Developmental S...

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“…These studies showed that β1 integrins activate Jagged1 transcription through ILK-mediated stabilization of β-catenin. Stabilized β-catenin directly binds and activates Jagged1 promoters upon translocation into the nucleus (Estrach et al, 2006;Rallis et al, 2010). This is consistent with our observations that Notch signaling is active in cells that express Fn1, and that Notch signaling declines concomitant with the decrease in Fn1 levels.…”

“…Thus, ablation of Fn1 or integrin α5 in the NC could interfere with Notch signaling by interfering with actin polymerization, formation of basal filopodia, or by altering the trafficking/ processing of Notch. An indirect role of β1-containing integrin heterodimers in the activation of Notch was demonstrated in the chick (Rallis et al, 2010). These studies showed that β1 integrins activate Jagged1 transcription through ILK-mediated stabilization of β-catenin.…”

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“…Wnt signaling upregulates Jag1 transcription via -catenin in the hair follicle (Estrach et al, 2006), increases Dll4 transcription during vascular remodeling (Corada et al, 2010) and induces Notch2 expression in colorectal cancer cells (Ungerback et al, 2011). During somite differentiation, 1-integrin activity controls both Wnt and Notch signaling, and activation of both signaling mechanisms is required for activation of the downstream gene cMESO1/mesp2 (Rallis et al, 2010). With regard to interaction between core components, Dishevelled (Dvl), an intracellular DEVELOPMENT 3604 mediator of all Wnt signaling pathways described to date, binds to Notch ICD (Axelrod et al, 1996;Munoz-Descalzo et al, 2010), and interactions of Notch ICD with several components of the -catenin destruction complex have also been described (Fig.…”

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“…6,7 Wnt signaling is also actively involved in myogenesis 5,[8][9][10][11] and embryonic somite patterning. 12,13 The Wnt pathway is crucial in cell proliferation, cell migration, polarity and cell death. 14,15 Dysregulation of Wnt/b-catenin signaling facilitates cancer invasion and metastases in various tumours.…”