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Background information. T-box transcription factors are a large family of transcriptional regulators involved in many aspects of embryonic development. In a previous report, we described the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula: a Brachyury homologue, Sd-Bra, and a Tbx2 homologue, Sd-Tbx2. Elucidation of the genomic structure of Sd-Bra allowed us to demonstrate the existence of two different isoforms, resulting from alternative splicing. Moreover, we demonstrated that the shorter isoform exists in two different glycosylation states.
Results. In the present study, we demonstrate a differential subcellular localization of the three Sd-Bra isoforms, suggesting that its differential nuclear import could be an important mechanism for its functional regulation. Furthermore, we demonstrate that Sd-Tbx2 exists only in one isoform, which is mainly localized in the nucleus. The pattern of expression of Sd-Bra and Sd-Tbx2 genes is analysed in sponge tissue, in gemmules and in cultured cells.
Conclusion. These results suggest a conserved role for Sd-Bra in the control of morphogenetic movements through the regulation of cell-adhesion properties and the involvement of Sd-Tbx2 in the determination of cell identity in the early stages of differentiation, reminiscent of the function of Tbx2-3-4-5 in vertebrates during limb specification. Also, the fact that a Brachyury and a Tbx2 homologue exist in S. domuncula suggests that the first divergence from the ancestral Brachyury-like gene might be a Tbx2-like gene and not a Tbrain-like gene as had been previously suggested [Adell, Grebenjuk, Wiens and Müller (2003) Dev. Genes Evol. 213, 421–434].
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- Materials and methods
T-box transcription factors are a large family of transcriptional regulators involved in developmental processes such as specification of the mesoderm, anterior—posterior axis patterning or specification and outgrowth of muscle and limbs (reviewed in Smith, 1997; Papaioannou, 2001). More than 50 members of this family have been identified from metazoans (Herrmann and Kispert, 1994; Papaioannou, 1997; Papaioannou and Silver, 1998; Smith, 1999; Tada and Smith, 2001). Interestingly, in contrast with other transcription factors, e.g. members of the homeobox gene family, T-box genes have not been found in plants or fungi. The characteristic DNA-binding (T-box) domain is composed of a highly conserved stretch of 180–190 amino acids (Kispert and Herrmann, 1993), which have been taken as the basis to group T-box genes into five different subfamilies: T or Brachyury, Tbrain, Tbx2, Tbx1 and Tbx6. In general, the different subfamilies are also distinguished by their pattern of expression and their function (Papaioannou and Silver, 1998). In a previous report, we described the isolation and genomic characterization of two T-box genes from the siliceous sponge Suberites domuncula (Adell et al., 2003a). The phylogenetic analysis classified one into the subfamily of Brachyury, Sd-Bra, and the other into the Tbx2 subfamily, Sd-Tbx2.
Brachyury, the founding member of the family, is characteristically expressed in the notochord and tail bud of chordates (Herrmann et al., 1990). Its function in mesoderm and notochord morphogenesis in chordates was elucidated from mouse and Xenopus mutant embryos lacking the notochord and tail (Wilkinson et al., 1990; Conlon et al., 1996). The finding of Brachyury homologues in all metazoan phyla studied and its phylogenetic position within the T-box genes suggest that Brachyury genes are the closest relatives of the common ancestral T-box (Wattler et al., 1998; Adell et al., 2003a). Despite its different pattern of expression in the embryos of different animals (Wilkinson et al., 1990; Smith et al., 1991; Kusch and Reuter, 1999; Arendt et al., 2001; Gross and McClay, 2001; Spring et al., 2002), it is accepted that the dominant expression of Brachyury in all metazoans occurs in the blastopore, which develops further into the fore- and hindgut (Arendt et al., 2001; Technau, 2001).
The vertebrate Tbx2 subfamily comprises four closely related paralogues (Tbx2-3-4-5), which evolved from a single ancestral locus by a tandem-duplication event followed by a duplication of the derived gene pair (Agulnik et al., 1996). They are involved in the patterning of several structures as notochord, somites, eyes, heart and limbs (Hayata et al., 1999; Yamada et al., 2000; Takabatake et al., 2002). Their roles in the developmental specification and growth of limbs have been extensively studied (Gibson-Brown et al., 1998; Rodriguez-Esteban et al., 1999; Simon, 1999; Takeuchi et al., 1999; Ahn et al., 2002). Besides the chordate genes and the two Tbx2 members from echinoderms (Croce et al., 2003; Gross et al., 2003), only two Tbx2 genes from protostomians and one from Placozoa have been published (Martinelli and Spring, 2003).
The elucidation of the genomic structure of Sd-Bra has allowed us to demonstrate the existence of two different isoforms corresponding to alternatively spliced isoforms (Adell et al., 2003a). Furthermore, analysis with a polyclonal antibody raised against the Sd-Bra protein showed that the shorter isoform exists in two different glycosylation states. In the present study, we demonstrate that differential splicing is important for the functional regulation of Sd-Bra, since it affects its transport to the nucleus. Moreover, using an antibody raised against the Sd-Tbx2 protein, we demonstrate that only one isoform, Sd-Tbx2, exists and that it is localized in the nucleus of a few cells scattered throughout the sponge tissue.
Sponges are diploblastic animals, which constitute the most basal animal phylum. They are composed of an epithelial-like layer, the pinacoderm, surrounding a mesohyl, which contains the specialized cells (spherulous cells, sclerocytes and choanocytes) and the archaeocytes, totipotent cells capable of differentiating into all the other cell types (Simpson, 1984). Sponges can reproduce sexually and asexually, by releasing embryos or by budding respectively.
The expression and functional analysis of the developmental genes, such as the T-box, should be performed on the corresponding embryos. However, because it is not yet possible to obtain sponge embryos, we have analysed the expression of Sd-Bra and Sd-Tbx2 in sponge gemmules and under different sponge cell-culture conditions. Gemmules are a peculiar type of buds formed during asexual reproduction in many sponge species, such as in S. domuncula. They are composed of undifferentiated and quiescent cells, the amoebocytes, covered by a hard coat of collagen reinforced by spicules. When conditions are favourable for germination, the amoebocytes escape and differentiate into the various cell types needed to build a complete young sponge.
Regarding the cell cultures, two types were compared: primmorphs and adherent aggregates. Primmorphs are 1–5 mm round-shaped aggregates, formed in suspension from dissociated single sponge cells after approx. 5 days of incubation on a moving platform. They have an organized tissue-like structure, with a central zone composed primarily of spherulous cells, surrounded by a pinacoderm-like epithelium (Müller et al., 1999; Le Pennec et al., 2003). In contrast, the dissociated sponge cell cultures on coated plastic dishes without movement first produce small aggregates in suspension, which, after a few hours, adhere to the substrate and grow as adhered aggregates during subsequent days. Although the cell—cell contacts in primmorphs are established mostly during the first hours of culture, reaching a balance around day 5, the interactions among cells and between cells and the matrix continuously remodel the growth and shape of the adherent aggregates.