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Fig. 4. An evolutionarily conserved site in egl-1 is required for
programmed cell death of specific cells in the P11 and P12 lineages.
(A) Light-gray boxes in the egl-1 genes of C.
briggsae and C. elegans indicate regions with evolutionarily
conserved sequences. The dark-gray boxes indicate the egl-1 open
reading frame. Numbered asterisks indicate the locations of four
evolutionarily conserved matches to the TGATNNAT Hox/Hox co-factor consensus.
F23B12.1 encodes a predicted phosphatase that is not present in the C.
briggsae (or C. remanei) egl-1 region. (B)
C. elegans genomic DNA sequence is shown flanked by nucleotide
positions relative to the egl-1 ATG. The positions of candidate
binding sites are indicated. Nucleotides conserved in C. briggsae are
indicated by black boxes. (C) Site 1 at position +5995 from C.
elegans is shown. Mutated nucleotides are underlined. (D) The
percentage of transgenic animals with the indicated number of corpses among
the descendants of specific P cells. The diameters of the spots are
proportional to the percentage of animals with the indicated number of
corpses. Transgenic animals were constructed by biolistic transformation
(Praitis et al., 2001) of
ced-1(e1735); unc-119(ed3); egl-1(n1084n3082) mutants with the 7.6 kb
genomic DNA of C. elegans egl-1 (see Materials and methods).
Wild-type (WT) indicates introduction of wild-type genomic DNA. In general, 15
animals were scored for each independently derived transgenic line and the
data were pooled (11 independent transgenic lines for the wild-type construct;
13 transgenic lines for the Site 1 NcoI construct; and six transgenic
lines for the others). The F23B12.1 phosphatase was not required for the
effects on programmed cell deaths of transgenic animals, and mutations
affecting sites 2, 3 and 4 did not alter the pattern of programmed cell deaths
in the ventral nerve cord (see Fig. S1 in the supplementary material).
Deletion of the 470 nucleotide evolutionarily conserved region, including Site
1 (sequences 3' of an XhoI site), resulted in a phenotype like
that of mutations in Site 1. (E) DIC (a,c) and epifluorescence (b,d)
images of some of the P11.a descendants of transgenic
egl-1(n1084n3082) mutants carrying either a (a,b) wild-type
Pegl-1histone:gfp reporter or a (c,d) mutant reporter in
which Site 1 was changed to an NcoI site. Thirty out of 30 transgenic
animals with a wild-type reporter expressed gfp in P11.aaap, and 29
of 30 expressed gfp in P11.aap. By contrast, of 90 descendants of
three independent transgenic lines with a Site1 NcoI mutant reporter,
only 11 expressed gfp in P11.aaap, while 83 out of 90 expressed
gfp in P11.aap. P11.aap undergoes programmed cell death in wild-type
animals and in mab-5 and ceh-20 mutants.
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