© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2473-2482 doi:10.1242/dev.103499 RESEARCH ARTICLE Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion Alicia M. Ebert1, Sarah J. Childs2, Carrie L. Hehr1, Paula B. Cechmanek1 and Sarah McFarlane1,* ABSTRACT morphogenetic events of embryogenesis in the absence of a Organs are generated from collections of cells that coalesce and physical barrier? remain together as they undergo a series of choreographed Two simple molecular mechanisms that result in the formation of movements to give the organ its final shape. We know little about borders between like and non-like tissues help explain how tissue the cellular and molecular mechanisms that regulate tissue cohesion integrity is promoted (Krens and Heisenberg, 2011). The first during morphogenesis. Extensive cell movements underlie eye involves selective adhesion, whereby two distinct cell populations development, starting with the eye field separating to form bilateral express different levels or types of adhesion molecules. Homotypic vesicles that go on to evaginate from the forebrain. What keeps eye interactions between like cells then separate and sort the two cell cells together as they undergo morphogenesis and extensive types (Nandadasa et al., 2009). The second mechanism relies on one proliferation is unknown. Here, we show that plexina2 (Plxna2), a population of cells expressing a repellent membrane-associated member of a receptor family best known for its roles in axon and cell ligand and the other expressing the receptor (Krens and Heisenberg, guidance, is required alongside the repellent semaphorin 6a 2011). For example, transmembrane ephrins and their Eph receptors (Sema6a) to keep cells integrated within the zebrafish eye vesicle mediate repellent interactions that prevent mixing between the cells epithelium. sema6a is expressed throughout the eye vesicle, of adjacent hindbrain rhombomeres (Cooke et al., 2005; Kemp whereas plxna2 is restricted to the ventral vesicle. Knockdown of et al., 2009). Ephrin/Eph signaling also segregates somitic cells Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse (Watanabe and Takahashi, 2010), ectodermal and mesodermal microscopy showing that eye progenitors either fail to enter the tissue (Rohani et al., 2011), and anterior neural plate domains evaginating vesicles or delaminate from the eye epithelium. Explant (Cavodeassi et al., 2013). Other than these few examples, however, experiments, and rescue of eye vesicle integrity with simultaneous we know little of the molecular mechanisms that mediate tissue knockdown of sema6a and plxna2, point to an eye-autonomous cohesion during development. requirement for Sema6a/Plxna2. We propose a novel, tissue- In this regard, members of the semaphorin (Sema) family and their autonomous mechanism of organ cohesion, with neutralization of plexin (Plxn) receptors are interesting (Zhou et al., 2008). They serve repulsion suggested as a means to promote interactions between as repellents for migrating neurons, axonal growth cones and cells within a tissue domain. endothelial cells (Yazdani and Terman, 2006). Furthermore, certain Sema proteins are membrane associated and thus would be ideal KEY WORDS: Plexin, Semaphorin, Morphogenesis, Repulsion, candidates to mediate signaling between cells in contact (Zhou et al., Zebrafish 2008). Indeed, Sema6a helps organize chick cardiac development (Toyofuku et al., 2004) and Plx2 controls C. elegans epidermal INTRODUCTION morphogenesis (Nakao et al., 2007). During development, cells are specified to become part of a given In this paper, we provide evidence that Sema6a and Plxna2 tissue, and then sorted so that like cells coalesce and are kept promote cohesion of the zebrafish eye primordia, though not segregated from non-like cells in adjacent tissues. Over time, primarily through a boundary mechanism between adjacent tissues. physical barriers, such as extracellular matrix or epithelium, Sema6a is expressed by progenitors throughout the eye vesicle, develop to prevent heterotypic interactions between cells of whereas cells in the ventral eye vesicle express its known receptor neighboring domains. A period of extensive change in both cell Plxna2. With Sema6a or Plxna2 knockdown, many morphant eye and tissue shape, however, usually precedes the establishment of a progenitors fail to associate with the eye epithelium and exit the eye physical barrier. For example, eye formation involves several vesicle into either the vesicle lumen or the surrounding mesenchyme. distinct phases of integrated cell movements (Graw, 2010). Eye We propose a novel and third mechanism of tissue cohesion, which precursors are separated away from non-retinal cells through a involves dampening of tissue-autonomous repellent signaling. Our motile sorting mechanism, ultimately to collect together as a single data argue that Sema6a/Plxna2 act eye autonomously to establish and contiguous eye field (Moore et al., 2004; Cavodeassi et al., 2005). maintain the position and integration of cells within the eye vesicle The eye field separates to produce the bilateral optic vesicles, which epithelium, processes we postulate involve both repulsive signaling evaginate from the forebrain. A key question then is how do cells of and selective neutralization of Sema6a activity within the ventral a specified tissue retain close associations through the complex eye vesicle. 1Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, Calgary, RESULTS Alberta T2N 4N1, Canada. 2Department of Biochemistry and Molecular Biology, plxna2 is required for eye vesicle development University of Calgary, Calgary, Alberta T2N 4N1, Canada. Several class 6 semaphorin genes (Ebert et al., 2012) and plxna2,a *Author for correspondence ([email protected]) known receptor for Sema6a in mammals (Suto et al., 2007; Renaud et al., 2008; Tawarayama et al., 2010), are expressed in the 72 hours Received 3 October 2013; Accepted 24 April 2014 post fertilization (hpf) zebrafish retina (supplementary material DEVELOPMENT 2473 RESEARCH ARTICLE Development (2014) 141, 2473-2482 doi:10.1242/dev.103499 Fig. S1). To investigate plxna2 expression earlier in zebrafish eye however, we focused our studyon understanding the unexpected early development, we used RNA in situ hybridization. Interestingly, role for Plxna2 in eye development. plxna2 is expressed transiently as the eye vesicles first form. At 4-12 somites, plxna2 is present in the ventral (future temporal) eye Plxna2 is required for eye vesicle integrity vesicle and the surrounding mesenchyme (Fig. 1A-D). Expression To visualize eye vesicle development, we used the Tg(rx3:GFP) continues through 18 somites and is absent from the optic cup at transgenic line, in which GFP is expressed by developing eye and 24 hpf (data not shown). hypothalamic progenitors (Rembold et al., 2006). In a 20-somite To investigate whether Plxna2 plays a role in early eye control, GFP+ cells are closely packed in the eye epithelium, development, we designed an antisense morpholino oligonucleotide whereas in plxna2 morphants the packing of GFP+ cells is less tight (MO) against plxna2 ( plxna2e3i3) that alters mRNA splicing and, as (Fig. 2A-D). In fact, significant numbers of GFP+ cells are ectopic seen by RT-PCR, excludes exon3 to create a frameshift mutation to plxna2 morphant eyes as the eye vesicles evaginate and elongate (Fig. 1F). Zebrafish eye fields are specified during gastrulation, and from the neural keel (8-12 somites) (Fig. 2E,F,H). By 12 somites, eye-field separation into the bilateral eye vesicles initiates during the GFP+ eye vesicles of plxna2 morphants are smaller than control neurulation (Rembold et al., 2006; Fuhrmann, 2010) (Fig. 1G). To (Fig. 2E-G), consistent with the decrease in the meis1+ eye domain determine whether these events occur normally in plxna2 morphants, (Fig. 1Q). Importantly, a similar loss of GFP+ cells from the eye we examined the expression of keyeye-field transcription factors: Rx3 vesicles is seen with a second plxna2 MO directed against the ATG is required for vesicle evagination (Loosli et al., 2003) and precursor start site, showing specificity (Fig. 2I,J). Subsequent analyses are proliferation (Stigloher et al., 2006); Six3 for specification (Liu et al., performed with the e3i3 MO. Moreover, the small eye vesicle 2010); and Meis1 for patterning (Erickson et al., 2010). Induction of observed with the plxna2 e3i3 MO is partially rescued by co- the eye field, as marked by rx3 and six3 at 4-6 somites, appears normal injection of human PLXNA2 mRNA, which is not targeted by the in plxna2 morphants (Fig. 1H-K). By 12 somites, in both control and splice MO (Fig. 2M-O). To verify that the loss of eye cells and small plxna2 morphant embryos, the eye field separates into bilateral eye size did not occur as a result morpholino off-target effects due to vesicles that evaginate, as seen by meis1 expression (Fig. 1L,M). The p53 activation (Robu et al., 2007), we compared eye size in plxna2 size of the meis1 domain, however, is smaller in the plxna2 morphants injected with or without a p53 MO. In both groups, eye morphants, and lagging meis1+ cells that fail to segregate into the size is reduced when compared with control eyes (data not shown). eye vesicles form a bridge of cells in the midline (Fig. 1L,M,P,Q). These data argue strongly for the specificity of the loss of eye One possibility is that cells destined