Summary An essential role of Xenopus Foxi 1 a for ventral specification of the cephalic ectoderm during gastrulation

In vertebrate gastrula embryos, the ectoderm is subdivided into various regional tissues by complex inductive processes. Along the dorsoventral (DV) axis, the ectoderm becomes subdivided into the dorsal (CNS or neural plate), intermediate (e.g. presumptive neural crest, placodes and cement gland) and ventral (epidermal) ectoderm. Although it is generally believed that the early DV specification is controlled by a Bmp activity gradient in Xenopus (Wilson et al., 1997; LaBonne and BronerFraser, 1998; Marchant et al., 1998; Tríbulo et al., 2003), how the exact subdivision boundaries are determined remains largely elusive. Along the anteroposterior (AP) axis, the neural plate is finely regionalized into the forebrain, midbrain, hindbrain and spinal cord. Recent molecular studies implicated Wnts, Nodal, Fgfs and RA in the AP regionalization of the CNS (McGrew et al., 1995; Kengaku and Okamoto, 1995; Piccolo et al., 1999; Gavalas and Krumlauf, 2000; Kiecker and Niehrs, 2001; Kudoh et al., 2002; Onai et al., 2004). In contrast to the CNS, relatively little is known about the AP specification of the non-CNS (intermediate and epidermal) ectoderm. In Xenopus, the non-CNS ectoderm of the head region (referred to as ‘cephalic non-neural ectoderm’ hereafter) has several characteristic features. For example, unlike that of the trunk, the non-neural ectoderm of the cephalic region differentiates into the cranial placodes and special exocrine glands such as the cement gland and hatching gland, in addition to the epidermis and neural crest. The cranial placodes, which develop within the preplacodal field of the head intermediate ectoderm, give rise to a number of sensory tissues (reviewed by Baker and Bronner-Fraser, 2001). To date, the molecular mechanism underlying the determination of the cephalic nonneural ectoderm (versus the CNS and the trunk ectoderm) remains largely to be elucidated. In this study, we have investigated the molecular control of the initial specification of the cephalic non-neural ectoderm by focusing on the roles of a Foxi1 family gene in Xenopus. The winged-helix transcription factor Foxi1 plays an essential role for the formation of placode-derived ectodermal tissues such as the otic vesicle (Hulander et al., 1998; Nissen et al., 2003; Solomon et al., 2003a) in mice and zebrafish. In Xenopus, three Foxi1-related genes have been reported: Xfoxi1a, Xfoxi1b (pseudoalleles generated by the pseudotetraploidy of Xenopus laevis, see alignment of Xfoxi1a and Xfoxi1b in Fig. 1M) and Xfoxi1c (which is not an Xfoxi1a pseudoallele) are expressed in the preplacodal area at the neurula stage (Lef et al., 1994; Pohl et al., 2002). Interestingly, Xfoxi1a and Xfoxi1b are also expressed even earlier than the establishment of the preplacodal expression at the neurula stage; they are expressed widely in the animal side of the embryo at the late blastula stage and in the anteroventral ectoderm at the late gastrula stage. By contrast, Xfoxi1c is expressed only after the gastrula stage and not during the blastula and gastrula stages (Pohl et al., 2002). The expression of a foxi1 gene in a broad domain of the gastrula ectoderm has been reported also in zebrafish (Nissen et al., 2003; Riley and During gastrulation in Xenopus, the head ectoderm is subdivided into the central nervous system (CNS) anlage (neural plate) and the non-CNS ectoderm (i.e. epidermis, placodes and neural crest). The winged-helix transcription factor Xfoxi1a is one of the earliest markers for the preplacodal region at the mid-neurula stage. Interestingly, before the establishment of the preplacodal region, Xfoxi1a expression is detected in the entire cephalic non-neural ectoderm at the midand late gastrula stages. The present study focuses on the role of Xfoxi1a particularly at the gastrula stages. The early Xfoxi1a expression in the anteroventral ectoderm is dependent on Bmp signals and suppressed by Wnt signals. Inhibition of Xfoxi1a activities by injection of antisense oligonucleotides leads to suppression of non-CNS ectodermal markers (e.g. keratin) and expansion of the anterior expression domain of the CNS marker Sox2. Conversely, misexpression of Xfoxi1a suppresses Sox2 and induces keratin in the anterior neural plate. In the animal cap, Xfoxi1a overexpression antagonizes the neuralizing activity of Chordin (Chd). Studies using an inducible Xfoxi1a construct (GR-Xfoxi1a) show that the ventralizing function of Xfoxi1a is confined to the gastrula stage. Thus, Xfoxi1a is an essential regulator of ventral specification of the early head ectoderm during gastrulation.