William C. Skarnes
Wellcome Trust Sanger Institute
Wellcome Trust Genome Campus, Hinxton
Cambridge, CB10 1SA
1998 Searle Scholar
Mechanisms of Early Mouse Development
We endeavor to understand the molecular and cellular mechanisms that underlie early mouse development. Using a gene trap approach in mouse embryonic stem cells, we are searching for genes expressed during gastrulation that are critical for the formation and patterning of the primary germ layers of the developing mouse embryo.
Cell:cell interactions are predicted to play a central role in establishing the body axis of the mammalian embryo. This prediction is based on the fact that the entire embryo develops from a small group of functionally equivalent progenitor cells of the inner cell mass in the absence of determinants known to initiate pattern in the unfertilized egg of other vertebrates and invertebrates. Mouse embryonic stem (ES) cells, derived from the inner cell mass, may be used to introduce random insertional mutations in the genome of mice, providing a unique opportunity to carry out genetic screens for genes important in normal mouse development and physiology. In addition, ES cells may be differentiated in culture into a wide variety of cell types thus offering a potential in vitro model system to define the biochemical, cellular and genetic pathways involved in the early steps of embryonic development.
Screen for cell surface molecules important for gastrulation
To focus on molecules involved in cell:cell interactions, we have modified the gene trap approach to selectively capture genes encoding membrane and secreted proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to produce an active beta-galactosidase fusion gene.
In producing beta-gal fusions, gene trap insertions create mutations by interrupting the coding sequence of the endogenous gene at the site of vector insertion. Furthermore, a portion of endogenous gene may be cloned directly from the fusion transcript and expression of the tagged gene may be followed by monitoring beta-gal activity in embryos. Thus far, we have generated insertional mutations in three cell adhesion molecules, two extracellular matrix components, a receptor tyrosine kinase and two receptor-linked protein tyrosine phosphatases. The identity of the first eight genes trapped by this method has proven the validity of the approach and has encouraged us to proceed with a large-scale screen currently in progress. ES cells carrying secretory trap insertions will be introduced into the germline of mice and each line will be examined for patterns of _gal expression and phenotypes that perturb normal gastrulation. From a large screen we hope to identify genes critical for the formation and subsequent diversification of the primary germ layers.
Formation and patterning of somites
Somites are the most obvious segmented structure of the early vertebrate embryo from which a wide variety of cell types emerge, notably cells of the vertebral column, axial and limb muscles and dermis. Somites arise from a condensation of paraxial mesoderm cells located on either side of the neura l tube to form an epithelial ball of cells which is progressively subdivided into regions that give rise to distinct differentiated cell types. Extrinsic factors produced first by the notochord and then by the floorplate of the neural tube are known to be essential for the correct patterning of somites. Our aim is to establish in vitro culture conditions for ES cells that recapitulate the program of events leading to the formation of somitic mesoderm and its subsequent differentiation. Using a variety of growth factors, chemical reagents and extracellular matrix components, ES cell cultures will be assayed for the expression of various somite markers. Once suitable conditions are found, a variety of molecular and biochemical approaches will be used to dissect the signaling pathways important for the formation and patterning of somites.
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