Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells

Abstract

Apical constriction typically accompanies inward folding of an epithelial sheet. In recent years there has been progress in understanding mechanisms of apical constriction and their contribution to morphogenetic processes. Sea urchin embryos form a specialized region of ectoderm, the ciliary band, which is a strip of epithelium, three to five cells wide, encircling the oral ectoderm and functioning in larval swimming and feeding. Ciliary band cells exhibit distinctive apical-basal elongation, have narrow apices bearing a cilium, and are planar polarized, so that cilia beat away from the mouth. Here, we show that filamentous actin and phosphorylated myosin light chain are uniquely distributed in ciliary band cells. Inhibition of myosin phosphorylation or actin polymerization perturbs this distribution and blocks apical constriction. During ciliary band formation, Sp-Ephrin and Sp-Eph expression overlap in the presumptive ciliary band. Knockdown of Sp-Eph or Sp-Ephrin, or treatment with an Eph kinase inhibitor interferes with actomyosin networks, accumulation of phosphorylated FAK (pY 397 FAK), and apical constriction. The cytoplasmic domain of Sp-Eph, fused to GST and containing a single amino acid substitution reported as kinase dead, will pull down pY 397 FAK from embryo lysates. As well, pY 397 FAK colocalizes with Sp-Eph in a JNK-dependent, planar polarized manner on latitudinal apical junctions of the ciliary band and this polarization is dissociable from apical constriction. We propose that Sp-Eph and pY 397 FAK function together in an apical complex that is necessary for remodeling actomyosin to produce centripetal forces causing apical constriction. Morphogenesis of ciliary band cells is a unique example of apical constriction in which receptor-mediated cell shape change produces a strip of specialized tissue without an accompanying folding of epithelium.

PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling

“…As a default response to ephrin contact, cell contraction and cell-cell segregation rely on intact Eph tyrosine kinase activity and ADAM10-mediated proteolytic shedding of the interacting cell surface ephrin (Hattori et al, 2000;Janes et al, 2005Janes et al, , 2009, initiating signalling that modulates cytoskeletal and focal adhesion proteins (Nievergall et al, 2012;Pasquale, 2008). Emerging evidence suggests that Ephs can also be directly linked to the cortical actin cytoskeleton and to actin stress fibres (Salaita et al, 2010), and are involved in actinmyosin polymerisation (Krupke and Burke, 2014). However, molecular details of potential interactions between Ephs and cytoskeletal proteins, and mechanisms involved in relaying the effects of Eph clustering and transient kinase activation into a cytoskeletal reorganisation, remain largely unclear.…”

Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos

“…However, urchins have the simplest set: a single receptor and ligand. In urchins, Eph and Ephrin are expressed in broad domains of ectoderm, where they function at the interface to mediate apical contractility (Krupke and Burke, 2014). We now add to this a role in mediating aspects of dispersal and epithelial transition of migratory cells.…”

“…Ectopic expression of Sp-Efn was achieved using synthetic, capped mRNAs derived from the full length S. purpuratus Sp-Efn gene cloned in pCS2+ and transcribed using the SP6 mMessage mMachine kit (Ambion). 2–4 pL of a 0.2 μM injection solution was injected into each freshly fertilized egg as previously described (Krupke and Burke, 2014). Single blastomere injections followed Krupke et al (2014).…”

“…In addition, urchins are basal deuterostomes, so they provide an opportunity to study the range of morphogenetic mechanisms employed by embryos that share a common ancestor with vertebrates (Drescher, 2002; Mellott and Burke, 2008). In S. purpuratus , embryos express Sp-Eph and Sp-Efn in two overlapping domains of ectoderm and Eph/Ephrin signaling appears to have a role in morphogenesis of the ciliary band (Krupke and Burke, 2014). …”

The characterization of larval homology: transcriptomic insights into the origin and evolution of animal life cycles

“…Additionally, I identified six TFs that are differentially expressed in four different species, some of which imply eumetazoan-specific (such as orthologs of TCF/Par), bilaterian-specific (such as Thr), or metazoan-specific conservation (such as Six1/2, Klf, Ezh, and Arx) , Otim et al 2004, Burke et al 2006, Peter and Davidson 2011, Tu et al 2012, Cui et al 2014, Krupke and Burke 2014, Barsi and Davidson 2016, which I identified as differentially expressed in all sampled deuterostome taxa, and is known to be important for larval ciliary bands in Strongylocentrotus (Barsi et al 2015, Barsi andDavidson 2016).…”