Evolution of the Pax-Six-Eya-Dach network: the calcisponge case study

Abstract

Background: The Pax-Six-Eya-Dach network (PSEDN) is involved in a variety of developmental processes, including well documented roles in determination of sensory organs and morphogenesis in bilaterian animals. Expression of PSEDN components in cnidarians is consistent with function in sensory organ development. Recent work in demosponges demonstrated the presence of single homologs of Pax and Six genes, and their possible involvement in morphogenesis, but the absence of the remaining network components. Calcisponges are evolutionarily distant from demosponges, and the developmental toolkits of these two lineages differ significantly. We used an emerging model system, Sycon ciliatum, to identify components of the PSEDN and study their expression during embryonic and postembryonic development.

Results: We identified two Pax, three Six and one Eya genes in calcisponges, a situation strikingly different than in the previously studied demosponges. One of the calcisponge Pax genes can be identified as PaxB, while the second Pax gene has no clear affiliation. The three calcisponge Six genes could not be confidently classified within any known family of Six genes. Expression analysis in adult S. ciliatum demonstrated that representatives of Pax, Six and Eya are expressed in patterns consistent with roles in morphogenesis of the choanocyte chambers. Distinct paralogues of Pax and Six genes were expressed early in the development of the putative larval sensory cells, the cruciform cells. While lack of known photo pigments in calcisponge genomes precludes formal assignment of function to the cruciform cells, we also show that they express additional eumetazoan genes involved in specification of sensory and neuronal cells: Elav and Msi.

Conclusions: Our results indicate that the role of a Pax-Six-Eya network in morphogenesis likely predates the animal divergence. In addition, Pax and Six, as well as Elav and Msi are expressed during differentiation of cruciform cells, which are good candidates for being sensory cells of the calcaronean sponge larvae.

Keywords: Calcisponges; Eyes absent; Pax; Sensory cells; Six; Sycon.

Figure 1

Phylogenetic analyses of the Six, Eya and Pax genes. A, Bayesian phylogenetic tree of Pax genes inferred from the paired domain. The red dots indicate the different positions of SciPaxF: 1, position of SciPaxF when the complete the PD domain was used to infer the phylogeny; 2, position of PaxF when removing ctenophore sequences and 3, when using the RED domain (S6) and including CrPaxE and Arthropod Eyg genes. B, The maximum likelihood tree was inferred from the Six homeodomain. The Six tree was rooted using the TALE class homeodomain as an outgroup. B’, Bayesian tree inferred from the Six homeodomain without including Mnemiopsis Six genes. C, Bayesian tree of Eya ED domain. The EYA tree was rooted using Arabidopsis thaliana (At) Eya-like gene. ML bootstrap values greater than 500 (left) and posterior probabilities generated by MrBayes greater than 0.5 (right) are displayed. Asterisks indicate the differences in the position of a given gene. Names are prefixed as follows: Porifera: Calcisponges, Sca = Sycon calcavaris; Sci = Sycon ciliatum and Lco = Leucosolenia complicata. Demosponges, Amq = Amphimedon queenslandica; Ef = Ephydatia fluviatilis; Chl = Chalinula loosanoffi. Ctenophora: Ml = Mnemiopsis leidyi and Cw = Coeloplana willeyi. Cnidaria: Anthozoa, Nv = Nematostella vectensis and Hydrozoa Cr = Cladonema radiatu; Hv = Hydra vulgaris; Ami = Acropora millepora. Bilateria: Protostomia, Tc = Tribolium castaneum.; Dm = Drosophila melanogaster. Deuterostomia: Bf = Branchiostoma floridae and Mm = Mus musculus.

Figure 2

Expression of Sycon Pax and Six genes during embryogenesis. A, Summary of embryogenesis in Sycon, with consecutive stages depicted from left to right: oocyte, cleavage, pre-inversion, post-inversion, larva. Abbreviations: pin, pinacocytes; ch, choanocytes; ac, accessory cells; cc, cruciform cells; mi, micromeres ma, macromeres; and mc, maternal cells. B-G, SciPaxB expression is detectable in the oocytes (B), and in all blastomeres during late cleavage (C). During early pre-inversion it is detectable in the micromeres and cruciform cells and in late pre-inversion (E), post-inversion (F) and in larvae (G) SciPaxB expression is restricted to the equatorial micromeres. H-M, SciPaxF expression is detectable in the oocytes (H) and is gradually restricted to become predominant in the cross cells (J to K), and then the equatorial micromeres in the larva (M). N-S, SciSixC expression is present in the oocytes (N) and then all blastomeres (O), but strongest in the cruciform cells by early pre-inversion (P). During late pre-inversion (Q) the expression is localized to the cruciform cells and the macromeres, and becomes limited to the macromeres in the post-inversion embryos and larva (R,S). T-Z, SciSixA is expressed in the oocytes (T), but not in cleavage stages or early pre-inversion stage embryos (U, V), when strong expression in a ring of accessory cells is apparent (U). Transient expression in the anterior micromeres is detectable in post-inversion stage embryos (W-Y) but not larvae (Z). All images are of whole mounted, glycerol-cleared specimens, except Y, which is a resin section. Embryos in pre-inversion, post-inversion and larvae were isolated from the tissue, except from K. The asterisk indicates macromeres at the posterior pole of the embryo and larva. Black arrows, white arrows and white arrowheads indicate the cruciform cells, micromeres and the accessory cells, respectively.

Figure 3

Expression of Pax, Six and Eya genes in adult sponges. A-C; SciPaxB is expressed in all choanocytes and a fraction of mesohyle cells. D-F, SciSixA and G-I, SciEya, are expressed in choanocytes of the radial chambers, but not choanocytes located in the upper region remaining in asconoid organization. A, D and G are upper parts of whole young sponges, B, E and H are magnifications of radial chambers, C, F and I are plastic sections through the radial chambers. Abbreviations: me, mesohyl cells; ch, choanocytes; arrows indicate strongest expression.

Figure 4

Phylogenetic tree and embryonic expression of S. ciliatum Msi and Elav genes. A, This is a Bayesian tree inferred from the RRM2 motif of the RNAbp, Elav and Musashi. ML bootstrap values greater than 500 and posterior probabilities generated by MrBayes greater than 0.5 are displayed. The tree was rooted with the Pabp subfamily. Names are prefixed as in Figure  1 except from: Ac, Aplysia californica; Ame, Apis mellifera; Mb, Monosiga brevicollis; Lg, Lottia gigantea and Ta, Trichoplax adhaerens. B–K Whole mount in situ hybridization: B-D, SciMsiA expression in sponges containing oocytes (B); embryos during pre-inversion showing expression in the cruciform cells (cc, arrows) and macromeres (C) and no expression in the larva (D). E-G, SciMsiB expression in oocytes (E), in cc and macromeres in embryos during post-inversion (F), and expression in the macromeres in the larva (G). H-K, SciElav whole mount in situ of sponges containing oocytes (H); during pre-inversion SciElav is seen in cruciform cells and weak in macromeres (I, J); and by post-inversion SciElav expression is predominant in macromeres (K). The asterisk indicates macromeres at the posterior pole of the embryo and larva.

Figure 5

Summary of the expression patterns. A, SciPaxB in cruciform cells and equatorial micromeres during pre-inversion and micromeres in the larvae; B, SciPaxF in cruciform cells during pre-inversion and equatorial micromeres of the larva; C, SixA in anterior micromeres, D, SciSixC, SciElav, SciMsiA and SciMsiB in the cruciform cells and in the macromeres of the post-inversion stage embryos and/or larvae; E, Gradients of SciSixA and SciEya in choanocyte cells organized in chambers, SciPaxB in all choanocytes and in scattered mesohyl cells.