Bs-pitx and the Chordate Pitx Family
In the present work, we cloned and characterized an ortholog of Pitx homeobox genes in the compound ascidian, B. schlosseri. A single full-length cDNA sequence was reconstructed from overlapping 5′ and 3′ rapid amplification of cDNA ends (RACE) fragments. It encodes the complete ORF of a putative Pitx-type homeoprotein. However, given the limits of the technique used, we cannot rule out the possibility that alternative Pitx isoforms might be present in B. schlosseri as is the case in vertebrates (Gage et al., 1999) and Ciona species (Christiaen et al., manuscript in preparation).
Phylogenetic analysis was carried out using other paired-related homeodomains. The tree clearly displays four defined families: Goosecoid, Otx/Otd, two main groups of the Aristaless-related subfamily, and Pitx (Fig. 2; Christiaen et al., 2002). Bs-Pitx was positioned at the base of the Pitx cluster, in both the homeodomain tree (Fig. 2) and the tree constructed with additional blocks of sequence conservation (data not shown). These observations reinforce the hypothesis of orthology, but at the same time, Bs-pitx divergence was pointed out.
Nevertheless, the B. schlosseri and Halocynthia roretzi Pitx homeodomain sequences cluster with a high bootstrap value. This observation fits the taxonomical position of the two species, which belong to the Pleurogona order, while C. intestinalis is a member of the Enterogona order (Burighel and Cloney, 1997; Van Name, 1945; Kott, 1969). According to morphological (Kott, 1969) and molecular data (Wada and Satoh, 1994; Wada, 1998; Swalla et al., 2000), these two orders diverged early in tunicate evolution.
Finally, the topology of the Pitx dendrogram exhibits some discrepancies with the current deuterostome phylogeny (Cameron et al., 2000). Indeed, we found Ci-pitx clustering with other chordates, although with a rather low bootstrap value; whereas other ascidian sequences are set apart together. This finding could be explained by the phylogenetic position of C. intestinalis among ascidians; actually, none of the authors accord on the position of this species, which is considered to belong to Phlebobranchia or Aplousobranchia (Stach and Turbeville, 2002). In addition to a likely long branch artifact, this thought would explain the former discrepancy.
Pitx Expression in Chordate Evolution
Pitx genes are stomodeal markers in vertebrates (Drouin et al., 1998), and they have already been used in ascidians to examine the conservation of the anterior neural boundary that gives rise to stomodeum (Christiaen et al., 2002; Boorman and Shimeld, 2002b). Briefly, Ci-pitx was found to be expressed in the stomodeum primordium of C. intestinalis from the mid tail bud stage onward. This finding was hypothesized to constitute a conserved expression domain that would reflect ancestry of the stomodeum in vertebrates and ascidians (Christiaen et al., 2002; Boorman and Shimeld, 2002b). Our results on B. schlosseri embryogenesis support this view and give new contributions to define the modality of differentiation of an ectodermal unit, located anteriorly to neuroectoderm, that could correspond to the stomodeal ectomere of the vertebrates (Lanctot et al., 1997).
We observed, in both embryogenesis and blastogenesis, comparable Bs-pitx expression domains during morphogenesis of the stomodeal/adenohypophyseal territory and its derivatives. Early on, Bs-pitx expression splits into three separate stomodeal domains: two of them are in the stomodeal area and coincide with the ciliated duct primordium and the oral siphon primordium. The third domain is represented by several neurons of the cerebral ganglion.
The dual Bs-pitx stomodeal expression domain suggests that the ascidian stomodeum is of dual nature. This apparently contrasts with the situation reported for C. intestinalis where the different stomodeal domains were not recognized (Christiaen et al., 2002). However, it should be considered that heterochrony exists between C. intestinalis and B. schlosseri during the development of larva and juvenile structures (Manni et al., 1999; Willey, 1893).
Although the adenohypophyseal nature of a subpopulation of stomodeal cells cannot be strictly inferred from Pitx expression alone, the persistence in successive developmental stages of Pitx expression at the level of the ciliated duct rudiment suggests that this gene participates to the specification of the adenohypophysis primordial territory in tunicates as well as in vertebrates (Lanctot et al., 1997) and cephalochordates (Manni et al., 1999; Boorman and Shimeld, 2002b). Another aspect of Pitx expression is maintained in the rudiment of the tentacles after oral perforation, where the secondary sensory cells (hair cells) are differentiating (Burighel et al., 2003; Manni et al., 2004).
Finally, our results reveal that Bs-pitx mRNAs are transcribed in the pioneer nerve cells. These are known to delaminate and migrate, from the wall of the neurohypophyseal duct in the embryo and its analogous structure, the dorsal tube in the bud, to form the cerebral ganglion (Manni et al., 2001). The third domain of Bs-pitx expression is maintained in the anterior and ventral portions of the cerebral ganglion during the entire morphogenesis and in the filtering oozooids and blastozooids. The latter detailed observations show Pitx expression during a morphogenetic process that involves both neural and non-neural (stomodeal) ectodermal cells. This finding is reminiscent of the Rathke's pouch morphogenesis during adenohypophyseal development (Kioussi et al., 1999). Therefore, we propose that these data provide additional support to the hypothesis of homology between the ascidian neural gland complex and the vertebrate pituitary.
Another striking Bs-pitx expression domain, when considered under the light of evolution, is the left-restricted asymmetric expression in both the perivisceral leaflet and dorsal lamina. In B. schlosseri, the perivisceral leaflet constitutes one of the first recognizable asymmetric structures. Bs-pitx is asymmetrically expressed in the rudiment of this structure during both embryogenesis and blastogenesis, and it is maintained in the adult zooid. It is of note that early asymmetric expression actually precedes the structural asymmetry of the gut (Fig. 7C), thus making Bs-pitx a likely molecular determinant implicated in the establishment of structural asymmetry in B. schlosseri.
Bs-pitx asymmetric expression is recognizable also in late blastogenetic and sexual development, on the left-hand side of the dorsal lamina. In the filtering zooids, this structure is bent to the right-hand side along the branchial roof, forming a ciliated groove that conveys the food cord to the esophagus. The dorsal lamina is a likely tunicate innovation that seems to have no counterpart in the vertebrate anatomy. Nonetheless, the left/right asymmetric expression of Pitx genes is a conserved character in chordates, as part of the ancestral Nodal/Pitx genetic pathway (Ryan et al., 1998; Ryan and Izpisua Belmonte, 2000; Boorman and Shimeld, 2002a, b). Thus, Bs-pitx asymmetric expression in the dorsal lamina might have suggested cooption of this ancestral left/right asymmetry-determining cassette.
In all the three subphyla of the extant chordates (vertebrates, cephalochordates, and tunicates) Pitx expression is left-sided in multiple germ layers. However, it must be noted that the tissues expressing Pitx asymmetrically in ascidians and cephalochordates are not all homologous. This observation raises the attractive hypothesis that the control of visceral organization was an ancestral role of Pitx as suggested by Boorman and Shimeld (2002b) and that the chordates coopted the ancestral left/right asymmetry determining nodal/pitx cassette wherever and whenever requested by evolutionary constraints.
Similar Bs-pitx Expression Patterns During Embryogenic and Blastogenic Organogenesis
Comparing gene expression patterns between embryogenesis and blastogenesis is an awkward task, because profound differences in the initial stages and deep heterochrony obscure likenesses between developmental processes. Nevertheless, as soon as the body pattern is established and the rudiments of the main organs are formed, several similarities are recognizable between the two developmental processes in Botryllus schlosseri. The stomodeum, the developing neural complex, the dorsal lamina, and the perivisceral leaflet can easily be identified on a morphological basis. This information provided us with a valuable morphological framework to carefully compare Bs-pitx expression in blastozooids and oozooids. As mentioned in the previous sections, Bs-pitx is expressed in the stomodeum derivatives and asymmetrically in the perivisceral epithelium and dorsal lamina in both blastozooids and oozooids. Although this observation might seem predictable, given that we consider the same gene, in the same species and at comparable stages, this is not a straightforward conclusion as the two zooids arise from drastically different developmental situations. Indeed, in ascidian embryos, development is initiated in a so-called mosaic manner, using molecular determinants maternally delivered to the single egg cell during oogenesis. On the other hand, blastogenesis is regulative by nature, as the buds arise from a homogenous population of trans-differentiated epithelial cells, and patterning thus involves progressive cell fate determination by extensive cell–cell communication (Kawamura and Fujiwara, 1995). In the light of these considerations, it seems that the similar expression patterns of Bs-pitx we observed at comparable stages of development in embryogenesis and blastogenesis might be controlled by the same upstream mechanisms. This is not a straightforward conclusion; given the previously described expression in young buds and in zooid organogenesis, one must take into account that gene expression pattern maintenance and variation might both rely on the evolution of gene regulation.
Evidence for Bs-pitx Recruitment in Early Blastogenesis
Despite the similarities described in the previous section, Bs-pitx expression itself exemplifies differences between early stages of the two developmental sequences. During the earliest embryonic stages observed (gastrula and neurula), no transcripts were detected and expression starts in a restricted dorsoanterior area at the tail bud stage; whereas in the early phases of blastogenesis, Bs-pitx expression is localized on the whole domain of the bud arising from peribranchial leaflet. It is noteworthy that Bs-pitx expression is maintained in the whole inner vesicle of the early bud (stage 3), a stage that could be compared with the gastrula (Brien, 1968) or the blastula stage (Rinkevich et al., 1995) for its ontogenetic significance. Thus, the early widespread Bs-pitx expression in budding constitutes a relevant difference with the situation observed in embryogenesis.
So far, the functional relevance of this early expression is unclear. Considering the cell types from which the bud is derived, the multipotent cells play a fundamental role, inasmuch as every kind of cell constituting the parent body is not incorporated into the bud. In vertebrates, pitx2 was shown to be expressed in hematopoietic progenitors (Degar et al., 2001), to control cell proliferation during adenohypophyseal and cardiac organogenesis (Kioussi et al., 2002; Clevers, 2002; Baek et al., 2003), and to regulate cell-shape changes in HeLa cells (Wei and Adelstein, 2002). All the above-mentioned cellular processes are likely to be important for early bud morphogenesis.
Therefore, regardless of the actual mechanism that triggers Bs-pitx expression in early budding stages, a likely evolutionary explanation for this peculiar observation is that Bs-pitx was recruited to this particular developmental mechanism for its specific effects on cell activity.
Our study, for the first time analyzed side by side, in the same species, the expression of a regulatory gene in different modes of development. This strategy gives us a fascinating example of a similar developmental situation reached using the same genetic toolkit, but probably following different routes. Hopefully this data, as well as studies on regeneration and fission, might provide a conceptual framework for evolutionary developmental biology to explain how homologous features can be obtained following different developmental mechanisms. Future interesting studies in Botyllus might address this issue by focusing on genes known to be involved in early body plan organization and patterning.