Luke S. Lambeth

Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads.

DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminising enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds.

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.

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Short hairpin RNA-mediated gene silencing.

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficult-to-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different effector molecule formats, promoters, and vector types, has meant that experiments can be tailored to target specific cell types and minimize cellular toxicities. Through the application of combinatorial RNAi (co-RNAi), multiple shRNA delivery strategies can improve gene knockdown, permit multiple transcripts to be targeted simultaneously, and curtail the emergence of viral escape mutants. This chapter reviews the history, cellular processing, and various applications of shRNAs in mammalian systems, including options for effector molecule design, vector and promoter types, and methods for multiple shRNA delivery.

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.

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.

Characterisation and application of a bovine U6 promoter for expression of short hairpin RNAs

BackgroundThe use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin.ResultsTo develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The promoter was then used to express shRNAs, which resulted in the efficient knockdown of an exogenous reporter gene and an endogenous bovine gene.ConclusionWe have mined data from the bovine genome sequencing project to identify a functional bovine U6 promoter and used the promoter sequence to construct a shRNA expression vector. The use of this native bovine promoter in shRNA expression is an important component of our future development of RNAi therapeutic and transgenic applications in bovine species.

Transgenic Chickens Overexpressing Aromatase Have High Estrogen Levels but Maintain a Predominantly Male Phenotype

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-characterized steroidogenic pathway, which is a multistep 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. Ectopic overexpression of aromatase in male chicken embryos induces gonadal sex reversal, and male embryos treated with estradiol become feminized; however, this is not permanent. To test whether a continuous supply of estrogen in adult chickens could induce stable male to female sex reversal, 2 transgenic male chickens overexpressing aromatase were generated using the Tol2/transposase system. These birds had robust ectopic aromatase expression, which resulted in the production of high serum levels of estradiol. Transgenic males had female-like wattle and comb growth and feathering, but they retained male weights, displayed leg spurs, and developed testes. Despite the small sample size, this data strongly suggests that high levels of circulating estrogen are insufficient to maintain a female gonadal phenotype in adult birds. Previous observations of gynandromorph birds and embryos with mixed sex chimeric gonads have highlighted the role of cell autonomous sex identity in chickens. This might imply that in the study described here, direct genetic effects of the male chromosomes largely prevailed over the hormonal profile of the aromatase transgenic birds. This data therefore support the emerging view of at least partial cell autonomous sex development in birds. However, a larger study will confirm this intriguing observation.

The molecular genetics of avian sex determination and its manipulation

The chicken (Gallus gallus domesticus) has long been a useful model for developmental biologists. The developing avian embryo is easily accessible and fertile eggs are widely available. In addition, the embryo is also amenable to genetic manipulation allowing studies on many important morphological and cellular processes. More recently, the ability to directly manipulate gene expression through the production of transgenic or mutant chicken embryos by viral delivery methods has been useful to analyse gene function in a wide range of tissues, including the developing gonads. Chickens are amniotes and their development closely resembles that of mammals, implying underlying genetic conservation of key pathways, including sex development. Studies of sex determination and gonadal development in this model are providing insight into avian ovarian and testis developmental pathways and their evolution. Indeed, the chicken embryo is a suitable model for the functional analysis of genes implicated in human disorders of sex development, and studies in this model will complement those carried out in mammalian models such as the mouse. In this review we discuss the current knowledge of sex determination and sexual differentiation in avians, using chicken as model. We review how sex chromosomes contribute to this process and provide current information on our understanding of gonadal sexual differentiation at both the cellular and molecular level in the chicken embryo. Finally, we review the methods currently used to investigate the role of genes and signaling pathways during sexual differentiation, and discuss how these methods may contribute to further understanding of vertebrate gonadogenesis. genesis 51:325–336.

Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

BackgroundDespite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29).ResultsRNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue.ConclusionsThis study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29). RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue. This study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.