Timothy J. Doran

The avian Z-linked gene DMRT1 is required for male sex determination in the chicken

Sex in birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination has been a long-standing mystery. In the chicken and all other birds, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). Two hypotheses have been proposed for the mechanism of avian sex determination. The W (female) chromosome may carry a dominant-acting ovary determinant. Alternatively, the dosage of a Z-linked gene may mediate sex determination, two doses being required for male development (ZZ). A strong candidate avian sex-determinant under the dosage hypothesis is the conserved Z-linked gene, DMRT1 (doublesex and mab-3-related transcription factor 1). Here we used RNA interference (RNAi) to knock down DMRT1 in early chicken embryos. Reduction of DMRT1 protein expression in ovo leads to feminization of the embryonic gonads in genetically male (ZZ) embryos. Affected males show partial sex reversal, characterized by feminization of the gonads. The feminized left gonad shows female-like histology, disorganized testis cords and a decline in the testicular marker, SOX9. The ovarian marker, aromatase, is ectopically activated. The feminized right gonad shows a more variable loss of DMRT1 and ectopic aromatase activation, suggesting differential sensitivity to DMRT1 between left and right gonads. Germ cells also show a female pattern of distribution in the feminized male gonads. These results indicate that DMRT1 is required for testis determination in the chicken. Our data support the Z dosage hypothesis for avian sex determination.

Sexually Dimorphic MicroRNA Expression During Chicken Embryonic Gonadal Development

Abstract 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.

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.

Characterization and Comparison of Chicken U6 Promoters for the Expression of Short Hairpin RNAs

RNA interference (RNAi) is a powerful method of sequence-specific gene knockdown that can be mediated by DNA-based expression of short hairpin RNA (shRNA) molecules. A number of vectors for expression of shRNA have been developed with promoters for a small group of RNA polymerase III (pol III) transcripts of either mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and future development of specific therapeutics in the chicken species, we have developed shRNA expression vectors featuring chicken U6 small nuclear RNA (snRNA) promoters. These sequences were identified based on the presence of promoter element sequence motifs upstream of matching snRNA sequences that are characteristic of these types of pol III promoters. To develop suitable shRNA expression vectors specifically for chicken functional genomic RNAi applications, we compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and silencing the reporter gene encoding the enhanced green fluorescent protein (EGFP). Plasmids containing one of four identified chicken U6 promoters gave a similar degree of knockdown in DF-1 cells (chicken); although, there was some variability in Vero cells (monkey). Because the chicken promoters were not stronger than the benchmark mouse U6 promoter, we suggest that the promoter sequence and structure is more important in determining efficiency in vitro rather than its species origin.

A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.

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.

Inhibition of Henipavirus infection by RNA interference

Abstract Nipah virus (NiV) and Hendra virus (HeV) are recently emerged zoonotic paramyxoviruses exclusively grouped within a new genus, Henipavirus. These viruses cause fatal disease in a wide range of species, including humans. Both NiV and HeV have continued to re-emerge sporadically in Bangladesh and Australia, respectively. There are currently no therapeutics or vaccines available to treat Henipavirus infection and both are classified as BSL4 pathogens. RNA interference (RNAi) is a process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. Small interfering RNAs (siRNAs) mediate RNAi by inhibiting gene expression of homologous mRNA and our preliminary studies suggest RNAi may be a useful approach to developing novel therapies for these highly lethal pathogens. Eight NiV siRNA molecules (four L and four N gene specific), two HeV N gene specific, and two non-specific control siRNA molecules were designed and tested for their ability to inhibit a henipavirus minigenome replication system (which does not require the use of live virus) in addition to live virus infections in vitro. In the minigenome assay three out of the four siRNAs that targeted the L gene of NiV effectively inhibited replication. In contrast, only NiV N gene siRNAs were effective in reducing live NiV replication, suggesting inhibition of early, abundantly expressed gene transcripts may be more effective than later, less abundant transcripts. Additionally, some of the siRNAs effective against NiV infection were only partially effective inhibitors of HeV infection. An inverse correlation between the number of nucleotide mismatches and the efficacy of siRNA inhibition was observed. The demonstration that RNAi effectively inhibits henipavirus replication in vitro, is a novel approach and may provide an effective therapy for these highly lethal, zoonotic pathogens.

Genetic characterization of three unique operational taxonomic units of Eimeria from chickens in Australia based on nuclear spacer ribosomal DNA.

Coccidiosis of chickens is one of the commonest and economically most important parasitic diseases of poultry worldwide. Given the limitations of traditional approaches, molecular tools have been developed for the specific diagnosis of coccidiosis. Recently, a polymerase chain reaction (PCR)-based capillary electrophoresis (CE) method, employing genetic markers in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA, was established for both analytical and diagnostic purposes. The application of this method to investigate the epidemiology of coccidiosis and genetic structures of Eimeria populations on commercial chicken establishments has discovered genetic variants of Eimeria (i.e., new operational taxonomic units OTU-X, OTU-Y and OTU-Z) which were (based on CE analysis) distinct from those of species of Eimeria identified previously in chickens in Australia. The present characterization of these OTUs, based on their ITS-2 sequences and phylogenetic analyses of selected sequence data, provides first evidence to support that OTU-X represents a population variant of Eimeria maxima, and that OTU-Y and OTU-Z represent cryptic species of Eimeria. Further biological and genetic studies are needed to rigorously test these proposals and establish the specific status of these OTUs and their importance as pathogens in chickens. An understanding of the epidemiology of these population variants or cryptic species in Australia is central to designing and implementing effective vaccination and control strategies.

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

BackgroundRNA 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.ResultsWe 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.ConclusionIn 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.