Genome-wide analysis of the sox family in the calcareous sponge Sycon ciliatum: multiple genes with unique expression patterns

Abstract

Background: Sox genes are HMG-domain containing transcription factors with important roles in developmental processes in animals; many of them appear to have conserved functions among eumetazoans. Demosponges have fewer Sox genes than eumetazoans, but their roles remain unclear. The aim of this study is to gain insight into the early evolutionary history of the Sox gene family by identification and expression analysis of Sox genes in the calcareous sponge Sycon ciliatum.

Methods: Calcaronean Sox related sequences were retrieved by searching recently generated genomic and transcriptome sequence resources and analyzed using variety of phylogenetic methods and identification of conserved motifs. Expression was studied by whole mount in situ hybridization.

Results: We have identified seven Sox genes and four Sox-related genes in the complete genome of Sycon ciliatum. Phylogenetic and conserved motif analyses showed that five of Sycon Sox genes represent groups B, C, E, and F present in cnidarians and bilaterians. Two additional genes are classified as Sox genes but cannot be assigned to specific subfamilies, and four genes are more similar to Sox genes than to other HMG-containing genes. Thus, the repertoire of Sox genes is larger in this representative of calcareous sponges than in the demosponge Amphimedon queenslandica. It remains unclear whether this is due to the expansion of the gene family in Sycon or a secondary reduction in the Amphimedon genome. In situ hybridization of Sycon Sox genes revealed a variety of expression patterns during embryogenesis and in specific cell types of adult sponges.

Conclusions: In this study, we describe a large family of Sox genes in Sycon ciliatum with dynamic expression patterns, indicating that Sox genes are regulators in development and cell type determination in sponges, as observed in higher animals. The revealed differences between demosponge and calcisponge Sox genes repertoire highlight the need to utilize models representing different sponge lineages to describe sponge development, a prerequisite for deciphering evolution of metazoan developmental mechanisms.

Figure 1

Sycon ciliatum: morphology and embryonic development. (A) Environmental sample of multiple specimens of Sycon growing on stipe of the kelp Laminaria hyperborea. (B) Transverse section of Sycon ciliatum demonstrating radial symmetry. (C) Schematic representation of Sycon body plan. (D) Schematic representation of key stages in embryogenesis (after [17]): top; oocyte, early and late cleavage stage; bottom, pre-inversion stage, inversion and post-inversion. (E) Confocal image of an embryo during pre-inversion stage showing four cruciform cells (cc) among micromeres. Actin cytoskeleton is labeled green, DNA is blue. (F) Larvae. Cell types are abbreviated as follows: ac, accessory cells; cc, cruciform cells; ch, choanocytes; ma, macromeres; mi, micromeres; pin, pinacocytes.

Figure 2

Phylogenetic analysis of Sycon Sox genes based on the HMG domain sequences and schematic representation of motif conservation within groups B, C, E, and F. Maximum likelihood tree using LG + G model of protein evolution is shown. Support values of posterior probabilities (bottom) and bootstrap (top) are displayed, BT values below than 10% and PP values below 0.5 were discarded. A root was placed in the out-groups. P values for Sycon motifs: Group B motif, 9.30E-07; conserved region I, 1.16E-04; conserved region II, 1.99E-05; Group C conserved region, 2.26E-07; Group E conserved region I, 9.03E-10; conserved region II, 4.83E-13; Group F conserved region, 1.40E-14 (SciSoxF1) and 1.10E-10 (SciSoxF2). Ami, Acropora millepora; Amq, Amphimedon queenslandica; Ce, Caenorhabditis elegans; Cin, Ciona intestinalis; Hsa, Homo sapiens; Lco, Leucosolenia complicata; Nv, Nematostella vectensis; Sci, Sycon ciliatum.

Figure 3

Expression of SciSoxB and SciSoxC during embryogenesis.SciSoxB is strongly expressed in oocytes and during cleavage (A); the expression gradually decreases from the late cleavage (B, top right) to pre-inversion (B, bottom left), becoming limited to the cruciform cells (cc) in pre-inversion stage embryos (B and C) and macromeres (asterisk) of pre-inversion stage embryos (C) and larvae (D). SciSoxC is expressed in oocytes (E) and during cleavage (F); in pre-inversion stage embryos expression is limited to macromeres (asterisk) (G, H) and becomes undetectable in the larvae (I). All images are of glycerol-cleared slices of sponges containing developmental stages, except of D, demonstrating an isolated larva.

Figure 4

Expression of SciSoxE ,SciSoxF1 ,SciSoxF2 ,SciSox6 , and SciSox7 .SciSoxE is expressed in choanocytes (ch) (A, B) and a fraction of mesohyle cells, particularly prominent in the apical (oscular) part (A). The expression is undetectable in the oocytes and accessory cells (ac) (B). SciSoxF1 expression is limited to choanocytes (C, D) and accessory cells (D), and absent in the embryos (D). SciSoxF2 is weakly expressed in the choanocytes (E) and strongly in large spindle shape cells surrounding the osculum (E, magnified on F). SciSox6 is strongly expressed in choanocytes (ch), pinacocytes (pin), and a variety of mesohyle cells, especially those in the apical part (G, H), but not in embryos (H). SciSox7 is expressed in choanocytes (I, J) and strongly expressed in the oocytes (J). Top row: oscular parts of young sponges. Bottom row: B - plastic section of sponge containing oocytes. D- thick slice of sponge containing embryos during pre-inversion. F- magnification of the tip of the osculum. H- Plastic sections of sponge containing post-inversion stage embryo. J - thick slice of sponge containing small oocytes.

Figure 5

Expression of SciSoxL1 and SciSoxL2. SciSoxL1 is strongly expressed in the oocytes (A) and during cleavage (B); in pre-inversion stage embryos the expression is limited to cruciform cells (cc) (C); the transcripts are also present in the choanocytes (D). SciSoxL2 is strongly expressed in oocytes (E) and cleavage stage embryos (F); in pre-inversion stage embryos expression in the macromeres (asterisk) is higher than in the micromeres and is undetectable in the cruciform cells (cc) (G); the transcripts are also present in choanocytes and a fraction of mesohyle cells (H). A and E - thick slices of sponge containing oocytes. B - isolated mid-cleavage stage embryo. F - thick slice of sponge containing cleavage stage embryos. C, G - isolated pre-inversion stage embryos. D, H - oscular parts of young sponges.