Expression of Hr-Pitx in papilla-forming region is regulated by Hr-Otx
In the present study, we examined the expression pattern and the transcription regulatory mechanism of the Pitx gene in the ascidian H. roretzi. We established that Hr-Pitx is expressed in the papilla-forming region, in addition to the left epidermis, ocellus photoreceptors and the stomodeum in the development of H. roretzi. We identified an enhancer region that drives expression in the papilla-forming region and found out that both putative Otx and Fox transcriptional factor binding sites were required for the activity of the enhancer. Next, we showed that Hr-Otx was expressed in the cells expressing Hr-Pitx first in the papilla-forming region at the neurula stage and knocking down of Hr-Otx caused downregulation of Hr-Pitx expression in the papilla-forming region. We further showed that the activity of the Hr-Pitx papilla enhancer was restored by injection of Hr-Otx mRNA together with Hr-Otx MO. These results suggest that expression of Hr-Pitx in the papilla-forming region is downstream of Hr-Otx function.
At the mid neurula stage, Hr-Pitx was expressed only in the eight cells of the papilla-forming region. By contrast, Hr-Otx is expressed in the prospective sensory vesicle region and in the anterior trunk epidermis, in addition to the papilla-forming region by the neurula stage (Wada et al. 1999). Thus, it is suggested that there is an additional factor that activates region specific expression of Hr-Pitx at the neurula stage. The most probable candidate would be Fox transcription factor, because putative Fox binding sites are required for activating the Hr-Pitx papilla enhancer. In H. roretzi, three groups of Fox genes, FoxA/HNF-3, FoxB and FoxD, have been identified. Hr-FoxA/HNF3-1 is expressed in the endoderm and notochord lineage cells from the 16-cell stage through the mid tailbud stage (Shimauchi et al. 1997) and it is required for notochord formation (Kumano et al. 2006). Hr-FoxA is also expressed in the lineage of the ventral layer of the central nervous system from the late gastrula stage to the mid tailbud stage (Shimauchi et al. 1997). Hr-FoxB has been shown to be expressed in the nerve cord precursors (A7.4 and A7.8 pairs) and muscle lineage cells (B7.4 pair) at the 64-cell stage and their descendant cells at the 110-cell stage, and has been shown to repress the expression of the Brachyury gene (Hashimoto et al. 2011). Two types of FoxD, designated as Hr-FoxDa and Hr-FoxDb, have been found in H. roretzi (Kumano et al. 2006). At the 16-cell stage, Hr-FoxDa is expressed in A5.1, A5.2 and B5.1 pairs, which give rise to the notochord, endoderm and other tissues. Although its expression is restricted to the endoderm precursors at the 32- and 64-cell stage, function of Hr-FoxDa is necessary for the notochord formation. Expression of Hr-FoxDb was detectable from the neurula stage in several domains but not in the papilla-forming region (Kumano et al. 2006). Thus, at present, it remains unclear which Fox factor is involved in activating expression of Hr-Pitx in the papilla-forming region.
Conserved gene expression in the adhesive organ development in chordate
The cement gland is a transient adhesive organ of the Xenopus larva and it is derived from the ventral-most portion of the extreme anterior domain that is mesoderm-free, with ectoderm and endoderm directly juxtaposed, and defined by expression of Pitx genes (Dickinson & Sive 2007). During the development of Xenopus, Otx2 is expressed in the cement gland primordium. It has been shown that overexpression of Otx2 induces ectopic cement gland formation in the ventrolateral epidermis (Blitz & Cho 1995; Pannese et al. 1995; Gammill & Sive 1997). Conversely, the cement gland formation is prevented by blocking Otx2 function using dominant negative construct (Gammill & Sive 2001). In the cement gland, Pitx1 and Pitx2c are expressed during embryogenesis (Campione et al. 1999; Hollemann & Pieler 1999; Schweickert et al. 2001b), and it has been reported that ectopic expression of Pitx1 induces ectopic cement gland formation (Chang et al. 2001). Furthermore, knocking down of Pitx1 and Pitx2c inhibits ectopic cement gland formation induced by Otx2 (Schweickert et al. 2001a). Additionally, it has been suggested that Pitx genes act downstream of Otx2 during the development of the cement gland (Dickinson & Sive 2007). Bone morphogenetic protein (BMP) signaling is also involved in the inducing and positioning the cement gland. BMP4 is expressed in the cement gland primordium together with Otx2 (Gammill & Sive 2000). Although high concentrations of BMP4 induce epidermis in ectodermal explants, lower concentrations promote specification of the cement gland (Wilson et al. 2002). It has been suggested that BMP signaling and Otx2 work together to activate cement gland formation and that adequate levels of BMP signaling define the permissive region for cement gland formation (Gammill & Sive 2000).
Cement gland-like structures have been described in teleost fishes (Pottin et al. 2010). Larvae of the Mexican tetra, A. mexicanus, have a transient adhesive organ called the casquette on the anterior head. The casquette has similar properties to the cement gland of Xenopus: it is derived from the ectoderm; is innervated by the trigeminal ganglion; and has an inhibitory function on larval swimming behavior. Moreover, BMP4, Pitx1 and Pitx2 are expressed in the casquette during embryogenesis, however, the expression pattern of Otx in the development of A. mexicanus has not been examined (Pottin et al. 2010). In the bichir, Polypterus senegalus, which belongs to the basal actinopterygians, single Otx1, Otx2 and Otx5 genes have been identified, and among them, PsOtx1 and PsOtx5 are expressed in the developing attachment gland (Suda et al. 2009).
The ascidian papilla shares some structural properties with the adhesive organ in vertebrate embryos in the development. Both the papillae and the cement gland of Xenopus consist of morphologically similar tall cuboidal epithelial cells that originate from the region anterior to the field of neural fate at the gastrula stage (Keller 1975; Nishida 1987). The papillae develop in the anterior-most part of the trunk where the ectoderm and endoderm lie adjacent to each other (this is apparent in C. intestinalis, according to sagittal sections of tailbud embryos in the Four-dimensional Ascidian Body Atlas (FABA) database, http://chordate.bpni.bio.keio.ac.jp/faba/1.4/top.html; Hotta et al. 2007) like in the extreme anterior domain of Xenopus larva (Dickinson & Sive 2007). It has been reported that papillae contain glutamatergic neurons in C. intestinalis larvae (Horie et al. 2008). Although Xenopus cement gland has no neurons, it is innervated by trigeminal neurons that release an excitatory amino acid (Boothby & Roberts 1992). In the development of H. roretzi, Hr-Otx is expressed in the cells of the papillae as well as in the sensory vesicle lineages (a6.5 pair) from the 32-cell stage. Although expression of Hr-Otx in the sensory vesicle continues to the swimming larva stage, the expression of Hr-Otx in the papillae becomes undetectable by the tailbud stage (Wada et al. 1999). Knockdown of Hr-Otx using antisense morpholino oligonucleotide causes the loss of the papillae and the anterior part of central nervous system (Wada et al. 1999). In H. roretzi, two BMP genes, Hr-BMPa and Hr-BMPb, which have been identified as homologues of BMPs5–8 and BMP2/4, respectively, are expressed in the papilla-forming region of the tailbud embryo (Miya et al. 1996, 1997). Overexpression of Hr-BMPb inhibits papillae formation in the larva and induces ectopic expression of Hr-EpiC, a marker for the epidermis, in the anterior-most region of the neurula, which would give rise to papillae (Miya et al. 1997). On the other hand, overexpression of Hr-Chordin causes abnormal formation of the adhesive organ that consists only of one large papilla (Darras & Nishida 2001b). These observations suggest that BMP signaling of an adequate level is required for correct formation of the adhesive organ in the ascidian development, which further supports the notion that the papilla is the ascidian homologue of the adhesive organ of vertebrate larvae.
In this study, we have shown that Hr-Pitx is expressed in the papilla-forming region and that this expression is under control of Hr-Otx gene function. Taking the previous observations made in Xenopus (Schweickert et al. 2001a; Dickinson & Sive 2007) and our results into account, it is suggested that the involvement of Pitx and Otx in the adhesive organ development, with the former being located downstream of the latter, is conserved between H. roretzi and X. laevis. Previously, Pottin and colleagues proposed that the adhesive organ developmental module, in which functions of Bmp, Otx and Pitx genes are involved, is an ancestral characteristic of the larval development of chordates (Pottin et al. 2010; Retaux & Pottin 2011). The results of the present study are consistent with their proposal and provide further insights into the adhesive organ GRN, in which the involvement of Otx as the upstream gene in the expression of Pitx may constitute a core part, in the larval chordate development. However, in another ascidian species, C. intestinalis, Pitx gene is not expressed in the papilla-forming lineages during its development (Boorman & Shimeld 2002; Christiaen et al. 2005), suggesting that the involvement of Pitx and Otx in the papilla formation is not conserved in this species. This is not necessarily surprising because there are several examples showing that conserved cell lineage and morphologies are controlled by different regulatory networks between H. roretzi and C. intestinalis (Lemaire 2009). For instance, the mechanism of secondary notochord induction appears to differ between these two species. In H. roretzi, BMP2/4 has been shown to act as an inducer of the secondary notochord cells (Darras & Nishida 2001a). In C. intestinalis, however, inhibition of BMP signaling has no effect on the secondary notochord induction (Hudson & Yasuo 2006; Lemaire 2009). In future studies, the comparison of underlying mechanisms for papilla development between H. roretzi and C. intestinalis will provide insights into the evolutional stability and plasticity of the GRN for adhesive organ formation.