Western blot analysis of whole-embryo protein extracts produced from wild type and elav mutants (stage 16) revealed that elav mutant embryos produce a larger amount of Ubx Ia and reduced levels of Ubx IVa compared with wild-type embryos, closely matching the ELAV-dependent changes observed in Ubx mRNA isoforms (Fig. 1C-H and Fig. 3B).
Wild-type and mutant embryos were produced by pair mating, the embryos were then collected for RNA isolation and fixed for in situ hybridization experiments at the specified stages.
Consistent with the phenotype of the corresponding mutant, and that of embryos produced by females expressing antisense RNA targeting pgc (Nakamura et al. 1996; Martinho et al. 2004), we observed a severe reduction in pole cell number in pgc knockdown embryos; pole cells were absent in 20% of embryos and present in reduced numbers in the remaining 80%.
Our results differ from observations of embryos produced by a Tao hypomorphic mutant where reduced numbers of pole cells were present (Sato et al. 2007).
For this reason, we examined Nos accumulation in syncytial and cellular blastoderm stage in both antisense embryos (see supporting information, Figure S1) and embryos produced by two different pgc mutant alleles.
As shown in Figure 1, we see penetrant defects in embryos produced by homozygous tba-1 or594 tba-1 or594homozygous tsb-2(or600 ts) mutand worms raised at 26°C from the L4 stage to adulthood (hereafter called mutant embyyos).
Similarly, injection of trpm7-E/I-MO into WT embryos produced a relatively small exocrine pancreas resembling that in the swd and trpm7 mutants (supplementary material Fig. S2).
Such anomaly of the spindle was previously reported in cleavage embryos produced by FILIA deficient females but the first mitosis was not affected in the corresponding mutant zygotes [23].
Western blot analysis confirmed that embryos produced by either method lacked detectable PLP (Fig. 3A; data not shown), and both sets of embryos exhibited the same phenotypes that we describe below; for ease of presentation we simply refer to these embryos collectively as plp mutants (plp mut).
Given that FCs and FCMs come from a common progenitor pool, we reasoned that the ectopic FC-like cells in ttk mutant embryos might be produced at the expense of FCMs.
