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.

Public Health Relevance

Germ line cell determination is essential to produce gametes and for reproduction. In addition, germ line genes have been implicated in the induction of pluripotency and tumorigenesis. Here we propose to study in detail cellular mechanisms involved in the segregation of specialized determinants that confer the germ cell fate. Such knowledge may allow the manipulation of cellular processes leading to cell fate determination, and will provide advances relevant to reproductive, regenerative, and cancer biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065303-10A1
Application #
8639222
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Gindhart, Joseph G
Project Start
2002-03-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
10
Fiscal Year
2014
Total Cost
$286,953
Indirect Cost
$94,217
Name
University of Wisconsin Madison
Department
Genetics
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Pelegri, F; Mullins, M C (2016) Genetic screens for mutations affecting adult traits and parental-effect genes. Methods Cell Biol 135:39-87
Eno, Celeste; Solanki, Bharti; Pelegri, Francisco (2016) aura (mid1ip1l) regulates the cytoskeleton at the zebrafish egg-to-embryo transition. Development 143:1585-99
Ge, Xiaoyan; Grotjahn, Danielle; Welch, Elaine et al. (2014) Hecate/Grip2a acts to reorganize the cytoskeleton in the symmetry-breaking event of embryonic axis induction. PLoS Genet 10:e1004422
Welch, Elaine; Pelegri, Francisco (2014) Cortical depth and differential transport of vegetally localized dorsal and germ line determinants in the zebrafish embryo. Bioarchitecture 5:13-26
Nair, Sreelaja; Marlow, Florence; Abrams, Elliott et al. (2013) The chromosomal passenger protein birc5b organizes microfilaments and germ plasm in the zebrafish embryo. PLoS Genet 9:e1003448
Nair, Sreelaja; Lindeman, Robin E; Pelegri, Francisco (2013) In vitro oocyte culture-based manipulation of zebrafish maternal genes. Dev Dyn 242:44-52
Nguyen, Phuong A; Ishihara, Keisuke; Wuhr, Martin et al. (2012) Pronuclear migration: no attachment? No union, but a futile cycle! Curr Biol 22:R409-11
Lindeman, Robin E; Pelegri, Francisco (2012) Localized products of futile cycle/lrmp promote centrosome-nucleus attachment in the zebrafish zygote. Curr Biol 22:843-51
Nair, Sreelaja; Pelegri, Francisco J (2011) Practical approaches for implementing forward genetic strategies in zebrafish. Methods Mol Biol 770:185-209

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