View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Available online at www.sciencedirect.com Developmental Biology 312 (2007) 245–257 www.elsevier.com/developmentalbiology A conserved role for FGF signaling in chordate otic/atrial placode formation ⁎ Matthew J. Kourakis, William C. Smith Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106, USA Received for publication 23 April 2007; revised 12 September 2007; accepted 13 September 2007 Available online 22 September 2007 Abstract The widely held view that neurogenic placodes are vertebrate novelties has been challenged by morphological and molecular data from tunicates suggesting that placodes predate the vertebrate divergence. Here, we examine requirements for the development of the tunicate atrial siphon primordium, thought to share homology with the vertebrate otic placode. In vertebrates, FGF signaling is required for otic placode induction and for later events following placode invagination, including elaboration and patterning of the inner ear. We show that results from perturbation of the FGF pathway in the ascidian Ciona support a similar role for this pathway: inhibition with MEK or Fgfr inhibitor at tailbud stages in Ciona results in a larva which fails to form atrial placodes; inhibition during metamorphosis disrupts development of the atrial siphon and gill slits, structures which form where invaginated atrial siphon ectoderm apposes pharyngeal endoderm. We show that laser ablation of atrial primordium ectoderm also results in a failure to form gill slits in the underlying endoderm. Our data suggest interactions required for formation of the atrial siphon and highlight the role of atrial ectoderm during gill slit morphogenesis. © 2007 Elsevier Inc. All rights reserved. Keywords: Ascidian; Chordate; Placode; Atrial siphon primordium; Gill slits; FGF signaling Introduction 2004; Mazet et al., 2005), implying that these structures predate vertebrates. Proposed phylogenies in which cephalochordata Vertebrate neurogenic placodes give rise to specialized and vertebrata are sister taxa – e.g., those based on morphology features of the sensory system including the organs of the inner and 18S rDNA, 28S rDNA and mitochondrial gene sequence ear, the lateral line system and cranial sensory ganglia. (Berril, 1955; Cameron et al., 2000; Glenner et al., 2004; Neurogenic placodes, along with neural crest, have been Turbeville et al., 1994; Winchell et al., 2002) – are consistent regarded as important innovations unique to the vertebrate with the appearance of placodes in the chordate ancestor, clade and, in fact, as defining characters which allowed for a followed by an independent loss in the cephalochordate lineage. wide range of adaptive behaviors including an increasingly Recent phylogenetic models based on concatenated protein predatory lifestyle (Northcutt and Gans, 1983). Phylogenies sequences conflict with this view and suggest that tunicates, not which had placed the cephalochordates as the sister group to cephalochordates, are the true sister group to vertebrates vertebrates, which together with the tunicates comprise the (Bourlat et al., 2006; Delsuc et al., 2006; Philippe et al., chordate phylum, were consistent with this claim as placodes 2005; Vienne and Pontarotti, 2006). Under this scenario, have not been observed in the filter feeding amphioxus. An neurogenic placodes were present before the vertebrate– increasing body of morphological and molecular evidence tunicate divergence, while their apparent absence in cephalo- shows, however, that neurogenic placodes are present in the chordates may represent the ancestral state. tunicata (Bassham and Postlethwait, 2005; Manni et al., 2001, Tunicate structures which have been homologized to vertebrate placodes show the same basic morphology, thicken- ings within the embryonic ectoderm, and include the paired ⁎ Corresponding author. atrial siphon primordia, the oral or stomodeal siphon primor- E-mail address: [email protected] (W.C. Smith). dium, the neurohypophyseal organ and the adhesive organ 0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2007.09.020 246 M.J. Kourakis, W.C. Smith / Developmental Biology 312 (2007) 245–257 precursor or rostral placode (Manni et al., 2004). In vertebrates, (Bassham and Postlethwait, 2005; Jefferies, 1986; Katz, 1983; different inductive cues for different placodes lead to the Manni et al., 2004; Mazet and Shimeld, 2005; Schlosser, 2005; upregulation of a similar suite of genes (Baker and Bronner- Wada et al., 1998). Both otic and atrial siphon precursors appear Fraser, 2001; McCabe et al., 2004). Vertebrate placodes are during development as paired rings of columnar cells, at about defined by combinatorial gene expression patterns including the level of the hindbrain in vertebrates and its equivalent in genes from the six, pax, eya, pitx, fox and dll families. Similar ascidians. A similar morphogenesis also characterizes these gene expression patterns have been observed in putative structures. The otic placode invaginates and pinches off placode homologs of ascidians and in the larvacean Oiko- forming the otic vesicle. The process which initiates the pleura (Bassham and Postlethwait, 2005; Mazet et al., 2005; formation of the atrial siphon also begins with invagination of Mazet and Shimeld, 2005). the atrial primordium, and, as described in the colonial species A one-to-one correspondence between tunicate and verte- Botryllus, the invagination pinches off as a vesicle separate brate placodes may not always be easy to ascertain— from the overlying ectoderm (Manni et al., 2002). The ascidian vertebrates have more placodes than tunicates, so homology atrial primordium is also neurogenic, a feature of most relationships are not likely to be direct in all cases. Vertebrate vertebrate cranial placodes: Bone and Ryan (1978) described placode number may be the result of morphological duplica- the cupular organ of the atrial siphon of Ciona, a structure tions of any one of the ancestral placodes, in which case a similar to the vertebrate mechanosensory hair cell, except that single ascidian placode may share homology to more than one the ascidian cupular organ is not secondarily innervated as are vertebrate placode, as has been proposed for the neurohypo- vertebrate hair cells; Mackie and Singla (2003) describe the physial duct which may be homologous to vertebrate olfactory, hydrodynamic capsular organ of Chelyosoma as consistent with adenohypophysial and hypothalamic placodes (Manni et al., an atrial-otic homology, but like the cupular organ, the capsular 2001). Alternatively, ascidians may have fewer placodes than organ is not secondarily innervated. In the larvacean tunicate the common ancestor, a loss perhaps explained by a com- Oikopleura, however, the proposed otic placode homolog, the paratively simplified larval body plan and a sessile adult phase. Langerhans organ, receives mechanosensory stimulation Finally, certain authors posit the existence of a pan-placodal through ciliated cells that are secondarily innervated (Bassham field from which all neurogenic placodes develop—some and Postlethwait, 2005). vertebrate placodes may have evolved through specification of In vertebrates, functional studies have shown that the novel territories within this field (Baker and Bronner-Fraser, fibroblast growth factor (FGF) pathway plays a role in the 2001; Schlosser, 2002). specification and formation of the otic placode and, as well, that Combined morphological, positional and expression data this pathway is important after placode formation during and can provide at least a first approximation of homology following the invagination phase, including the morphogenetic between vertebrate placodes and tunicate structures (Bassham movements which give rise to the inner ear. Before otic placode and Postlethwait, 2005; Manni et al., 2001; Mazet and formation, members of the Fgf family are expressed in the Shimeld, 2005; Schlosser, 2005). Several authors have periotic mesenchyme beneath the future site of the otic proposed homology between the atrial siphon primordia of placodes. Studies in fish, mouse and chick have shown that, ascidians, which form the adult atrial siphon, or exhalant in particular, members of the Fgf3/7/10 and Fgf8/17/18 families siphon, out of which water, waste and gametes are directed, play a prominent role during placode specification and and the otic placodes of vertebrates, which contribute to induction (Ladher et al., 2005; Leger and Brand, 2002; Liu structures of the ear (Bassham and Postlethwait, 2005; et al., 2003; Maroon et al., 2002; Phillips et al., 2001; Solomon Jefferies, 1986; Katz, 1983; Manni et al., 2004; Mazet and et al., 2004; Wright and Mansour, 2003). Later in vertebrate Shimeld, 2005; Schlosser, 2005; Wada et al., 1998). In Ciona embryogenesis, the placode invaginates and forms the otic cyst, savignyi and Ciona intestinalis, the atrial siphon primordia are and FGF from the hindbrain helps to direct patterning and evident in larvae as paired ring-shaped structures of morphogenesis of ear formation (Liu et al., 2003; Phillips et al., approximately six cells in the lateral ectoderm of the trunk, 2001; Wright and Mansour, 2003). just anterior to the notochord and posterior to the sensory In this report, we test the hypothesis that the otic placode vesicle (Katz, 1983). During metamorphosis each