Genetic Regulation of Avian Testis Development.

As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.

The homeobox gene TGIF1 is required for chicken ovarian cortical development and generation of the juxtacortical medulla.

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells: Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations: the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted misexpression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation.

Insights into Gonadal Sex Differentiation Provided by Single-Cell Transcriptomics in the Chicken Embryo.

Although the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell sequencing in the embryonic chicken gonad during sexual differentiation. The data show that chicken embryonic-supporting cells do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, they derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes and derivation of embryonic steroidogenic cells from a differentiated supporting-cell lineage. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

FOXL2 antagonises the male developmental pathway in embryonic chicken gonads.

FOXL2 is a conserved transcription factor with a central role in ovarian development and function. Studies in humans and mice indicate that the main role of FOXL2 is in the postnatal ovary, namely folliculogenesis. To shed light on the function and evolution of FOXL2 in the female gonad, we examined its role in embryonic avian gonads, using in ovo over-expression and knockdown. FOXL2 mRNA and protein are expressed female-specifically in the embryonic chicken gonad, just prior to the onset of sexual differentiation. FOXL2 is expressed in the medullary cord cells, in the same cell type as aromatase (CYP19A1). In addition, later in development, expression also becomes localised in a subset of cortical cells, distinct from those expressing estrogen receptor alpha. Mis-expression of FOXL2 in the male chicken embryonic gonad suppresses the testis developmental pathway, abolishing local expression of the male pathway genes, SOX9, DMRT1 and AMH, and repressing Sertoli cell development. Conversely, knockdown of FOXL2 expression allows ectopic activation of SOX9 in female gonads. However, mis-expression of FOXL2 alone was insufficient to activate aromatase expression in male gonads, while FOXL2 knockdown did not affect aromatase expression in females. These results indicate that FOXL2 plays an important role in embryonic differentiation of the avian ovary via antagonism of SOX9, but may be dispensable for aromatase activation at embryonic stages. The data suggest that FOXL2 has different roles in different species, more central for embryonic ovarian differentiation in egg-laying vertebrates.