Among the range of animal model systems used in developmental biology, most do not possess an extensive capability to differentiate new tissues from multipotent somatic cells as occurs during regeneration and asexual reproduction (see Allen and Nollen, 1991; Galliot and Schmid, 2002; Alvarado, 2003; Holstein et al., 2003).
In particular, the developmental mechanisms of asexual reproduction remain largely unexplored, especially in deuterostomes. Therefore, there exists a gap in our understanding of the cellular and molecular mechanisms involved in nonembryonic development. Whereas it has been supposed that organisms that can asexually reproduce have secondarily evolved from animals with high regenerative capabilities, many animals that can regenerate tissues do not possess the ability to propagate asexually, suggesting that fundamental differences exist between these two processes and highlighting the derived nature of vegetative propagation (Vorontsova and Liosner, 1960). Although clonal reproduction apparently counteracts genetic variation and harmful mutations removal (Wuethrich, 1998), it has been successful in evolution and evolved independently in and within many phyla (Blackstone and Jaske, 2003).
Tunicates are the closest relatives to vertebrates known to use asexual reproduction. In ascidians, solitary species propagate exclusively sexually and display regenerative capabilities to some extent, whereas compound ascidians evolved extensive and varied modes of bud formation, allowing them to reproduce both sexually and asexually (Berrill, 1975).
In the first case, the oozooids arise after an egg–embryo–larva–metamorphosis–juvenile developmental sequence known as embryogenesis. In the latter case, the blastozooids of a colony form from the initial juvenile, or their sibling zooids, in a process generally called blastogenesis (Satoh, 1994).
In the colonial species Botryllus schlosseri (Fig. 1), the two developmental sequences deeply differ at early steps: the embryo develops from an oligolecithic egg into a swimming larva, that metamorphoses into a filtering juvenile, while each bud is initially formed by a bilayered somatic protrusion of the parent body wall (Brien, 1968). Nevertheless, the oozooid and the blastozooid are very similar, differing mainly regarding the shape of the branchial stigmata. Moreover, recent work shed light on organogenetic events that are similar in the two pathways: the epithelial fusion and perforation during gill slit formation (Manni et al., 2002) and the mechanisms of neurogenesis (Manni et al., 1999). Considering that these two developmental processes converge to form similar functional zooids, we investigated developmental gene expression during crucial phases of the two pathways.
To this aim, Pitx (pituitary homeoboxgene), a paired-related homeobox gene, was chosen as an informative genetic marker to examine similarities and differences in gene expression between different developmental sequences. Among other ontogenetic roles, Pitx genes in vertebrates participate to the establishment of left/right asymmetries in viscera (Ryan and Izpisua Belmonte, 2000; Hamada et al., 2002; Boorman and Shimeld, 2002a). They are also necessary for the differentiation of adenohypophyseal endocrine cells, mesencephalic GABAergic and dopaminergic neurons, and for posterior limb development (Westmoreland et al., 2001). Pitx genes were also reported in the protostome species Caenorhabditis elegans and Drosophila melanogaster (McIntire et al., 1993; Vorbruggen et al., 1997), in the basal chordate amphioxus (Boorman and Shimeld, 2002b; Yasui et al., 2000), and the ascidians Ciona intestinalis and Ciona savignyi (Christiaen et al., 2002), Halocynthia roretzi (Morokuma et al., 2002), and a short fragment from B. schlosseri (Christiaen et al., 2002).
Here, we report isolation of a full-length Pitx cDNA of the compound ascidian B. schlosseri and assess its expression during embryogenesis and blastogenesis. Our results show comparable expression patterns during organogenesis in both developmental pathways, suggesting that a common genetic circuit controls similar organogenetic events. On the other hand, the widespread Bs-pitx expression observed in early blastogenesis suggests a recruitment of this gene in this original developmental strategy.
Characterization of a Full-Length Bs-pitx cDNA
The obtained contig sequence contained a single open reading frame (ORF), starting with an ATG embedded in a bona fide Kozak consensus, and encoding a putative protein of 233 amino acids. In contrast with the situation observed in C. intestinalis (Christiaen et al., 2002), we found no obvious leader sequence at the 5′ cDNA end (Vandenberghe et al., 2001). The cDNA sequence obtained was deposited into GenBank under accession no. AY665612.
BLAST search identified the putative protein as a likely Pitx ortholog. This protein, henceforth, will be referred to as Bs-Pitx (for B. schlosseri Pitx).
The alignment of Bs-Pitx with other Paired-class homeoproteins allowed us to identify the four main Pitx molecular features. The homeodomain is 80% (48 of 60) identical to mouse Pitx2 homeodomain and contains a lysine residue at position 50, typical of bicoid-type homeodomains (Fig. 2). Nevertheless, it is of note that the level of conservation is higher (56 of 60, 93.3%) when comparing the Ci-Pitx and Mm-Pitx2 homeodomains. The C-terminal region of the protein exhibits an Aristaless (ALL) domain (Meijlink et al., 1999) 76.9% to 92.3% identical to orthologous domain of several other proteins from a variety of organisms (except for C. elegans UNC30, in which the ALL domain appears more divergent). The alignment between homeo- and Aristaless domains was difficult to align due to the abundance of proline (P) and serine (S) residues. Although positional homology cannot be confirmed, the presence of such a P/S-rich region might be of some functional relevance for Pitx proteins. Downstream to the Aristaless domain, Bs-Pitx also displays a CQY motif, encountered so far only in all the Pitx proteins. All these features make Bs-Pitx a likely Pitx ortholog.
A phylogenetic analysis was carried out using the homeodomains of several paired-class proteins. Members of the Aristaless, Goosecoid, and Otx/Otd families were included in the analysis, whereas Ubx and Antp were used as an outgroup. In the neighbor-joining tree, Bs-Pitx clearly clusters with other Pitx homeodomains, this topology being strongly supported by bootstrap analysis (99%, Fig. 3). However, Bs-Pitx is set at the base of Pitx cluster, together with Hr-Pitx and Unc-30 (Pitx proteins from Halocynthia roretzi and C. elegans, respectively), showing a high level of divergence from other Pitx proteins, including Ci-pitx (53.6% identity with Ci-pitx homeodomain).
For that reason, we performed further analysis using additional blocks of amino acids conserved among Pitx proteins, e.g., the homeodomain together with Aristaless domain and CQY conserved motif (data not shown). Neighbor-joining and parsimony trees display the same result as tree in Figure 3, placing Bs-Pitx together with Hr-Pitx and Ce-Unc30 at the base of the Pitx dendrogram. Comparing the amino acid sequences, the major differences in homeodomain were found on the first helix and between the end of second and the beginning of third helix.
Bs-pitx Expression During Embryogenesis
We analyzed the localization of Bs-pitx transcripts from the gastrula stage onward. At this stage, and during neurulation, no staining was observed. At the early tail bud stage (stage II) expression appears in one domain. Comparison with semithin serial sections of specimen at the same stage (not shown) allowed us to localize the expression domain in the dorsal portion of the embryo (arrow in Fig. 4B).
In stage III embryos, when major organogenetic events occur, signals could clearly be assigned to different structures being put in place, in particular to the neurohypophyseal duct (ND; Manni et al., 1999). Thus, the neural complex rudiment (neural gland and cerebral ganglion) is derived from an ectodermal area associated to the neural tube and appears as an anterodorsal outgrowth of the larval nervous system. Posteriorly, the lumen of the duct is continuous with the left ganglionic vesicle, a small counterpart of the large right sensory vesicle (Manni et al., 1999), and extends anteriorly to meet the dorsal wall of the pharynx, which forms the rudiment of the ciliated duct. A careful analysis of histological sections revealed the presence of Bs-pitx transcripts in this latter region and maintenance of Bs-pitx expression until the ND fuses with the wall of the pharynx and looses the original contact with the left ganglionic vesicle. Dorsally, there is another wide point of contact between the dorsal groove, a dorsal invagination of the epidermis, and the stomodeum, which participates to the formation of the oral siphon. Also in this region, transcripts were detected: probes label the pharyngeal epithelium on the future internal lips of the oral siphon (Fig. 4C–I). This labeled area is visible in the stomodeal region, from stage III to the “swimming larva,” as a ring that surrounds the region of contact between dorsal groove and ND (Fig. 4F–I). In the metamorphosing larva, Bs-pitx expression continues in the inner wall of the oral siphon, where the rudiment of the velum and the oral tentacles are forming (Fig. 4L), and strongly labels the anterior portion of the cerebral ganglion (Fig. 4M).
From the stage III onward, we also observed a permanent asymmetric expression in the perivisceral epithelium, which accompanies the growth of the stomach and the intestine on the left side of the branchial basket (Fig. 4N–P). Asymmetric staining was also detected along the left side of the developing dorsal lamina of the metamorphosing larva (Fig. 4M). Settling larvae usually present one, occasionally two, early bud of the double vesicle stage, in which expression is ubiquitous in the inner vesicle epithelium (see below).
Bs-pitx Expression During Blastogenesis
In B. schlosseri, the buds arise as a thickening of the peribranchial epithelium accompanied by the epidermis. At this stage, transcripts are widely distributed in the inner epithelium. During the successive stages 2 and 3, the expression remains ubiquitous, labeling the entire inner vesicle that is forming (Fig. 5A,A′,B,B′). Bs-pitx expression is observed in the bud at stage 3 of both the blastozooids and the metamorphosing larva or juvenile. Between stages 4 and 6, the inner vesicle undergoes intense morphogenesis, and Pitx expression becomes restricted to two different domains: first, the transcripts are detected on the left peribranchial chamber, in the epithelium delimiting the stomach and the intestine (Fig. 5C,C′). The presumptive ciliated duct area, at the roof of the branchial chamber, constitutes a second expression domain (Figs. 5C,C′, 6A–E). This rudiment originates during the above-mentioned stages as a dorsoposterior evagination, the dorsal tube, from the roof of the inner vesicle, and extends anteriorly as a blind tube destined to open into the branchial rudiment. Then, the original posterior aperture closes (Burighel et al., 1998). The signal appears clearly restricted to the region of contact between the blind tube and the dorsal side of the branchial basket rudiment.
From late stage 6 onward, Bs-pitx expression remains in the ciliated duct (Fig. 6I) and is also detected in the oral siphon rudiment, represented by a dorsoanterior outgrowth of the branchial wall toward the epidermis. Here, the staining appears on the branchial evagination as a ring that surrounds the future siphon aperture (Fig. 6H). At stages 8 and 9, this expression persists in the inner wall of the oral siphon, its velum, and the tentacles (Fig. 7A). At the same time, strong Bs-pitx expression is detected in the forming cerebral ganglion and is restricted to its anterior part, on the ventral and lateral sides (Fig. 7B). Expression starts at the initiation of ganglion formation (stage 7, Fig. 6L) and persists during subsequent developmental stages.
Two asymmetric expression domains are detected. The first is restricted to the epithelium of the left peribranchial chamber adhering to stomach and intestine at early blastogenetic stages (from 4 to 6), and persists at all the successive developmental stages (Fig. 7C,D). The staining seen on the perivisceral epithelium extends to the pyloric gland, which encrusts the central portion of the intestine (Fig. 7E). The second is localized along the left side of the dorsal lamina (Fig. 7F), following an asymmetric pattern maintained during the subsequent developmental stages analyzed (Fig. 7G).
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.
Animals and Embryos
B. schlosseri (Styelidae, Stolidobranchia) colonies were collected in the lagoon of Venice and cultured by adhering to glass in the laboratory (at 18°C), according to Sabbadin's method (1955). Each colony contains several zooids organized in star-shaped systems and embedded in a common tunic, with a vascular network running throughout. Three blastogenetic generations generally coexist in the same colony: the filtering adults, their buds, and the buds they themselves produce. The developmental stages of the two generations of buds are closely correlated with those of the parent and are synchronous for all the individuals of the same blastogenetic generation. According to Sabbadin (1955), bud development can be subdivided as follows: the bud rudiment appears as a thickened disc of the mantel (peribranchial leaflet and the overlying epidermis; stage 1) that folds, forming an arc (stage 2). At stage 3, the young bud forms a double vesicle that remains connected to and receives blood from the parent. During stage 4, the wall of the inner vesicle folds and a central branchial chamber delaminates, flanked by two symmetrical peribranchial chambers. At stage 5, the gut rudiment appears at the dorsal posterior end of the branchial chamber. At stage 6, the three chambers are well delimited and the intestine arises from the stomach wall. At stage 7, the stigmata rudiments begin to form and the rudiment of the new bud appears on the mantle of the bud. The heart starts beating at stage 8. This phase is then characterized by a considerable growth rate and cell differentiation. Finally, stage 9 refers to the filtering adult blastozooid (Fig. 1). Thus, the stage of the colony can be indicated by three numbers corresponding to the stage of each generation: 9/7/1, 9/8/2, 9/8/3, and so on, the first number indicates the adult, whereas the latter numbers indicate the stages of the two successive generations of buds.
After several blastogenetic generations, gonads do mature and adult blastozooids reproduce sexually. Eggs are spawned and fertilized in the parental atrial chamber, where embryos develop into swimming tadpole larvae in approximately 5 days. Next, larvae are released from the colony, settle on a substrate, and undergo metamorphosis. Referring to the developmental stage of the colony and to the gross embryonic and larval anatomy, we analyzed the embryos and larvae at successive development stages: embryos at the end of neurulation (stage I), in which the rudiment of the tail begins to form; embryos with tail surrounding one quarter of the trunk, early tail bud (stage II); embryos with tail encircling the trunk 1.5 times (stage III); “swimming larvae;” and “metamorphosing larvae.” The swimming larva bears an early bud.
Bs-pitx cDNA Cloning and Sequence Analysis
cDNA pools were obtained from total RNA extracted from mixed developmental stages of fresh B. schlosseri colonies and were screened by nested polymerase chain reaction (PCR) using degenerated primers as described (Christiaen et al., 2002). The sequence obtained (130 bp) was used to design homologous primers for subsequent 5′- and 3′-RACE. Nested PCR were performed according to manufacturer's instructions (SMART RACE cDNA Amplification Kit, Clontech). The primers used to amplify Bs-Pitx cDNA extremities were as follows: PEBSPTX5 5′-TATGAGCACGCGTGAAGATATGTCG-3′ and PIBSPITX5 5′-GTTCAAGAACCGCCGAGCAAAGTGG-3′ for 3′-RACE (external and nested primers respectively); and BSPitXR1 5′-CAGGATGCCGTTGATACCGTTGAAG-3′ and BSPitXR2 5′-GACCATCTGGTTTCTTTCGCGCTTG-3′ for 5′-RACE (external and nested primers, respectively). Touchdown program was used for both, first, and nested PCR as described (Christiaen et al., 2002). Overlapping 5′ and 3′-RACE fragments of approximately 1 kb and 2.2 kb, respectively, were gel purified (Qiaquick gel extraction kit, Qiagen), cloned into pCRII TOPO vector (Invitrogen), and sequenced on both strands. Several sequences for each RACE were used to build a single contig.
All sequences, obtained either from the sequencing or downloaded from GenBank, were handled with the VectorNTI Suite (Informax). Alignments were constructed using CLUSTALW (Thompson et al., 1994) and refined by eye. The homeodomain's distance trees were built by using the neighbor-joining method (Saitou and Nei, 1987) with MEGA2 program (Kumar et al., 2001). Alternatively, the BLOCKMAKER (Henikoff et al., 1995) program was used to define conserved blocks among three groups of paired-related families of proteins. The trees were constructed by MEGA2 using either the neighbor-joining or the maximum parsimony methods (data not shown). Bootstrap analysis was carried out for each phylogenetic analysis (1,000 iterations; Felsenstein, 1992).
Whole-Mount In Situ Hybridization
Colonies and embryos were anesthetized with MS222 (Sigma) to prevent muscle contractions; fixed in freshly prepared 4% paraformaldehyde (PFA), 0.1 M MOPS (pH 7.5), 0.5 M NaCl, 1 mM ethyleneglycoltetraacetic acid, 2 mM MgSO4 overnight at 4°C; dehydrated in graded series of ethanol; and stored in methanol at −20°C. Whole-mount in situ hybridization on B. schlosseri colonies and embryos was performed in an Insitupro (Intavis AG) automate, using a protocol modified from Nguyen et al. (2001). Briefly, colonies and embryos were treated at room temperature for 30 min in 50 μg/ml and 25 μg/ml proteinase K, respectively. Hybridization was carried out for 16 hr at 65°C; after extensive washing, staining was performed in alkaline phosphatase buffer supplemented with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP)for a few hours.
The reaction was stopped by washing twice in PBST. Samples were post-fixed 1 hr in phosphate buffered saline-buffered (pH 7.5) 4% PFA, dehydrated in graded series of ethanol to absolute ethanol, and embedded in Paraplast (Sherwood medical). Samples in various orientations were sectioned (4–7 μm), cleaned from Paraplast by xylene (15 min), mounted in Eukitt (Electronic Microscopy Sciences), and photographed with a light microscope.
We thank Prof. Bernie Degnan for critical reading of the manuscript. This investigation was supported by grants from Ministero della Universitá e Ricerca Scientifica e Tecnologica and by University of Padova to P.B. and L.M.