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Rho Signaling Pathway and Apical Constriction in the Early Lens Placode

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Rho Signaling Pathway and Apical Constriction in the Early Lens Placode ' 2011 Wiley-Liss, Inc. genesis 00:1–12 (2011) ARTICLE Rho Signaling Pathway and Apical Constriction in the Early Lens Placode Ricardo Moraes Borges,1 Marcelo Lazzaron Lamers,2 Fabio Luis Forti,3 Marinilce Fagundes dos Santos,1 and Chao Yun Irene Yan1* 1Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sa˜ o Paulo, Sa˜ o Paulo, SP, Brazil 2Departamento de Cieˆ ncias Morfolo´ gicas do Instituto de Cieˆ ncias Ba´ sicas da Sau´ de, Universidade Federal do Rio Grande do Sul, Brazil 3Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, Sao Paulo, Brazil Received 18 October 2010; Revised 5 January 2011; Accepted 16 January 2011 Summary: Epithelial invagination in many model sys- a classical experimental paradigm in developmental tems is driven by apical cell constriction, mediated by biology. The study of its development has contributed actin and myosin II contraction regulated by GTPase to our understanding of various issues pertinent to the activity. Here we investigate apical constriction during field. In particular, the various morphological changes chick lens placode invagination. Inhibition of actin poly- that the lens placode undergoes at different stages of de- merization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify velopment illustrate its usefulness as a model system for if lens placode invaginate through apical constriction, embryonic morphogenesis. we analyzed the role of Rho-ROCK pathway. Rho In brief, prior to the appearance of the placodes, GTPases expression at the apical portion of the lens the cells of the head ectoderm are cuboidal. After placode occurs with the same dynamics as that of the the molecular events that lead to the induction of the cytoskeleton. Overexpression of the pan-Rho inhibitor lens placode (reviewed in Donner et al., 2006), the C3 exotoxin abolished invagination and had a strong cells elongate and the placode can be identified as a effect on apical myosin II enrichment and a mild effect flat thickened pseudo-stratified columnar structure in on apical actin localization. In contrast, pharmacologi- the head ectoderm overlying the optic vesicle. There- cal inhibition of ROCK activity interfered significantly after, the cell apical/basal surface ratio decreases and with apical enrichment of both actin and myosin. These the cells assume a conical shape. This results in the results suggest that apical constriction in lens invagina- tion involves ROCK but apical concentration of actin inward bending of placode, forming an initial shallow and myosin are regulated through different pathways concavity which deepens into the lens pit as invagina- upstream of ROCK. genesis 00:1–12, 2011. VC 2011 tion progresses (Schook, 1980b). The pit expands dor- Wiley-Liss, Inc. sal-ventrally so as to involve the whole placode in an Key words: lens placode; epithelial Invagination; apical cell constriction; Rho GTPase; morphogenesis Additional Supporting Information may be found in the online version of this article. * Correspondence to: Chao Yun Irene Yan, Departamento de Biologia Celular e do Desenvolvimento, Instituto de Cieˆncias Biome´dicas, Universi- INTRODUCTION dade de Sa˜o Paulo, Sa˜o Paulo, SP 05508-000, Brazil. E-mail: [email protected] The developing eye is privileged amongst all the other Contract grant sponsor: FAPESP; Contract grant number: 2007/57450-7; embryonic tissues with an anatomical localization that Contract grant sponsor: CNPq; Contract grant number: 47.5752/06-6; Contract grant sponsor: FAPESP facilitates its experimental manipulation and imaging, Published online in Wiley Online Library (wileyonlinelibrary.com). properties that have contributed to its establishment as DOI: 10.1002/dvg.20723 2 BORGES ET AL. invaginating event. After the ingression of the whole lar to previously-identified AC-driven epithelial morpho- placode toward the underlying optic cup, the edges genesis, the distribution of both apical actin and myosin fuse and detach from the surface ectoderm, forming in lens placode also require ROCK activity. However, the lens vesicle (Schook, 1980b). further investigation of the signaling involved in the reg- A variety of mechanisms have been suggested to con- ulation of AC indicates that actin and myosin II are dif- tribute to the initial inward buckling of the placode, such ferentially regulated by components upstream to as differential cell proliferation or death, tissue interac- ROCK. Accumulation of apical myosin II is abnormal tion, planar pressure derived from lateral growth and api- with C3-exotoxin-mediated inactivation of RhoGTPases, cal constriction (Hendrix et al., 1993; Plageman et al., whereas actin is unaffected by the same treatment. 2010; Schook, 1980a; Wrenn and Wessels, 1969). Apical constriction (AC) is an evolutionary conserved phenom- ena that has been extensively characterized in a variety of RESULTS epithelial bending events in metazoan embryogenesis, such as in cell shape changes during sea-urchin, Drosoph- Actin Filaments Colocalize With Myosin II ila and Xenopus gastrulation, vertebrate neural plate and at Apical Portion Prior to and During placodal bending (Barrett et al., 1997; Beane et al., 2006; Lens Placode Invagination Dawes-Hoang et al., 2005; Fox and Peifer, 2007; Kinosh- Reduction of apical surface through myosin II-medi- ita et al., 2008; Lee and Harland, 2007; Nikolaidou and ated contraction of apical actin network is associated Barrett, 2004; Plageman et al., 2010; Sai and Ladher, with specific changes in cytoskeletal architecture (Bau- 2008). The main driving force of AC is the contraction of mann, 2004; Dawes-Hoang et al., 2005; Hildebrand apically localized acto-myosin cytoskeleton. The mechani- 2005): First, actin and myosin accumulate in the apical cal force generated drives reduction of the apical surface region where reduction of cell surface area will occur. area and converts cuboidal/columnar cells into wedge- Specifically in AC-driven events, this accumulation pre- shaped cells. The coordinated occurrence of this single cedes onset of invagination. Finally, inhibition of actin- cell-shape change across the planar epithelium cell layer, myosin function interferes with epithelial invagination together with the increased intercellular adhesion con- by preventing apical constriction. ferred by the enrichment of adhesive junctions, results in To address whether chick lens placode goes through the bending of the flat epithelium. the same process during its invagination, we first inves- The molecular mechanisms that regulate apical con- tigated the dynamics of actomyosin cytoskeleton local- striction have been extensively studied in Drosophila ization prior to and during lens placode invagination. In mesodermal invagination (Lecuit and Lenne, 2007) and particular, we analyzed three different stages of lens pla- in vertebrate neural tube closure (Haigo et al., 2003; code morphogenesis: pre-placodal ectoderm, early lens Nishimura and Takeishi, 2008, Hildebrand and Soriano, placode prior to invagination and invaginating placode. 1999). In the former, the future mesoderm invaginates Pre-placodal cells are cuboidal, morphologically indis- and detaches from the ventral blastoderm epithelia. This tinguishable from the surrounding ectoderm and do not process is initiated with the binding of the secreted present a clear elongated apico-basal polarity (Byers and ligand Fog to its receptor Concertina, which in turn acti- Porter, 1964; Fig. 1). In contrast, the early placode is a vates Rho1/RhoA through RhoGEF2 (Barrett et al., 1997; prominently thickened region composed of a single Costa et al., 1994). Rho1/RhoA then activates the Rho- layer of pseudostratified columnar cells. Thereafter, the dependent kinase Drok/ROCK, which phosphorylates placode initiates the process of invagination. Morpho- and initiates myosin II contractile activity. Inhibition of logically this can be identified by an initial inward bend- Drok/ROCK activity interferes with myosin II recruit- ing step which forms a central dimple. ment to the apical region, and consequently abolishes Pre-placode ectoderm cells present both F-actin and mesodermal invagination (Dawes-Hoang et al., 2005; myosin II homogeneously distributed at the baso-lateral Nikolaidou and Barrett, 2004; Pilot and Lecuit, 2005). and apical domains (Fig. 1a–c). In the early placode, Apical constriction in vertebrates also depends on labeling for actin at the apical side is significantly ROCK (Haigo et al., 2003; Kinoshita et al., 2008). Inhi- increased relative to the basal signal. This increase was bition of ROCK activity reduces the apical levels of detectable prior to invagination (Fig. 1d–f), persisted at phosphorylated myosin II and impairs AC in avian neu- the initial bending step (Fig. 1g–i) and also throughout ral tube and MDCK kidney epithelial cell line (Hilde- its invagination (data not shown). In contrast, in the brand, 2005; Kinoshita et al., 2008; Nishimura and Take- ectoderm that surrounds the placode, which does not ichi, 2008). Here, we confirm that apical constriction invaginate, the pattern of F-actin and myosin II staining contributes toward the initial bending of the chick lens remained similar to that of pre-placode ectoderm and placode. Moreover, we show that GTPases play an im- did not present apical accumulation of contractile ele- portant role in the proper placement of apical actin and ments in any of the stages analyzed (Fig. 1j–l). Quantifi- myosin prior to AC in this experimental
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