Analysis of the basal chordate Botryllus schlosseri reveals a set of genes associated with fertility

Abstract

Background: Gonad differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. The colonial ascidian, Botryllus schlosseri is a chordate model organism which offers two unique traits that can be utilized to characterize the genes underlying germline development: a colonial life history and variable fertility. These properties allow individual genotypes to be isolated at different stages of fertility and gene expression can be characterized comprehensively.

Results: Here we characterized the transcriptome of both fertile and infertile colonies throughout blastogenesis (asexual development) using differential expression analysis. We identified genes (as few as 7 and as many as 647) regulating fertility in Botryllus at each stage of blastogenesis. Several of these genes appear to drive gonad maturation, as they are expressed by follicle cells surrounding both testis and oocyte precursors. Spatial and temporal expression of differentially expressed genes was analyzed by in situ hybridization, confirming expression in developing gonads.

Conclusion: We have identified several genes expressed in developing and mature gonads in B. schlosseri. Analysis of genes upregulated in fertile animals suggests a high level of conservation of the mechanisms regulating fertility between basal chordates and vertebrates.

Figure 1

Botryllus schlosseri sexual and asexual development (blastogenesis) and anatomy. A) Sexual cycle showing the larva stage and metamorphosis into oozoid. B) Asexual cycle – blastogenesis: Dorsal views of zooids (green frame), primary buds (yellow frame) and secondary buds (red frame). A secondary bud appears as a thickening of the epidermis and the peribranchial chamber leaflet of the primary bud (stage A1), which evaginates into a closed vesicle (stage B2), followed by organogenesis (stages C1-D). Gonadogenesis occurs in the secondary bud from mobile precursors (blue; stages B1-C2). During takeover (stages C2-D), the secondary bud becomes the primary bud and a new blastogenic cycle begins for the next secondary bud. After the second takeover event, the primary bud opens its siphons and becomes a functional adult (zooid). In fertile colonies (as illustrated here), the hermaphrodite gonad fully matures on both sides of the zooid. C) Schematic showing the anatomy of an adult fertile zooid with two primary buds. D and E) Ventral live image of both infertile and a fertile colonies (respectively). Arrows point to testes and arrowheads point to eggs. Panels A and B are modified from Brown et al. 2009, Laird et al. 2005 and Laird and De Tomaso 2005.

Figure 2

Fluorescent in situ hybridization (FISH) for selected differentially expressed genes on fertile colonies. FISH for selected genes with homologs in humans and other organisms was performed on whole mounts of adult fertile colonies. Nuclei were stained with DAPI (blue) and riboprobes are shown in green. Based on their expression patterns in developing gonads, these genes fall into four distinct categories: 1) (A-E) localized to testes or developing testes (otoa, tspan8, tsk1, vtg1); 2) (F-I) localized to maturing eggs (vtg1, ldlr, zp1, tll1); 3) (J-N) oocytes at stage 2 (hsd17β8, selp, tsk2, tspn8); and 4) (O-R) other tissues (zp1, selp, and ldlr). otoa = otoancorin, tspan8 = tetraspanin-8, tsk1 = testis-specific serine/threonine-protein kinase1, tsk2 = testis-specific serine/threonine-protein kinase2, vtg1 = vitellogenin, ldlr = Low-density lipoprotein receptor 1, zp1 = zona-pellucida sperm binding protein 1, tll1 = Tolloid-like protein1, hsd17β8 = Estradiol 17β dehydrogenase-8, selp = P-selectin. Scale bar 50 μm.

Figure 3

Fluorescent in situ hybridization (FISH) for selected differentially expressed genes on infertile colonies. FISH for selected genes was performed on whole mounts of adult infertile colonies (A-J). Nuclei were stained with DAPI (blue) and riboprobes are shown in green. Expression of genes detected in fertile colonies is absent in juveniles except for p-selectin (localizing to both oral and excurrent siphons) as indicated by arrows (B) and Zona-pellucida sperm binding protein1 (localizing to the endostyle) as indicated by the arrowhead (H). otoa = otoancorin, tspan8 = tetraspanin-8, tsk1 = testis-specific serine/threonine-protein kinase1, tsk2 = testis-specific serine/threonine-protein kinase2, vtg1 = vitellogenin, ldlr = Low-density lipoprotein receptor 1, zp1 = zona-pellucida sperm binding protein 1, tll1 = Tolloid-like protein1, hsd17b8 = Estradiol 17β dehydrogenase-8, selp = P-selectin. Scale bar 200 μm.