The isolation and characterisation of two genes required for tip formation in <italic>Dictyostelium discoideum</italic>

Following aggregation of starving Dictyostelium cells into a compacted mound, a tip forms at the mound apex and formation of a fruiting body containing spores ensues. It is well-documented that the tip is formed via the accumulation of prestalk cells to the apex and subsequent protrusion of this region, but the control of this process is poorly understood. In order to identify novel genes required for tip formation, an insertional mutagenesis technique known as restriction enzyme-mediated integration (REMI) was used to create mutants arrested prior to tip formation as tight mounds. Following a screen of five such mutants, two (TIPB and TIPC) were selected for further analysis (Chapter 3). A very low level of prestalk specialisation and an overall reduction and delay in prestalk differentiation occurs within TIPB mounds. Sequence analysis of the disrupted gene showed that the TIPB phenotype is caused by disruption of the previously characterised prestalk-specific tagB gene (Shaulsky et al., 1994) (Chapter 4). This gene is proposed to be required for normal DIF (the stalk cell morphogen) accumulation (Chapter 6). In TIPC, prestalk and prespore differentiation is unaffected prior to tip formation and prestalk cells aggregate to the mound apex. TIPC cells undergo spore cell formation but completely fail to differentiate as stalk cells under in vitro induction conditions. TipC, the disrupted gene, is not expressed in vegetative cells but is strongly expressed during aggregation and tip formation. Sequence analysis of tipC revealed homology to the cytoskeletal protein talin. Hence, tipC is proposed to encode a developmentally-regulated Dictyostelium talin homologue. Consistent with this identity, TIPC cells disaggregated from mounds show a rounded morphology and impaired movement (Chapter 5). Development is believed to be blocked because TIPC cells are unable to undergo the shape changes required for tip formation (Chapter 6)