Progress 10/01/03 to 09/30/07
Outputs
OUTPUTS: Funding allowed the mentoring of one undergraduate and two Ph.D. students. Results were presented in various meetings, including the Madison Zebrafish Biology meeting, the Madison Cellularization meeting and the 2006 International Zebrafish Biology Strategic Initiative meeting.
PARTICIPANTS: Individuals Elizabeth Theusch, undergraduate student, worked on the analysis of germ plasm RNAs in the early embryo and the involvement of cytoskeletal rearrangements in their segregation Kimberly Brown, Ph.D. candidate, determined compartmentalized nature of germ plasm and studied function of dazl in embryo Emily Putiri, Ph.D. candidate, studied mechanisms of segregation conserved between germ plasm components and other maternal determinants
TARGET AUDIENCES: Investigators in the fields of germ plasm segregation and germ cell determination
PROJECT MODIFICATIONS: Our studies determined that there is no obvious effects of reducing the function of dazl in germ cell development. In addition, we discovered new mechanisms of RNA segregation and the global cytoskeletal rearrangements that drive them. We therefore increased our focus on the analysis of the subcellular mechanisms required for the segregation of dazl RNA and other germ plasm and maternal mRNAs
Impacts
We have analyzed in detail the functional requirements for the segregation of the zebrafish germ plasm during the early cleavage divisions, which involves the recruitment of germ plasm components to the forming furrows of the first and second cell cleavage cycles and the subsequent compaction of the recruited germ plasm during furrow maturation. Because in the zebrafish the segregation of germ cell fate determinants is intimately linked to the process of cellular division, we have also focused on the analysis of genes and subcellular events required for cytokinesis. Our studies have involved on one hand the analysis of subcellular events necessary for cytokinesis and germ plasm segregation, such as cytoskeletal and motor requirements, and on the other, the molecular genetic analysis of maternal-effect genes required for these processes. We previously found that, while some germ plasm mRNAs, such as nanos, dead end and vasa, are originally present in the animal cortex
and are recruited to the forming furrow through the mechanisms described above, other mRNAs such as dazl are originally localized to the vegetal pole of the egg and are recruited to the germ plasm through an animally-directed cortical movement (Theusch et al., 2006). Remarkably, mRNA germ plasm components that are recruited through each of these two separate mechanisms eventually occupy different regions of the germ plasm, and this arrangement persists during the cleavage stages. The subcompartmentalized germ plasm remains oriented in a stereotypic orientation during until the 32 cell stage, with mRNA germ plasm components originally present in the animal pole of the egg in the center of the blastodisc, and dazl mRNA in more distal regions of the embryo. We have done a time course of these compartmentalized germ plasm structure using double fluorescent hybridization and find that, at the 64 cell stage, the structure appears to split into 2 parts, one containing the animal mRNAs and
one the dazl RNA. Our studies suggest that the compartmentalization of the germ plasm may be involved in either the subcellular transport of the mRNA during the cleavage stages or, alternatively, may have a role in germ cell determination. We have also studied germ plasm segregation in a number of situations where early cell division is affected. In particular, we tested the recruitment of germ plasm mRNAs to the furrow in mutants affected in the gene cellular island, which we have shown encodes the zebrafish aurora B kinase homologue. Interestingly, germ plasm mRNAs do not become recruited to the furrow in this mutant, even though some events that occur in furrow initiation, such as the formation of the furrow microtubule array, appear normal in the mutant. This suggests a specific role for cellular island/aurora B kinase in the recruitment of germ plasm components to the furrow.
Publications
- Theusch, E.V., Brown, K.J., Pelegri, F. (2006) Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm, Developmental Biology 292, 129-141.
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Progress 01/01/06 to 12/31/06
Outputs
We have analyzed in detail the functional requirements for the segregation of the zebrafish germ plasm during the early cleavage divisions, which involves the recruitment of germ plasm components to the forming furrows of the first and second cell cleavage cycles and the subsequent compaction of the recruited germ plasm during furrow maturation. Because in the zebrafish the segregation of germ cell fate determinants is intimately linked to the process of cellular division, we have also focused on the analysis of genes and subcellular events required for cytokinesis. Our studies have involved on one hand the analysis of subcellular events necessary for cytokinesis and germ plasm segregation, such as cytoskeletal and motor requirements, and on the other, the molecular genetic analysis of maternal-effect genes required for these processes. Through the visualization of germ plasm mRNA components in relation to cytoskeletal networks, as well as drug inhibition studies, we
have found that global changes in the cytoskeleton during the first cell cycle facilitate the recruitment of germ plasm components to the forming furrow (Theusch et al., 2006). In addition, we have found that, while some germ plasm mRNAs, such as nanos, dead end and vasa, are originally present in the animal cortex and are recruited to the forming furrow through the mechanisms described above, other mRNAs, such as daz-l, are originally localized to the vegetal pole of the egg and are recruited to the germ plasm through an animally-directed cortical movement (Theusch et al., 2006). Remarkably, mRNA germ plasm components that are recruited through each of these two separate mechanisms eventually occupy different regions of the germ plasm, and this arrangement persists during the cleavage stages. Our studies suggest that separate pathways of segregation may have evolved to facilitate the compartmentalization of the germ plasm, and that this compartmentalized structure may have a function
in germ cell determination. We have also studied germ plasm segregation in a number of situations where early cell division is affected. The mutation cellular atoll affects a centriolar component and leads to one-cycle delays in the early cell divisions, thus allowing us to uncouple cell division count and developmental time in the early embryo. We have found that germ plasm recruitment fails to be recruited at the delayed second cleavage furrow, indicating a time window that is optimal for germ plasm recruitment that corresponds to the first and second cell cycles. We are currently using fluorescent in situ hybridization in order to study germ plasm segregation under these altered conditions at the subcellular level. Using cea mutant embryos, we have also found a correlation between a delay in the recruitment of germ plasm mRNA and the enrichment of endoplasmic reticulum (ER) at the furrow, which suggests that these two processes might be coupled during cell division. We are
currently further exploring the dependence of mRNA localization on ER redistribution in the early embryo.
Impacts
The gene deleted in Azoospermia has been found to be important for the development and differentiation of germ cells in a variety of organisms, including humans. The careful analysis of the segregation and expression pattern of this gene in a genetic model organism such as the zebrafish should provide important insights into our understanding of the molecular events required for the formation of the germ line. The knowledge we gain in this system may be applicable to studies in fertility in humans and livestock, as well as in the formation and maintenance of stem cell lines.
Publications
- Theusch, E.V., Brown, K.J. and Pelegri, F. (2006). Separate pathways of RNA recruitment lead to the compartmentalization of the zebrafish germ plasm. Developmental Biology, 292, 129-141.
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Progress 01/01/05 to 12/31/05
Outputs
Our work has shown that the mRNA for the gene Deleted in Azoospermia (DAZ) is recruited in regions of the early embryo, specifically in the forming early cellular furrows, that contain the zebrafish germ plasm, a specialized cytoplasm that confers the germ cell fate. Other mRNAs thought to be important in germ cell development, for the genes nanos, vasa and dead end, are also located in the same region. We have carried out careful time course analysis using whole mount in situ hybridization to detect the DAZ mRNA in the early embryo and its movement from its original vegetal location in the oocyte to the site of furrow formation in the animal region of the embryo. Careful comparison of the localization pattern of other mRNAs components of the germ plasm during these same stages have revealed multiple pathways used for localization. In addition, we have tested the cytoskeletal requirements for these segregation movements of the DAZ mRNA, again in comparison with those
of other mRNAs components of the germ plasm, during these early stages. by testing the effect of drugs that destabilize or stabilize microfilament or microtubule networks on the mRNA localization patterns. We have found that different mRNA components are initially present in different regions of the eggs, and localize to the germ plasm by different mechanisms. We have also shown a requirement of the microfilament network in the local enrichment of some of these mRNAs around the first cell during the first cellular cycle, and a requirement for a microtubule-based network in the aggregation of these enriched mRNAs. Our data also supports a model in which these aggregates are further recruited at the forming furrow by their movement along the cortex of the egg. Furthermore, one of the mRNAs, for the gene DAZ, appears to become recruited to the germ plasm in the furrow after all other components have been incorporated in the germ plasm. These two pathways result and likely facilitate the
recruitment of germ plasm components in different regions of the germ plasm. showing for the first time a compartmentalized nature of this important cytoplasmic structure. We are currently pursuing the dynamics and role of this compartmentalization during the early cleavage stages. These data reveal complex patterns of segregation of various germ plasm determinants and novel roles for cytoskeletal networks in these movements.
Impacts
The gene Deleted in Azoospermia has been found to be important for the development and differentiation of germ cells in a variety of organisms, including humans. The careful analysis of the segregation and expression pattern of this gene in a genetic model organism such as the zebrafish should provide important insights into our understanding of the molecular events required for the formation of the germ line. The knowledge we gain in this system may be applicable to studies in fertility in humans and livestock, as well as in the formation and maintenance of stem cell lines.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Analysis of the early segregation of DAZ-like mRNA within the germ plasm. Our work has shown that the mRNA for the gene Deleted in Azoospermia (DAZ) is recruited in regions of the early embryo, specifically in the forming early cellular furrows, that contain the zebrafish germ plasm, a specialized cytoplasm that confers the germ cell fate. Other mRNAs thought to be important in germ cell development, for the genes nanos, vasa and dead end, are also located in the same region. We have carried out careful time course analysis using whole mount in situ hybridization to detect the DAZ mRNA in the early embryo and its movement from its original vegetal location in the oocyte to the site of furrow formation in the animal region of the embryo. Careful comparison of the localization pattern of other mRNAs components of the germ plasm during these same stages have revealed multiple pathways used for localization. In addition, we have tested the cytoskeletal requirements for
these segregation movements of the DAZ mRNA, again in comparison with those of other mRNAs components of the germ plasm, during these early stages. We have shown a requirement of the microfilament network in the local enrichment of some of these mRNAs around the first cell during the first cellular cycle, and a requirement for a microtubule-based network in the aggregation of these enriched mRNAs. Our data also supports a model in which these aggregates are further recruited at the forming furrow by their movement along the cortex of the egg. The mRNA for the gene DAZ, appears to become recruited to the germ plasm in the furrow after all other components have been incorporated in the germ plasm. These data reveal complex patterns of segregation of various germ plasm determinants and novel roles for cytoskeletal networks in these movements.
Impacts
The gene Deleted in Azoospermia has been found to be important for the development and differentiation of germ cells in a variety of organisms, including humans. The careful analysis of the segregation and expression pattern of this gene in a genetic model organism such as the zebrafish should provide important insights into our understanding of the molecular events required for the formation of the germ line. The knowledge we gain in this system may be applicable to studies in fertility in humans and livestock, as well as in the formation and maintenance of stem cell lines.
Publications
- No publications reported this period
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