Transcriptional upregulation of both egl-1 BH3-only and ced-3 caspase is required for the death of the male-specific CEM neurons

Abstract

Most of the 131 cells that die during the development of a Caenorhabditis elegans hermaphrodite do so approximately 30 min after being generated. Furthermore, in these cells, the pro-caspase proCED-3 is inherited from progenitors and the transcriptional upregulation of the BH3-only gene egl-1 is thought to be sufficient for apoptosis induction. In contrast, the four CEM neurons, which die in hermaphrodites, but not males, die approximately 150 min after being generated. We found that in the CEMs, the transcriptional activation of both the egl-1 and ced-3 gene is necessary for apoptosis induction. In addition, we show that the Bar homeodomain transcription factor CEH-30 represses egl-1 and ced-3 transcription in the CEMs, thereby permitting their survival. Furthermore, we identified three genes, unc-86, lrs-1, and unc-132, which encode a POU homeodomain transcription factor, a leucyl-tRNA synthetase, and a novel protein with limited sequence similarity to the mammalian proto-oncoprotein and kinase PIM-1, respectively, that promote the expression of the ceh-30 gene in the CEMs. On the basis of these results, we propose that egl-1 and ced-3 transcription are coregulated in the CEMs to compensate for limiting proCED-3 levels, which most probably are a result of proCED-3 turn over. Similar coregulatory mechanisms for BH3-only proteins and pro-caspases may function in higher organisms to allow efficient apoptosis induction. Finally, we present evidence that the timing of the death of the CEMs is controlled by TRA-1 Gli, the terminal global regulator of somatic sexual fate in C. elegans.

Fig. 1. Cloning of unc-86(bc151), lrs-1(bc155) and unc-132(bc159)

(A) The gene dpy-17 used for mapping bc151 is indicated on the genetic map (LG III). The two mutations identified in the C30A5.7/unc-86 gene in bc151 animals are indicated in red. (B) Genes and SNPs used for mapping bc155 on LG III are indicated on the genetic map. Cosmids and fosmids tested for bc155 rescue are shown below the genetic map. The bc155 mutation in R74.1/lrs-1 is indicated in red. (C) dpy-11 and H10D18:28865 used for mapping bc159 on LG V are indicated on the genetic map. Cosmids tested for bc159 rescue are shown below. pBC430 and pBC431 represent rescuing subclones of cosmid W08A12. The bc159 mutation in W08A12.1/unc-132 is indicated in red. (D) Alignment of the protein sequences of UNC-132 (isoform a), D. melanogaster CG30118 and human PIM-1. The alignment was done using the EMBOSS algorithm. Identical amino acids have a black background. Amino acids with similar biochemical properties have a gray background. The blue boxes indicate the P-loop domains and the orange box the kinase domain. The amino acid that is changed as a result bc159 (M200I) is indicated by a red asterisk.

Fig. 2. unc-86, lrs-1 and unc-132 are required for ceh-30 expression in the CEMs in masculinized hermaphrodites

(A) DIC and fluorescence images (P_ceh-30ceh-30∷gfp) of CEM corpses (white arrow heads) or CEMs (white arrows) in wild-type hermaphrodites (+/+), males (XO +/+), sel-10(n1077) hermaphrodites or lrs-1(bc155); sel-10(n1077) hermaphrodites. (B) Summary of data obtained on P_ceh-30ceh-30∷gfp expression. Green bars indicate the percentage of CEMs that were GFP-positive and green numbers above indicate the fraction of CEMs analyzed that were GFP-positive. Blue bars indicate the percentage of CEMs that acquired a corpse-like morphology and blue numbers above indicate the fraction of CEMs analyzed that had a corpse-like morphology. All strains analyzed were homozygous for unc-76(e911) and carried the extrachromosomal array nEx1171 (P_ceh-30ceh-30∷gfp). The wild-type hermaphrodites and males analyzed were homozygous for him-5(e1467ts). The strains unc-86(bc151); sel-10(n1077) and lrs-1(bc155); sel-10(n1077) were also homozygous for dpy-17(e164).

Fig. 3. egl-1 is transcribed in surviving CEMs in masculinized unc-86(bc151), lrs-1(bc155), or unc-132(bc159) hermaphrodites

(A) DIC and fluorescence images (P_egl-1his-24∷gfp) of CEMs (white arrows) or CEM corpses (white arrow heads) in wild-type hermaphrodites (+/+), sel-10(n1077), sel-10(n1077); ceh-30(bc272) or lrs-1(bc155); sel-10(n1077) hermaphrodites. (B) Summary of data obtained on P_egl-1his-24∷gfp expression. Green bars indicate the percentage of CEMs that were GFP-positive and blue bars indicate the percentage of CEMs that acquired a corpse-like morphology. All strains analyzed were homozygous for the integrated P_egl-1his-24∷gfp array bcIs37. The strain ceh-30(bc272); sel-10(n1077) was also homozygous for the integrated array bcIs9. The strains unc-86(bc151); sel-10(n1077) and lrs-1(bc155); sel-10(n1077) were homozygous for dpy-17(e164).

Fig. 4. Analysis of ced-3 transcription in the CEMs

(A) DIC and fluorescence images (P_ced-3gfp) of CEM corpses (white arrow heads) or CEMs (white arrows) in wild-type (+/+), sel-10(n1077), sel-10(n1077); ceh-30(bc272) or lrs-1(bc155); sel-10(n1077) hermaphrodites. (B) Summary of data obtained on P_ced-3gfp expression. Green bars indicate the percentage of CEMs that were GFP-positive and blue bars indicate the percentage of CEMs that acquired a corpse-like morphology. All strains analyzed were homozygous for the integrated P_pkd-2gfp array bcIs9 and carried extrachromosomal arrays of P_ced-3gfp (+/+ [bcEx834, bcEX836], sel-10(n1077) [bcEx839, bcEx840], ceh-30(bc272); sel-10(n1077) [bcEx876], unc-86(bc151); sel-10(n1077) [bcEx870, bcEx871], lrs-1(bc155); sel-10(n1077) [bcEx872, bcEx873], unc-132(bc159); sel-10(n1077) [bcEx874, bcEx875]).

Fig. 5

Genetic pathway of the CEM death. Upper panel. In wild-type hermaphrodites (XX), tra-1 is active in the CEMs, thereby blocking ceh-30. Thus the pro-apoptotic genes egl-1 and ced-3 are active, which promotes CEM death. See text for details. Lower panel. In males (XO) or masculinized hermaphrodites (sel-10(n1077)), tra-1 is not active in the CEMs, which results in the activation of ceh-30, and, consequently, the repression of egl-1 and ced-3. Thus, CEM death is inhibited. See text for details.

Fig. 6. ceh-30, egl-1 and ced-3 expression in embryonic CEMs in wild-type hermaphrodites

Schematic representation of time course analyses of the expression of the reporters P_ceh-30ceh-30∷gfp, P_egl-1his-24∷gfp, and P_ced-3gfp in the ventral left embryonic CEM (CEMVL) in wild-type hermaphrodites. Time (min) is indicated on the left. Three embryos were analyzed. White dots represent the CEM mother cells, green dots indicate CEMs expressing a particular gfp reporter. Green dots surrounded by purple circles indicate CEMs at the time when expression of the gfp reporter was first detected. (See Figure S1 for corresponding DIC and fluorescent images.) Gray dots indicate CEMs lacking reporter expression. Embryos were prepared for 4-D microscopy and lineaged starting at the 2- or 4-cell stage as described in Materials and Methods. Fluorescent stacks were taken every ~20 min after the CEM mother cell was born. No expression was detected for any of the reporters in the CEM mother cell.

Fig. 7. Model of the life-versus-death decision in the CEMs

Top panel. Most cells programmed to die do so ~30 min after being generated. In these cells, proCED-3 protein inherited from the progenitor is sufficient for apoptosis induction in response to egl-1 transcriptional activation. Bottom panel. The CEMs in hermaphrodites die ~150 min after being generated (~470 min after the first cell division). Increasing TRA-1 activity results in decreasing CEH-30 activity, which is required for the de-repression of egl-1 and ced-3 transcription. The activation at the transcriptional level of the ced-3 gene compensates for decreased levels of proCED-3. See text for details.