The cisd gene family regulates physiological germline apoptosis through ced-13 and the canonical cell death pathway in Caenorhabditis elegans

Abstract

Programmed cell death, which occurs through a conserved core molecular pathway, is important for fundamental developmental and homeostatic processes. The human iron-sulfur binding protein NAF-1/CISD2 binds to Bcl-2 and its disruption in cells leads to an increase in apoptosis. Other members of the CDGSH iron sulfur domain (CISD) family include mitoNEET/CISD1 and Miner2/CISD3. In humans, mutations in CISD2 result in Wolfram syndrome 2, a disease in which the patients display juvenile diabetes, neuropsychiatric disorders and defective platelet aggregation. The C. elegans genome contains three previously uncharacterized cisd genes that code for CISD-1, which has homology to mitoNEET/CISD1 and NAF-1/CISD2, and CISD-3.1 and CISD-3.2, both of which have homology to Miner2/CISD3. Disrupting the function of the cisd genes resulted in various germline abnormalities including distal tip cell migration defects and a significant increase in the number of cell corpses within the adult germline. This increased germ cell death is blocked by a gain-of-function mutation of the Bcl-2 homolog CED-9 and requires functional caspase CED-3 and the APAF-1 homolog CED-4. Furthermore, the increased germ cell death is facilitated by the pro-apoptotic, CED-9-binding protein CED-13, but not the related EGL-1 protein. This work is significant because it places the CISD family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery.

Fig. 1

The cisd-1(tm4993) animals displays germline abnormalities. a Left: schematic diagram of the C. elegans adult hermaphrodite gonad; shown is one of two gonads to note the meiotic regions, cell death zone, and location of the distal tip cell (DTC) [20, 61]. Right: representative images of the gonad arm for the N2 wild-type or the cisd-1(tm4993) 1-day old adult hermaphrodite. The oocytes are outlined and the number of oocytes present in the gonad are represented. b Left: Schematic diagram of the C. elegans L4 larvae gonad; shown is one of two gonads to note the gonad structure and location of the DTC. Right: representative images of the gonad arm for the N2 wild-type or the cisd-1(tm4993) L4 larvae hermaphrodite. The cisd-1(tm4993) animal displays a DTC migration (Mig) defect. The dashed line indicates the distal tip cell migration pattern and black half-moon outlines the DTC. c Left: schematic diagram of a 1-day old adult hermaphrodite showing the migration pattern of the DTC (black arrows). Right: representative images of the gonad arm for the N2 wild-type or the cisd-1(tm4993) 1-day old adult hermaphrodite. The cisd-1(tm4993) animal displays a DTC migration (Mig) defect. The dashed line indicates distal tip cell migration pattern and black half-moon outlines the DTC. For a, b and c, the scale bar = 20 µm. d There is a significant decrease in the number of maturing oocytes in the gonad of cisd-1(tm4993) one-day old adult hermaphrodite relative to the N2 wild-type (* indicates P < 0.005, two-tailed unpaired t-test). Three independent experiments for a total of 30 animals were analyzed. e There is a significant increase in the proportion of animals that displayed a Mig phenotype in the cisd-1(tm4993) 1-day old adult hermaphrodite gonad relative to N2 wild-type (* indicates P < 0.0005, two-tailed unpaired t-test). f The average number of progeny produced in the 1- to 5-day old cisd-1(tm4993) adult relative to the N2 wild-type of the same developmental stage. There is a significant decrease in the number of progeny produced by the 1- to 3-day old cisd-1(tm4993) adult relative to N2 wild-type adults (bar indicates P < 0.05, two-way ANOVA, Sidak’s multiple comparisons test). For d, e and f, error bar equals standard deviation.

Fig. 2

Disruption of the cisd-3.1 and cisd-3.2 gene functions lead to germline defects. a Representative images of one gonad arm in cisd-3.1(RNAi), cisd-3.2(RNAi), or cisd-3.1(RNAi); cisd-3.2(RNAi) in N2 wild-type or cisd-1(tm4993) 1-day old adult hermaphrodites. The RNAi control is when the respective genotype is fed HT115 bacteria with empty vector. The knock-down of the cisd-3.1 and/or cisd-3.2 gene function results in germline defects. Defects include abnormal oocyte number (left panel) and DTC migration (Mig) defects (right panel). The oocytes are outlined and the number of oocytes present in the gonad are represented. The dashed line indicates the DTC migration pattern and the black half-moon outlines the DTC. Scale bar = 20 μm. b There is a significant decrease in the number of maturing oocytes in the gonad arm of cisd-3.1(RNAi), cisd-3.2(RNAi) and cisd-3.1(RNAi); cisd-3.2(RNAi) animal relative to the respective control animal (N2 wild-type, black symbol or cisd-1(tm4993), gray symbol on scatter plot), (one-way ANOVA, Dunnett multiple comparison test, bar indicates P ≤ 0.05). c There is a significant increase in DTC migration defects in the cisd-3.1(RNAi), cisd-3.2(RNAi), and cisd-3.1(RNAi); cisd-3.2(RNAi) animal relative to N2 wild-type (one-way ANOVA, Dunnett multiple comparison test, bar indicates P < 0.05). For b and c, at least 30 animals from three independent experiments were examined; error bar equals standard deviation.

Fig. 3

The cisd-1 gene is expressed in the germline. a Illustration of the cisd-1 locus predicted to produce two isoforms from the cisd-1a and cisd-1b transcripts. CRISPR editing was used to produce a GFP transcriptional reporter strain (PM152) that disrupts the cisd-1 gene. The GFP^SEC^3xFlag sequence was inserted into the second exon of cisd-1b at 536 bp from the start site. b Representative images of L4 cisd-1(pnls27) animals and c 1-day old adult cisd-1(pnls27) animals exhibiting GFP expression in the gonad arm. The N2 wild-type animal is shown to communicate the typical autofluorescence (AFL) observed within the intestine of C. elegans. White arrows indicate transcriptional GFP expression in the germline of cisd-1(pnls27) animals. For b and c, the top panel shows fluorescent images and the bottom panel shows a fluorescent image merged with the DIC image; scale bar = 20 µm. Images are representative animals from 30 animals examined.

Fig. 4

The cisd-1(tm4993) animals exhibits an increase in germline cell corpses. a Representative DIC images of one gonad arm with cells undergoing apoptosis in the cisd-1(tm4993) and N2 wild-type animal. The boxed region shows an enlarged area containing the cell corpse(s); the white arrow points to an apoptotic cell appearing as a “button”. Scale bar = 20 µm. b The cisd-1(tm4993) animal has a significantly higher number of apoptotic cells relative to the N2 wild-type as determined by analysis of the gonad using DIC microscopy. c Representative fluorescent microscopy images of the germline for control or cisd-1(tm4993) animals. Reporters were used to mark specific aspects of programmed cell death (ACT-5::YFP, CED-1::GFP, or acridine orange). Shown are the fluorescent reporters individually or merged with the DIC image to better visualize the gonad anatomy. White arrows mark either germ cell corpses with surrounding actin bundles in the early stages of apoptosis (ACT-5::YFP), surrounding cells that have initiated engulfment (CED-1::GFP), or cells positive for cell surface acridine orange staining. Scale bar = 20 µm. d-f Relative to control populations, the cisd-1(tm4993) animals have a significantly higher number of germline apoptotic cells marked by d ACT-5::YFP, e CED-1::GFP positive cells, or f acridine orange. For b, d, e and f, the number of apoptotic corpses within the gonad was quantified in animals from three independent experiments for a total of at least 30 animals (*indicates P < 0.0001, two-tailed unpaired parametric t-test, or Mann–Whitney two-tailed nonparametric test). The error bar indicates standard deviation.

Fig. 5

The cisd-3.1(RNAi) and cisd-3.2(RNAi) animals exhibits an increase in germ cell corpses. a Representative images of the gonad arm of cisd-3.1(RNAi), cisd-3.2(RNAi) or cisd-3.1(RNAi); cisd-3.2(RNAi) in the background of control animal (left panel) or the cisd-1(tm4993) animal (right panel). The ACT-5::YFP fluorescent reporter is shown individually or merged with the DIC image. White arrows point to apoptotic cells within the gonad. Scale bar = 20 µm. b The cisd-3.1(RNAi), cisd-3.2(RNAi) and cisd-3.1(RNAi); cisd-3.2(RNAi) animals have a significantly higher number of cell death corpses relative to the control animal (bar indicates P < 0.001, Kruskal–Wallis test, Dunn’s multiple comparisons test). c The cisd-3.1(RNAi); cisd-1(tm4993), cisd-3.2(RNAi); cisd-1(tm4993) or cisd-3.1(RNAi); cisd-3.2(RNAi); cisd-1(tm4993) animals do not have a significantly higher number of cell death corpses relative to cisd-1(tm4993) animals (one-way ANOVA, Dunnett multiple comparison test). For b and c, the number of apoptotic corpses within the gonad were quantified in animals from three independent experiments for a total of at least thirty animals. Error bar represents standard deviation.

Fig. 6

The number of gonad cell corpses is reduced in the cisd-1(tm4993) animals with disrupted apoptotic core machinery processes. The number of apoptotic germ cells within the gonad was visualized and quantified, using the ACT-5::YFP reporter strain, for the respective animals: control, ced-3(RNAi), ced-9(n1950gf), cisd-1(tm4993), cisd-1(tm4993); ced-3(RNAi) and cisd-1(tm4993); ced-9(n1950gf)). a Representative images of the gonad arm in respective animals. The ACT-5::YFP reporter is shown individually and merged with the DIC image. White arrows point to the apoptotic cells within the gonad. Scale bar = 20 µm. b The knock-down of ced-3 using RNAi reduced the number of cell corpses in the gonad of cisd-1(tm4993) animals. Relative to control animals, the number of cell death corpses within the gonad was significantly reduced in the ced-3(RNAi) animals, indicating effective RNAi of ced-3. c The ced-9(n1950gf) allele reduced the number of cell corpses in the gonad of cisd-1(tm4993) animals. b, c Identical letters indicate groups with no significant differences; different letters indicate P < 0.05 (Kruskal–Wallis, Dunn’s multiple comparison test). The number of apoptotic corpses within the gonad was quantified in animals from three independent experiments for a total of at least 30 animals. Error bar represents standard deviation.

Fig. 7

The number of cell corpses within the gonad is reduced in the cisd animals with disrupted ced-13 function. Using the ACT-5::YFP reporter strain to visualize apoptotic cells, the number of apoptotic cells was quantified in the cisd-1(tm4993), cisd-3.1(RNAi) and cisd-3.2(RNAi) animals with ced-13 knock-down. a Representative images of the gonad arm of control, cisd-1(tm4993), cisd-3.1(RNAi), and cisd-3.2(RNAi) animals with or without knock-down of ced-13 by RNAi. The ACT-5::YFP reporter is shown individually and merged with the DIC image. White arrows point to apoptotic cells within the gonad. Scale bar = 20 µm. b Disruption of ced-13 function by RNAi reduced the number of cell death corpses within the gonad of the cisd-1(tm4993), cisd-3.1(RNAi), cisd-3.2(RNAi) animals. Identical letters indicate groups with no significant differences; different letters indicate P < 0.0001 (Kruskal–Wallis, Dunn’s multiple comparison test). The number of apoptotic corpses within the gonad were quantified in animals from three independent experiments for a total of at least thirty animals. Error bar represents standard deviation.

Fig. 8

Model of CISD function. We propose that the CISD protein family and CED-13 compete to regulate CED-9 function in the process of physiological germline apoptosis. a In the absence of a cellular signal that initiates physiological germline apoptosis, CISD functions in an anti-apoptotic/pro-survival manner. However, it is not known if this is by CISD protein(s) interacting with CED-9 and/or CED-13. b The dysregulation of any member of the cisd gene family results in increased germline apoptosis. This could be through increased CED-13 inhibition of pro-survival CED-9, given that ced-13(RNAi) suppress the increased germline apoptosis observed in animals with disrupted cisd function.