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First published online 18 January 2006
doi: 10.1242/dev.02234

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Direct regulation of egl-1 and of programmed cell death by the Hox protein MAB-5 and by CEH-20, a C. elegans homolog of Pbx1

Departments of Pediatrics and Molecular Biology, Division of Pediatric Hematology-Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75039-9148, USA.

View larger version (27K): [in a new window]   Fig. 1. ceh-20 mutants have defects in programmed cell death. X indicates cells that die in wild-type animals; X indicates programmed cell deaths of cells that survive in wild type animals; an open circle indicates cells that survive in ceh-20 mutants but undergo programmed cell death in wild-type animals. (A) In the P12 cell lineage of wild-type animals, the P12.a division axis is anterior dorsal left (adl) and posterior ventral right (pvr). (B) The P12 lineages of ceh-20 mutants did not show this evidence of transformation to the P11 fate. mab-5 lineage is from Kenyon (Kenyon, 1986). Cells in which MAB-5 protein is detected are indicated in blue, with data taken from Salser et al. (Salser et al., 1993).

View larger version (133K): [in a new window]   Fig. 2. Transcription of egl-1 correlates with programmed cell death. Nomarski optics (A,C,E) and epifluorescence (B,D,F) of the posterior ventral nerve cord of L3 stage larvae carrying an integrated Pegl-1histone:gfp reporter construct localized to nuclei. The descendants of P11.aaa are reproducibly located immediately posterior to the hypodermal cell P10.p. (A,B) In ced-3; Pegl-1histone:gfp transgenic animals (30 out of 30 animals), egl-1 is expressed in the two cells that undergo programmed cell death in the P11 lineage (P11.aap and P11.aaap). (C,D) In mab-5(n1384); ced-3; Pegl-1histone:gfp transgenic mutants, the egl-1 transcriptional reporter is not expressed (27 of 30 mutants) in P11.aaap, which survives in mab-5 mutants. (E,F) In ceh-20(ay42); ced-3; Pegl-1histone:gfp mutants, the egl-1 reporter is not expressed (58 out of 60 mutants) in P11.aaap, which survives in mab-5 and ceh-20 mutants. The P12 lineage descendants arise in the preanal ganglion, which contains other neuronal cells; consequently, we were unable to identify unambiguously P12.aaap. However, the Pegl-1histone:gfp reporter was expressed in two rather than three cells in the preanal ganglion of mab-5 and ceh-20 mutants.

View larger version (64K): [in a new window]   Fig. 3. lin-39 and ceh-20 are likely to act through egl-1 in the VC neurons. For all genotypes, the expression pattern of the Plin-11gfp reporter was scored in 50 animals. (A,B) The Plin-11gfp reporter is expressed in the six VC neurons of wild-type animals [average of 4.4 cells; two VC neurons (VC4 and VC5) are often obscured by vulval and sex muscle expression, and are not scored] and in the VC-like neurons that survive in an egl-1 mutant (average of 8.7 cells from P2-P12 descendants) (Cameron et al., 2002). (C,E) VC neurons undergo programmed cell death in lin-39 mutants (average of 0 cells). VC and VC-like neurons survive and express Plin-11gfp in lin-39; egl-1; Plin-11gfp mutants (average of 9.0 cells). In lin-39 mutants, the P cell descendants migrate abnormally far forwards (Clark et al., 1993). (D,F) Many VC neurons die in ceh-20(ay9) mutants. In ceh-20(ay9); egl-1; Plin-11gfp mutants, the VC and VC-like neurons survive and express Plin-11gfp but the fluorescence is of variable brightness, especially in the anterior nerve cord (average of 7.4 cells). (G) In ceh-20(ay42); egl-1; Plin-11gfp mutants, the VC and VC-like neurons survive but do not express Plin-11gfp (average of 0 cells). Direct observation of cell lineages in the ceh-20(ay42); egl-1; Plin-11gfp mutant confirmed that the VC neurons did not undergo programmed cell death. Arrowheads indicate nuclei of VC and VC-like neurons.

View larger version (42K): [in a new window]   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.

View larger version (74K): [in a new window]   Fig. 5. A CEH-20/MAB-5 complex binds the Hox/Hox co-factor site at +5995 in egl-1. (A) Electrophoretic mobility shift assays were performed with epitope-tagged CEH-20 and MAB-5 proteins using wild-type and mutant Site 1 32P-labeled oligonucleotides. The mutant oligonucleotide probe contains the NcoI mutation described in Fig. 4C. (B) CEH-20/MAB-5 proteins were used in an in vitro binding reaction with a 32P-radiolabeled oligonucleotide containing Site 1. Cold competitor oligonucleotide was included in the binding reactions at the indicated molar ratios. The NcoI-Mut, HOX-Mut and CEH-20-Mut mutant probes correspond to those in Fig. 4C.

View larger version (136K): [in a new window]   Fig. 6. CEH-20 is expressed in P11.aaap and may not be required for expression of a Pmab-5gfp reporter. DIC (A,C,E) and epifluorescence (B,D,F) images. (A,B) The posterior ventral nerve cord of a late L2 stage animal carrying a rescuing Pceh-20ceh-20:cfp fusion gene. (C,D) Of the 30 otherwise wild-type ced-3 mutants carrying the Pmab-5gfp reporter, 28 and 26 animals expressed gfp in P11.aaaa and P11.aaap, respectively. (E,F) Of the 60 ceh-20(ay42) mutants carrying the Pmab-5gfp reporter, 51 and 50 mutants expressed gfp in P11.aaaa and P11.aaap, respectively.

View larger version (16K): [in a new window]   Fig. 7. Site 1 at position +5995 in egl-1 is required for programmed cell death of P11.aaap and, in some animals, of P12.pp. P11 and P12 cell lineages were observed of seven egl-1 mutants carrying the egl-1 Site1 NcoI mutant transgene. The pattern of programmed cell deaths of this integrated line was representative of those of 13 other transgenic lines, some of which were also integrated, containing the Site 1 NcoI mutation. (A) P11 and P12 cell lineages of wild-type animals. (B) P11 and P12 cell lineages of mab-5 and ceh-20 mutants. (C) P11 and P12 cell lineages of transgenic animals. The fraction of animals displaying each lineage is shown.