Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance

The sex determining gene is divergent among different animal species. However, sox9 is up-regulated in the male gonads in a number of species in which it is the essential regulator of testis determination. It is therefore often discussed that the sex determining gene-sox9 axis functions in several vertebrates. In our current study, we show that sox9b in the medaka (Oryzias latipes) is one of the orthologues of mammalian Sox9 at syntenic and expression levels. Medaka sox9b affects the organization of extracellular matrices, which represents a conserved role of sox9, but does not directly regulate testis determination. We made this determination via gene expression and phenotype analyses of medaka with different copy numbers of sox9b. Sox9b is involved in promoting cellular associations and is indispensible for the proper proliferation and survival of germ cells in both female and male medaka gonads. Medaka mutants that lack sox9b function exhibit a seemingly paradoxical phenotype of sex reversal to male. This is explained by a reduction in the germ cell number associated with aberrant extracellular matrices. Together with its identified roles in other vertebrate gonads, a testis-determining role for Sox9 in mammals is likely to have been neofunctionalized and appended to its conserved role in germ cell maintenance.     

Homologues of sox8 and sox10 in the orange-spotted grouper Epinephelus coioides: sequences, expression patterns, and their effects on cyp19a1a promoter activities in vitro

Sox8 and Sox10 are members of group E Sox proteins involved in a wide range of developmental processes including sex determination and neurogenesis in vertebrates. The orange-spotted grouper sox8a and sox10a homologues were isolated and characterized in the present study. Both sox8a and sox10a genes contain three exons and two introns, and encode putative proteins with typical structures of group E Sox. Sox8a was expressed in diverse tissues including the central nervous system and some peripheral tissues. In contrast, sox10a mRNA was detected primarily in the central nervous system. During embryogenesis, sox8a mRNA seemed to be de novo synthesized in the embryos from otic vesicle stage. However, sox10a mRNA was only detectable in juvenile fish 35 days post hatching and thereafter. The mRNA levels of sox8a in the gonads were not significantly different among ovarian developmental stages but increased in the testis. In vitro transfection assays showed that the Sox10a but not Sox8a up-regulated cyp19a1a promoter activities. Taken together, these results suggested that the sox8a may play roles in diverse tissues and during embryogenesis, whereas sox10a may be mainly involved in the neural regulation of juvenile and adult fish, and that certain Sox homologues may regulate the orange-spotted grouper cyp19a1a promoter.     

Comparative analysis of duplicated sox21 genes in zebrafish

Sox21 is thought to function as a counteracting partner of SoxB1 (Sox1, 2, 3) genes and is involved in cell fate determination. In this study, we comparatively analyzed the expression patterns and conserved cis-regulatory elements of the duplicated sox21 genes in zebrafish. In embryogenesis, sox21b is predominantly expressed in the telencephalon, hypothalamus, mesencephalon and lens, and sox21a is solely expressed in the midbrain-hindbrain boundary, olfactory placode and lateral line, while both genes are expressed in the hindbrain, spinal cord and ear. In adult, sox21a is expressed in the brain, skin, ovary and intestine, while sox21b is expressed in the brain and testis. Interestingly, all 16 pan-vertebrate conserved non-coding elements (CNEs) are asymmetrically preserved in the sox21b locus, whereas two fish-specific elements are kept in the sox21a locus, and this is correlated with increased evolutionary rate of the sox21a protein sequence. Transient transgenic reporter analysis revealed that six sox21b CNEs and two sox21a CNEs drove green fluorescent protein (GFP) expression in tissues correlated with the partitioning of expression in two orthologues. These results indicate that sox21a and sox21b have reciprocally lost expression domains of the ancestral gene reflected by degeneration of certain CNEs in their genomic loci and provide clear evidence for evolution of the duplicated sox21 genes by subfunctionalization. In addition, our data suggest that some CNEs-based regulatory pathways have been predominantly preserved in the sox21b locus.     

A pair of Sox: distinct and overlapping functions of zebrafish sox9 co-orthologs in craniofacial and pectoral fin development

Understanding how developmental systems evolve after genome amplification is important for discerning the origins of vertebrate novelties, including neural crest, placodes, cartilage and bone. Sox9 is important for the development of these features, and zebrafish has two co-orthologs of tetrapod SOX9 stemming from an ancient genome duplication event in the lineage of ray-fin fish. We have used a genotype-driven screen to isolate a mutation deleting sox9b function, and investigated its phenotype and genetic interactions with a sox9a null mutation. Analysis of mutant phenotypes strongly supports the interpretation that ancestral gene functions partitioned spatially and temporally between Sox9 co-orthologs. Distinct subsets of the craniofacial skeleton, otic placode and pectoral appendage express each gene, and are defective in each single mutant. The double mutant phenotype is additive or synergistic. Ears are somewhat reduced in each single mutant but are mostly absent in the double mutant. Loss-of-function animals from mutations and morpholino injections, and gain-of-function animals injected with sox9a and sox9b mRNAs showed that sox9 helps regulate other early crest genes, including foxd3, sox10, snai1b and crestin, as well as the cartilage gene col2a1 and the bone gene runx2a; however, tfap2a was nearly unchanged in mutants. Chondrocytes failed to stack in sox9a mutants, failed to attain proper numbers in sox9b mutants and failed in both morphogenetic processes in double mutants. Pleiotropy can cause mutations in single copy tetrapod genes, such as Sox9, to block development early and obscure later gene functions. By contrast, subfunction partitioning between zebrafish co-orthologs of tetrapod genes, such as sox9a and sox9b, can relax pleiotropy and reveal both early and late developmental gene functions.     

sox4 And sox11 Function during Xenopus laevis Eye Development

SoxC genes are involved in many developmental processes such as cardiac, lymphoid, and bone development. The SoxC gene family is represented by Sox4, Sox11, and Sox12. Loss of either Sox4 or Sox11 function is lethal during mouse embryogenesis. Here, we demonstrate that sox4 and sox11 are strongly expressed in the developing eye, heart as well as brain in Xenopus laevis. Morpholino oligonucleotide mediated knock-down approaches in anterior neural tissue revealed that interference with either Sox4 or Sox11 function affects eye development. A detailed analysis demonstrated strong effects on eye size and retinal lamination. Neural induction was unaffected upon Sox4 or Sox11 MO injection and early eye field differentiation and cell proliferation were only mildly affected. Depletion of both genes, however, led independently to a significant increase in cell apoptosis in the eye. In summary, Sox4 and Sox11 are required for Xenopus visual system development.