2002 — 2021 |
Pelegri, Francisco J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Cytokinesis and Rna Segregation in Zebrafish Development @ University of Wisconsin Madison
Cytokinesis, the physical process that divides daughter cells, is of fundamental importance to development and growth regulation. In many instances, cytokinesis is coupled with the asymmetric segregation of cellular determinants, which in turn results in the functional diversification of cell types. During the systematic identification of maternal-effect mutations in the zebrafish, two mutations, in the genes aura and nebel, were identified which affect both of these processes. Mutations in these genes result in embryonic lethality due to defects in cell adhesion and membrane deposition. In both cases, the basis of this phenotype is a defect in the completion of cytokinesis, possibly in the addition or stabilization of new membrane at the cleavage plane. In addition, both of these mutations affect different steps in a pathway of segregation of the vasa RNA, a component of a specialized cytoplasm that confers the germ cell fate. Both types of defects may be based on the inability of the cytoskeleton to undergo specific rearrangements required for its function: aura-dependent microfilament rearrangements at multiple stages during early cleavage and nebel-dependent microtubule reorganization at the forming furrow. The proposed research will determine the precise roles of these genes in the process of cytokinesis and the segregation of the vasa RNA. For this purpose we will use cell biological techniques to visualize cellular components in wild-type and mutant embryos and inhibitor studies to determine the role of subcellular processes or networks. In addition, we will determine the molecular identity of the affected genes through a positional cloning approach. The understanding of these processes will provide insights on a variety of poorly understood subjects: i) the processes involved in the completion of cytokinesis, ii) the asymmetric segregation of RNA and other products within dividing cells, and iii) the functional relationship between the cellular mechanisms required for these two processes.
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2011 — 2012 |
Pelegri, Francisco J |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Single Centriole-Mediated Gynogenesis in the Zebrafish @ University of Wisconsin-Madison
DESCRIPTION (provided by investigator): 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. PUBLIC HEALTH RELEVANCE: 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.
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2014 |
Pelegri, Francisco J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Diversity Supplement: Cytokinesis and Rna Segregation in Zebrafish Development @ University of Wisconsin-Madison
DESCRIPTION (provided by applicant): Maternal inheritance of germ plasm ribonucleoparticles (GP RNPs) results in the activation of a conserved gene expression program for primordial germ cell specification, and we use the zebrafish as a vertebrate model system to study this process. Zebrafish maternally inherited GP RNPs have co-opted the cytoskeletal machinery to reach progressive levels of multimerization: aggregation prior to furrow initiation (pre- aggregation), recruitment to the furrow during its initiation, and distal compaction along th furrow undergoing maturation. These sequential processes result in the formation of four large masses of aggregated GP RNPs, which will eventually confer germ cell specification. The overarching hypothesis of this proposal is that increases in GP RNP multimerization prior to and during furrow formation are based on actomyosin-dependent rearrangements of the cytoskeleton, mediated by myosin II motors associated with GP RNPs. We hypothesize that rearrangements leading to various stages of this process, pre-aggregation, furrow recruitment and distal compaction, have a common underlying mechanistic basis. We will test models of actomyosin interactions that may result in GP RNP multimerization prior to and during furrow initiation (Aim 1) and during furrow maturation (Aim 2). We further hypothesize that these underlying mechanisms are modified differently to produce different cellular outputs. In Aim 1, we will test the hypothesis that, prior to and during furrow initiation, a GP RNP- associated f-actin network is modified by outwardly growing astral microtubules, a process that is coupled to the global activation of GP RNP-associated myosin II. In Aim 2, we will test the hypothesis that, during furrow maturation, slow calcium waves associated with cytokinesis confer a medial-to-distal bias of myosin II activity and/or cytoskeletal dynamics that result in GP RNP compaction at distal ends of the furrow. Our findings will provide insights into novel fundamental mechanisms for the segregation of cell fate determinants. Recent studies have shown a link between germ line genes and pluripotency and tumorigenicity, and understanding mechanisms of germ plasm segregation will provide knowledge applicable to reproductive, regenerative and cancer biology.
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