Animals in each subgroup of the phylum Chordata exhibit a similar process by which they form a tubular central nervous system (CNS). However, little is known about spatial relationship among the CNSs of chordates; vertebrates, cephalochordates and urochordates (tunicates). Ascidians constitute a major animal group in the subphylum Urochordata. In the present study, we examined the expression patterns of labial and orthodenticle related genes of the ascidian, Halocynthia roretzi, in the developing larval CNS. These homeobox genes exhibited region-specific expression patterns that are strikingly similar to those of murine Hoxb-1 and Otx2. The regionalization as characterized by the expression of these genes supports the division of the ascidian larval CNS suggested by the previous morphological studies. Furthermore, conservation of the expression pattern of the homeobox genes suggests that such regionalization occurred in the CNS of a putative common ancestor of chordates.
Ascidian embryos form a tubular CNS in a similar way to that of vertebrates. The ascidian larval CNS can be divided into two parts, a prosencephalon over a gut primordium and a deuterencephalon underlain by a notochord (Katz, 1983). Nicol and Meinertzhagen (1991) distinguished four regions in the larval CNS of Ciona based on the composition of cell types, which are the sensory vesicle and neck in the prosencephalon and the visceral ganglion and spinal cord in the deuterencephalon. The sensory vesicle is further subdivided into three regions. These descriptions show that the ascidian CNS has its own characteristic morphology and therefore the spatial relationship in the CNS between vertebrates and ascidians is difficult to be understood by morphology.
A number of homeobox genes, including HOM-C genes of Drosophila as excellent examples, have been found to play key roles in regional specification in developmental fields of metazoan embryos. In the developing murine CNS, Hox genes define segmental identities of the rhombomeres that are developmental subdivisions of the hindbrain (McGinnis and Krumlauf, 1992). In the more anterior regions, fore- and mid-brain, homeobox genes of other class such as Emx and Otx show spatially restricted expression along the anteroposterior (A-P) axis (Holland et al., 1992; Simeone et al., 1992). In the present study, we compare the expression pattern of Hroth, an ascidian homeobox gene related to Otx, with that of HrHox-1 in the developing larval CNS. In the light of the present findings, we discuss regionalization of the ascidian larval CNS and spatial relationship between the CNSs of ascidians and vertebrates.
MATERIALS AND METHODS
Embryos of the ascidian, Halocynthia roretzi, were raised as described previously (Katsuyama et al., 1995). Whole mount in situ hybridization was carried out according to the procedure described by Wada et al. (1995). Probes used in this study were the HrHox-1 cDNA clone, HH-1f (Katsuyama et al., 1995) and a 2.1 kb cDNA clone of Hroth (Wada et al., in preparation). Detailed procedures for isolation of Hroth gene will be described elsewhere (Wada et al., in preparation). Briefly, a gene fragment of Hroth was obtained by PCR using Halocynthia gastrula cDNA and primers for the bicoid class homeobox and used as a probe to isolate the clone from a 64-cell stage cDNA library.
RESULTS AND DISCUSSION
Previously we found that HrHox-1, the ascidian Hox gene structurally related to labial of Drosophila and Hox genes of paralogous subgroup 1 of vertebrates, shows the expression pattern similar to that of the murine Hoxb-1 gene in the developing ascidian CNS (Fig. 1) (Katsuyama et al., 1995). Then we isolated and examined the expression pattern of an ascidian homologue of orthodenticle, designated Hroth (Halocynthia roretzi orthodenticle homologue) (Fig. 1). Here our attention will be focused on expression of Hroth in the CNS. At the neurula and tailbud stages, Hroth is expressed in the anterior region of the neural fold, closing to form a neural tube (Fig. 1A). This expression is detected as two short stripes running parallel along the A-P axis (Fig. 1B). At the tailbud stage, these become a single stripe (Fig. 1C). At the larva stage, expression of Hroth is observed in the sensory vesicle, surrounding two types of pigment cells, the otolith and the ocellus (Fig. 1D, 1I, 1I′).
HrHox-1 expression in the CNS becomes detectable from the tailbud stage onward in the region posterior to the expression domain of Hroth (Fig. 1F). Thus, the two expression domains align along the A-P axis in the developing CNS from this stage (Fig. 1B, 1F). The anterior border of HrHox-1 expression remains fixed at the level rostral to the anterior tip of the notochord. Thus, the two expression domains do not overlap, keeping a space between them throughout the embryogenesis. The expression patterns of both genes in the CNS are schematically illustrated and compared with those of murine counterparts in Fig. 2. This comparison shows striking similarity between expression patterns of the two sets of related genes in the CNS of the ascidian and mouse. Expressions of the murine Otx genes are restricted to presumptive fore- and mid-brain region in the CNS (Simeone et al., 1992), while Hroth is expressed in the anterior part of the ascidian CNS. Similarly, expression of Hoxb-1 is restricted to the rhombomere 4 of 9.5 d.p.c. mouse embryo (Frohman et al., 1990; Murphy and Hill, 1991), while HrHox-1 expression is restricted to the region encompassing the level of the atrial primordium to the junction of the trunk and tail in the ascidian larval CNS (Fig. 1H, 1I, 1I′).
Expression of these homeobox genes characterizes four regions in the ascidian larval CNS; from rostral to caudal, the Hroth expressing region, the intervening region, the HrHox-1 expressing region and the spinal cord, the region posterior to these. Hroth and HrHox-1 expressing regions are included in the prosencephalon and the deuterencephalon of the ascidian larval CNS, respectively, as designated by Katz (1983). In vertebrates, a prosencephalon and a deuterencephalon are the regions where forebrain and hindbrain develop, respectively. In this regard, the expression pattern of Hroth and HrHox-1 in the CNS supports the designation by Katz (1983). Considering the histological division by Nicol and Meinertzhagen (1991), the HrHox-1 and Hroth expressing domains correspond to the visceral ganglion and the sensory vesicle except the posterior sensory vesicle that is the posterior most subdivision of the sensory vesicle. The intervening region which includes the posterior sensory vesicle and the neck, might be characterized by the expression of other genes such as an engrailed cognate.
Recent molecular analysis and morphological comparison have shown the phylogenetic relationship among subphyla of chordates (Brusca and Brusca, 1990; Wada and Satoh, 1994), which implies that chordates share a common ancestor and that ascidians diverged first from the lineage to vertebrates. Thus, similarity between the expression patterns of HrHox-1 and Hroth and those of their vertebrate counterparts suggests that such molecular regionalization in the CNS along the A-P axis occurred in a common ancestor of the ascidians and vertebrates, that is a putative earliest chordate.
We thank Drs. Peter W. H. Holland, Walter J. Gehring, Paul J. Scotting and Sadao Yasugi for comments and discussions. Thanks are also due to Yasuhiro Shimojima for his help in preparing Fig. 2.