The requirements for LET-60 Ras during later stages of development were determined by means of generating animals mosaic for let-60(sy101sy127), a likely null allele [the strain used has the genotype: ncl-1(e1865) unc-36(e251); let-60(sy101sy127); him-5(e1490); kuEx72(ncl-1+unc-36+let-60+)]. As previously reported, an analysis of hermaphrodites has revealed a requirement for let-60 activity in the sex myoblasts for their terminal positioning (Sundaram et al., 1996). Mosaic animals have also been examined for other properties, revealing several expected and unexpected observations, including a very restricted lethal focus for embryonic expression of the gene. An expectation based on laser ablations and genetic analyses is a requirement for let-60 in the vulval precursor cells. This expectation has been completely met: seven of seven larvae observed at the L3 stage as lacking the array in all six vulval presursor cells as a consequence of two independent losses had a complete failure of induction. Based on hermaphrodites in which the vulval precursor cells were themselves mosaic, let-60 activity is absolutely required for induction of the primary cell fate but not for the secondary fate, in complete agreement with mosaic analyses of let-23 (Simske & Kim, 1995; Koga & Oshima, 1995). An absence of let-60 activity in the germ line results in a failure of germ nuclei to exit pachytene during oogenesis, in agreement with mosaic analysis of the gene encoding MAP kinase (Church et al. 1995). The morphology of the copulatory spicules is affected in males lacking let-60 activity in parts of the cell lineage generating the blast cell B. In contrast, males in which B but not F retains the array have morphologically normal spicules, in agreement with a model proposed by Chamberlin and Sternberg (1994). Animals homozygous for let-60(sy101sy127) die in the first larval stage with a fluid-filled morphology. The cause of death has remained unknown, but the mosaic analysis gave the first indication that it results from an absence of an excretory duct cell, as follows. The lethal focus for embryonic expression of let-60 is very restricted in the cell lineage and exhibits lineal plasticity: the only losses not seen were AB and ABp, but animals with losses in ABpl or ABpr were readily obtained. This paradox was resolved by an examination of the excretory duct cell (normally ABplpaaaapa) in 54 animals with losses in ABpl or ABplp. The nucleolus of this cell was nonNcl in each of these animals, indicating that it had inherited the array and could not have derived from ABpl, as in normal development. A lack of let-60 activity in ABpl versus ABpr appears to result in a switch of cell fates reminiscent of the switch that occurs when ABplpaaaap, normally the mother of the duct cell, is ablated (Sulston et al., 1983). Moreover, animals homozygous for let-60(sy101sy127) lack a duct cell, whereas being homozygous for an activating allele (n1046) of let-60 frequently results in the presence of two duct cell nuclei in one animal. A discrepancy between the lethal focus for let-60 and those (ABplp and part of ABal) for let-23(mn23) (Koga & Oshima) might be explained by different degrees of maternally-provided activities. Chamberlin & Sternberg, Development 120: 2713 (1994); Church et al., Development 121: 2525 (1995); Koga & Oshima, Development 121: 2655 (1995); Simske & Kim, Nature 375: 142 (1995); Sulston et al., Dev. Biol. 100: 64 (1983); Sundaram et al., Development 122: 2823 (1996).

Affiliation:
- Dept. of MCD Biology University of Colorado Boulder, CO 80309-0347