miRNAs in gonadal development
(Derek McBride)
We recently initiated a project to identify miRNAs involved in tissue differentiation i.e. the structural reorganisation and cellular differentiation that occurs during organogenesis. We selected gonadal development as the ideal model system for this investigation, both for its unique features and for our previous experience with this system. The development of the gonads in early embryogenesis has been well characterised in terms of morphology, physiology and gene expression. The genital ridge develops on the ventral surface of the mesonephros and, initially, is considered to be identical in males and females. Within a short period (days 5-9 in chicken, days 10-14 in mouse) the genital ridge develops into an identifiable gonad – a testis in male and an ovary in female. A number of the protein-coding genes that regulate this structural and functional differentiation have been identified and we and others have shown that these are conserved across vertebrates (Reviewed in: Clinton and Haines, 2001;Morrish and Sinclair, 2002). The unique feature of this system is that a single tissue primordium has the capacity to differentiate into one of two possible developmental outcomes – an ovary or a testis. In one tissue the supporting and steroidogenic cells form a spherical structure around the female germ cell and produce a female-profile of steroids. In the other, these cells form tubular structures around the male germ cells and produce a male profile of steroids. We reasoned that during gonadal development the vast majority of miRNAs would be involved with growth and common developmental processes and would be expressed at equivalent levels in males and females, and that only miRNAs that are associated specifically with male/female structural reorganisation and function would be expressed in a sexually dimorphic fashion.
The chick was chosen as the model system for several reasons, in addition to the obvious welfare advantage over mammals. As an amniote, developmental processes in the chick embryo closely mimic those in mammalian species, including mouse and human. It is easily accessible for tissue collection, manipulation and functional studies, and the development of large numbers of synchronised embryos can be generated easily and inexpensively within a short time frame.
For our miRNA profiling analysis we generated miRNA libraries from the developing ovaries and testes. This analysis was extremely successful: we identified a large number of different miRNAs, most of which had not been previously validated in chickens. Over 50% of these were entirely novel miRNAs that have not been reported in any species. As predicted, the vast majority were expressed at similar levels in the developing ovary and testis, with a small number showing sexually dimorphic expression.
Preliminary data
This analysis identified 71 sequences that matched predicted chicken miRNAs with a further 29 sequences matched known miRNAs, previously identified in other species but not predicted for chicken on the basis of current genomic information. Most significantly, we also identified sequences that represent a further 127 novel miRNAs not previously identified in any species. The number of observations of each miRNA sequence were compared in male and female libraries. As predicted the majority (95%) of all miRNAs identified are present at similar levels in male and female libraries, validating both our overall approach and experimental technique. The remaining 5% of sequences are expressed in a sexually dimorphic fashion: up to 19 fold higher in either males or females. Differential expression was initially confirmed by Northern analysis and detailed expression profiles throughout gonadal development and in individual embryonic tissues have been obtained for all sexually dimorphic miRNAs.