Parthenogenesis, the ability to generate viable diploid offspring from a single organism, allows the direct homozygosis (in a single generation) of alleles present in the heterozygous form in a single individual. This potentially provides to the zebrafish model a power similar to that of the hermaphroditism-based C. elegans genetics. Such ability would be particularly useful for the analysis of adult traits of biomedical relevance, which are difficult to approach using forward genetics based solely on natural crosses. Current gynogenesis methods appear to be limited by the fact that they involve physical treatments to inhibit cell division, which are only partially effective and also result in a large fraction of developmental abnormalities. The objective of this proposal is to develop an efficient genetically-based method of gynogenesis (parthenogenesis from a female). We have found that paternal dysfunction in the centriolar biogenesis gene cellular atoll/sas-6 (cea) causes a one-cycle delay in the first embryonic cell division, presumably because the sperm provides a single centriole instead of the normal two-centriole component. This first cell division delay results in the exact duplication of the genome. When coupled to haploid production, cea-dependent genome duplication promotes gynogenetic development. Ploidy duplication based on a single-centriole paternal component has the potential to promote gynogenesis with a high efficiency and in the absence of embryonic syndromes. The objective of the proposed research is to optimize such Single Centriole-mediated Gynogenesis. We will attempt this through multiple approaches.
In Aim 1 we will test the effect of various genetic backgrounds on the penetrance of the paternal cea phenotype.
In Aim 2, we will use an F1 genetic screen to identify temperature-sensitive mutations that either enhance the paternal-effect cea phenotype or cause on their own a similar first cell division delay.
In Aim 3, we will combine our findings from previous Aims to implement Single Centriole-mediated Gynogenesis with a high efficiency. This combined approach is intended to provide a powerful, easily-applicable and widely- available new method to facilitate forward genetic screens and other genetic manipulations in the zebrafish. This work will also provide genetic entry points into pathways involved in spermatogenesis and early embryogenesis.
The proposed studies will develop a simple, effective and widely-available genetic tool kit to induce ploidy manipulation, specifically gynogenesis, in the zebrafish. The ability to use genetically-mediated gynogenesis will facilitate genetic analysis in this organism by promoting the direct homozygosis of alleles present in the heterozygous form in a single individual. In essence, effective gynogenesis should provide to the zebrafish a similar genetic power to that which hermaphroditism has provided to the Caenorhabditis elegans model system. The application of this technology will particularly impact the genetic analysis of adult traits of biomedical relevance. Knowledge gained on this process in the zebrafish could also be applied to generate similar methods in other vertebrate model systems. In addition, our studies will identify genes involved in spermatogenesis and early embryonic development.