Dachsous (Dchs), an atypical cadherin, is an evolutionarily conserved regulator of planar cell polarity, tissue size and cell adhesion. In humans, DCHS1 mutations cause pleiotropic Van Maldergem ...syndrome. Here, we report that mutations in zebrafish dchs1b and dchs2 disrupt several aspects of embryogenesis, including gastrulation. Unexpectedly, maternal zygotic (MZ) dchs1b mutants show defects in the earliest developmental stage, egg activation, including abnormal cortical granule exocytosis (CGE), cytoplasmic segregation, cleavages and maternal mRNA translocation, in transcriptionally quiescent embryos. Later, MZdchs1b mutants exhibit altered dorsal organizer and mesendodermal gene expression, due to impaired dorsal determinant transport and Nodal signaling. Mechanistically, MZdchs1b phenotypes can be explained in part by defective actin or microtubule networks, which appear bundled in mutants. Accordingly, disruption of actin cytoskeleton in wild-type embryos phenocopied MZdchs1b mutant defects in cytoplasmic segregation and CGE, whereas interfering with microtubules in wild-type embryos impaired dorsal organizer and mesodermal gene expression without perceptible earlier phenotypes. Moreover, the bundled microtubule phenotype was partially rescued by expressing either full-length Dchs1b or its intracellular domain, suggesting that Dchs1b affects microtubules and some developmental processes independent of its known ligand Fat. Our results indicate novel roles for vertebrate Dchs in actin and microtubule cytoskeleton regulation in the unanticipated context of the single-celled embryo.
The zebrafish locus one-eyed pinhead (oep) is essential for the formation of anterior axial mesoderm, endoderm and ventral neuroectoderm. At the beginning of gastrulation anterior axial mesoderm ...cells form the prechordal plate and express goosecoid (gsc) in wild-type embryos. In oep mutants the prechordal plate does not form and gsc expression is not maintained. Exposure to lithium, a dorsalizing agent, leads to the ectopic induction and maintenance of gsc expression in wild-type embryos. Lithium treatment of oep mutants still leads to ectopic gsc induction but not maintenance, suggesting that oep acts downstream of inducers of dorsal mesoderm. In genetic mosaics, wild-type cells are capable of forming anterior axial mesoderm in oep embryos, suggesting that oep is required in prospective anterior axial mesoderm cells before gastrulation. The oep gene is also essential for endoderm formation and the early development of ventral neuroectoderm, including the floor plate. The loss of endoderm is already manifest during gastrulation by the absence of axial-expressing cells in the hypoblast of oep mutants. These findings suggest that oep is also required in lateral and ventral regions of the gastrula margin. The sonic hedgehog (shh).gene is expressed in the notochord of oep animals. Therefore, the impaired floor plate development in oep mutants is not caused by the absence of the floor plate inducer shh. This suggests that oep is required downstream or in parallel to shh signaling. The ventral region of the forebrain is also absent in oep mutants, leading to severe cyclopia. In contrast, anterior-posterior brain patterning appears largely unaffected, suggesting that underlying prechordal plate is not required for anterior-posterior pattern formation but might be involved in dorsoventral brain patterning. To test if oep has a wider, partially redundant role, we constructed double mutants with two other zebrafish loci essential for patterning during gastrulation. Double mutants with floating head, the zebrafish Xnot homologue, display enhanced floor plate and adaxial muscle phenotypes. Double mutants with no tail (ntl), the zebrafish homologue of the mouse Brachyury locus, display severe defects in midline and mesoderm formation including absence of most of the somitic mesoderm. These results reveal a redundant function of oep and ntl in mesoderm formation. Our data suggest that both oep and ntl act in the blastoderm margin to specify mesendodermal cell fates.
Previously, a tissue-specific fate map was compiled for the gastrula stage of the zebrafish embryo, indicating that development subsequent to this stage follows a reproducible pattern. Here it is ...shown that each early zebrafish blastomere normally contributes to a subset of the gastrula and thus gives rise to a limited array of tissues. However, the final contribution that any early blastomere makes to the fate map in the gastrula cannot be predicted because of variability in both the position of the future dorsoventral axis with respect to the early cleavage blastomeres and the scattering of daughter cells as the gastrula is formed. Therefore, early cell divisions of the zebrafish embryo cannot reproducibly segregate determinants of tissue fates.
It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated ...and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.
Reverse genetics in zebrafish Lekven, A C; Helde, K A; Thorpe, C J ...
Physiological genomics,
03/2000, Volume:
2, Issue:
2
Journal Article
Peer reviewed
The zebrafish has become a popular model system for the study of vertebrate developmental biology because of its numerous strengths as a molecular genetic and embryological system. To determine the ...requirement for specific genes during embryogenesis, it is necessary to generate organisms carrying loss-of-function mutations. This can be accomplished in zebrafish through a reverse genetic approach. This review discusses the current techniques for generating mutations in known genes in zebrafish. These techniques include the generation of chromosomal deletions and the subsequent identification of complementation groups within deletions through noncomplementation assays. In addition, this review will discuss methods currently being evaluated that may improve the methods for finding mutations in a known sequence, including screening for randomly induced small deletions within genes and screening for randomly induced point mutations within specific genes.
Descendants of early blastomeres in the zebrafish come to populate distinctive regions of the fate map. We present a model suggesting that the distribution of cells in the early gastrula (the fate ...map stage) results from the passive response of cells to reproducible forces that change the overall shape of the blastoderm just prior to gastrulation. We suggest that one of the morphogenetic changes that accompanies epiboly, the upward doming of the yolk cell into the overlying blastoderm, could be responsible for cell mixing. In support of the model, we show that the timing, extent, and directions of cell mixing in the embryo accurately reflect the expectations of the model. Finally, we show that one portion of the gastrula, a marginal region that later gives rise to many of the mesendodermal derivatives, experiences little cell mixing during the doming process. As a result, this region in the gastrula is populated by the descendants of the subset of the early blastomeres that were originally at the margin. The finding that cytoplasm initially at the edge of the 1-celled blastodisc is transmitted specifically to mesendodermal precursors at the fate map stage raises the possibility that maternal determinants may contribute to initiation of embryonic patterning in the zebrafish embryo.
A recent paper has challenged the prevailing view that zebrafish blastomeres undergo extensive cell mixing and are unrestricted in their cell fates during early development. Here, we offer a model ...that resolves apparent contradictions between studies that have examined the origins of cell fate in the zebrafish embryo. The model reconsiders cell movements during epiboly, and how cell mixing, or the lack of it, can affect the predictability of cell fate.
We describe a simple method for producing androgenetic haploid embryos in zebrafish. Mature eggs are expressed, and the maternal genome is inactivated by irradiation with short-wave ultraviolet light ...prior to in vitro fertilization. We demonstrate the absence of maternal genome in the resultant embryos using assays based on morphology and polymerase chain reaction. This technique for the production of androgenetic haploids will be extremely useful for identifying and recovering mutations as a complement to, or in place of, generating gynogenetic haploids.
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