The potential role of microRNAs in regulating gonadal sex differentiation in the chicken embryo.

Differential gene expression regulates tissue morphogenesis. The embryonic gonad is a good example, where the developmental decision to become an ovary or testis is governed by female- or male-specific gene expression. A number of genes have now been identified that control gonadal sex differentiation. However, the potential role of microRNAs (miRNAs) in ovarian and testicular pathways is unknown. In this review, we summarise our current understanding of gonadal differentiation and the possible involvement of miRNAs, using the chicken embryo as a model system. Chickens and other birds have a ZZ/ZW sex chromosome system, in which the female, ZW, is the heterogametic sex, and the male, ZZ, is homogametic (opposite to mammals). The Z-linked DMRT1 gene is thought to direct testis differentiation during embryonic life via a dosage-based mechanism. The conserved SOX9 gene is also likely to play a key role in testis formation. No master ovary determinant has yet been defined, but the autosomal FOXL2 and Aromatase genes are considered central. No miRNAs have been definitively shown to play a role in embryonic gonadal development in chickens or any other vertebrate species. Using next generation sequencing, we carried out an expression-based screen for miRNAs expressed in embryonic chicken gonads at the time of sexual differentiation. A number of miRNAs were identified, including several that showed sexually dimorphic expression. We validated a subset of miRNAs by qRT-PCR, and prediction algorithms were used to identify potential targets. We discuss the possible roles for these miRNAs in gonadal development and how these roles might be tested in the avian model.

Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads.

Tissue-specific patterns of microRNA (miRNA) expression contribute to organogenesis during embryonic development. Using the embryonic chicken gonads as a model for vertebrate gonadogenesis, we previously reported that miRNAs are expressed in a sexually dimorphic manner during gonadal sex differentiation. Being male biased, we hypothesised that up-regulation of microRNA 202* (MIR202*) is characteristic of testicular differentiation. To address this hypothesis, we used estrogen modulation to induce gonadal sex reversal in embryonic chicken gonads and analyzed changes in MIR202* expression. In ovo injection of estradiol-17beta at Embryonic Day 4.5 (E4.5) caused feminization of male gonads at E9.5 and reduced MIR202* expression to female levels. Female gonads treated at E3.5 with an aromatase inhibitor, which blocks estrogen synthesis, were masculinized by E9.5, and MIR202* expression was increased. Reduced MIR202* expression correlated with reduced expression of the testis-associated genes DMRT1 and SOX9, and up-regulation of ovary-associated genes FOXL2 and CYP19A1 (aromatase). Increased MIR202* expression correlated with down-regulation of FOXL2 and aromatase and up-regulation of DMRT1 and SOX9. These results confirm that up-regulation of MIR202* coincides with testicular differentiation in embryonic chicken gonads.

Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase.

Many marine animals, ranging from corals to fishes, synchronise reproduction to lunar cycles. In the annelid Platynereis dumerilii, this timing is orchestrated by an endogenous monthly (circalunar) clock entrained by moonlight. Whereas daily (circadian) clocks cause extensive transcriptomic and proteomic changes, the quality and quantity of regulations by circalunar clocks have remained largely elusive. By establishing a combined transcriptomic and proteomic profiling approach, we provide first systematic insight into the molecular changes in Platynereis heads between circalunar phases, and across sexual differentiation and maturation. Whereas maturation elicits large transcriptomic and proteomic changes, the circalunar clock exhibits only minor transcriptomic, but strong proteomic regulation. Our study provides a versatile extraction technique and comprehensive resources. It corroborates that circadian and circalunar clock effects are likely distinct and identifies key molecular brain signatures for reproduction, sex and circalunar clock phase. Examples include prepro-whitnin/proctolin and ependymin-related proteins as circalunar clock targets.

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression.

BACKGROUND: RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter. RESULTS: We identified and characterised the ch7SK promoter sequence upstream of the full-length 7SK small nuclear RNA (snRNA) sequence in the chicken genome and used this to construct vectors to express shRNAs targeting enhanced green fluorescent protein (EGFP). We transfected chicken DF-1 cells with these constructs and found that anti-EGFP-shRNAs (shEGFP) expressed from the ch7SK promoter could induce efficient knockdown of EGFP expression. We further compared the efficiency of ch7SK-directed knockdown to that of chicken U6 (cU6) promoters and found that the efficiency of the ch7SK promoter was not greater than, but comparable to the efficiency of cU6 promoters. CONCLUSION: In this study we have demonstrated that the ch7SK promoter can express shRNAs capable of mediating efficient RNAi in a chicken cell line. However, our finding that RNAi driven by the ch7SK promoter is not more efficient than cU6 promoters contrasts previous comparisons of mammalian U6 and 7SK promoters. Since the ch7SK promoter is the first non-mammalian vertebrate 7SK promoter to be characterised, this finding may be helpful in understanding the divergence of pol III promoter activities between mammalian and non-mammalian vertebrates. This aside, our results clearly indicate that the ch7SK promoter is an efficient alternative to U6-based shRNA expression systems for inducing efficient RNAi activity in chicken cells.

Sexually dimorphic microRNA expression during chicken embryonic gonadal development.

MicroRNAs are a highly conserved class of small RNAs that function in a sequence-specific manner to posttranscriptionally regulate gene expression. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryogenesis, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a model, microarrays were used to profile the expression of chicken miRNAs prior to, during, and after the time of gonadal sex differentiation (Embryonic Day 5.5 [E5.5], E6.5, and E9.5). Sexually dimorphic miRNAs were identified, and the expression patterns of several were subjected to further validation by in situ hybridization and Northern blot analysis. Expression of one chicken miRNA, MIR202*, was observed to be sexually dimorphic, with upregulation in the developing testis from the onset of sexual differentiation. Additional data from deep sequencing of male and female embryonic gonad RNA samples also indicated upregulation of MIR202* in male gonads. These findings provide the first evidence of sexually dimorphic miRNA expression during vertebrate gonadal sex differentiation and suggest that MIR202* may function in regulating testicular development.