Bullock, Simon Lee; (1999) A genetic requirement for glycosaminoglycans during organogenesis revealed by a gene trap mutation in the gene encoding heparan sulphate 2-sulphotransferase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The acquisition of pattern and form by metazoan organisms relies on highly orchestrated cell-cell communications. In an attempt to learn more about the molecules that are required for these processes, a gene trap screen has been employed that is designed to identify, mutate and report on the embryonic expression pattern of genes expressed in mouse embryonic stem cells. This thesis deals with the initial characterisation of gene trap integrations from this screen and the detailed analysis of one of these insertions, termed ST125. The gene disrupted in this cell line encodes the previously unidentified mouse heparan sulphate 2-sulphotransferase (HS2ST). This enzyme catalyses a specific modification of the glycosaminoglycan chains of heparan sulphate proteoglycans (HSPGs), a class of molecule that has recently been implicated in the reception of a number of secreted signalling molecules important for development. Transgenic mice have been generated from embryonic stem cells harbouring this insertion. lacZ reporter gene activity in heterozygous embryos has demonstrated that the gene is expressed differentially during embryogenesis, presumably directing dynamic changes in heparan sulphate structure. Moreover, mice homozygous for the Hs2st gene trap allele die perinatally and exhibit bilateral renal agenesis and defects of the eye, skeleton and female genital system. Analysis of metanephric kidney development in Hs2st mutants reveals that the gene is not required for two early events; ureteric bud outgrowth from the Wolffian duct and initial induction of the metanephric mesenchyme. However, it is required subsequently for condensation of the mesenchyme around the ureteric bud and initiation of branching morphogenesis. Finally, the results of in vitro manipulations of Hs2st mutant kidneys are consistent with a crucial role for HSPGs in intercellular signalling during metanephric development. These data provide the first genetic evidence that specific glycosaminoglycan structures are required for highly selective morphogenetic events, presumably by facilitating the action of specific signalling molecules.

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