Mitochondrial maturation drives germline stem cell differentiation in Caenorhabditis elegans

Abstract

The C. elegans germline recapitulates mammalian stem cell niches and provides an effective platform for investigating key aspects of stem cell biology. However, the molecular and physiological requirements for germline stem cell homeostasis remain largely elusive. Here, we report that mitochondrial biogenesis and function are crucial for germline stem cell identity. We show that general transcription activity in germline mitochondria is highly compartmentalized, and determines mitochondrial maturation. RPOM-1, the mitochondrial RNA polymerase, is differentially expressed as germ nuclei progress from the distal to the proximal gonad arm to form oocytes. Mitochondria undergo changes from globular to tubular morphology and become polarized, as they approach the proximal gonad arm. Notably, this mitochondrial maturation trajectory is evolutionarily conserved. We find that a similar transition and temporal mitochondrial RNA polymerase expression profile characterizes differentiation of mammalian stem cells. In C. elegans, ATP, and ROS production increases sharply during maturation. Impaired mitochondrial bioenergetics causes gonad syncytium tumor formation by disrupting the balance between mitosis and differentiation to oocytes, which results in a marked reduction of fecundity. Consequently, compensatory apoptosis is induced in the germline. Sperm-derived signals promote mitochondrial maturation and proper germ cell differentiation via the MEK/ERK kinase pathway. Germ cell fate decisions are determined by a crosstalk between Insulin/IGF-1 and TGF-β signaling, mitochondria and protein synthesis. Our findings demonstrate that mitochondrial transcription activity determines a shift in mitochondrial bioenergetics, which in turn regulates germline stem cell survival and differentiation. Perturbation of mitochondrial transcription hinders proper germ cell differentiation and causes germline tumor development.

Fig. 1

RPOM-1 depletion causes germline tumor formation in C. elegans. a, b The brood size of rpom-1(RNAi)-treated hermaphrodites is significantly reduced (up to 50%) compared to their control counterparts. Unpaired t-test was used for the estimation of statistical significance (n > 40; ***P < 0.001). c Egg laying measurement of animals treated with control or rpom-1(RNAi) for two subsequent generations. d DAPI staining of day 1 control and rpom-1(RNAi)-treated WT animals. Inhibition of mitochondrial transcription results in germ nuclei arrest in the pachytene region of prophase I in the germline syncytium. The dashed lines surround the germline syncytium. e Phosphorylated histone H3 antibody staining of extruded gonads for the detection of mitotic nuclei in the distal gonad arm. Red dashed lines highlight the border between the mitotic region and the transition zone, marked by the appearance of crescent-shape nuclei, while the red asterisk marks the relative position of the distal gonad arm tip. f Quantification of phosphorylated histone H3 positive germ nuclei in control and rpom-1(RNAi)-treated hermaphrodites. g Representative images of EGG-1 positive oocytes in the proximal arm from control and RPOM-1-depleted gonads. h Quantification of EGG-1 positive oocytes in control and rpom-1(RNAi)-treated hermaphrodites. i Confocal image of day 1 transgenic worms expressing a cell membrane tagged GFP and a histone-54 fused mCherry. Animals treated with rpom-1(RNAi) display a lower number of nuclei in diplotene (compare dashed rectangles) and fewer mature oocytes in diakinesis. −1 denotes the most proximal oocyte. Unpaired t-test was used for the estimation of statistical significance (n > 40; ***P < 0.001). Error bars, s.e.m. Images were acquired using a ×40 objective lens. Scale bars, 20 μm

Fig. 2

Induction of apoptosis alleviates germline tumor development. a Apoptosis induction following rpom-1 downregulation, as monitored using the CED-1::GFP reporter in combination with DIC microscopy. Arrows highlight apoptotic corpses in the syncytium area. b A two-fold induction in the number of early apoptotic corpses can be detected upon RPOM-1 depletion. Hmg-5 and tfbm-1 downregulation also trigger apoptosis (n = 40; ***P < 0.001, one-way ANOVA was used for multiple comparisons). c RPOM-1 depletion in ced-3(n717)/caspase-deficient animals causes pronounced tumor formation, even more severe than in wild-type worms. Heterozygous ced-9(n2812)/BCL-2 animals exhibit no sign of germ nuclei arrest in pachytene. The red dashed lines surround the pachytene region of the gonads. −1 denotes the most proximal oocyte. Images were acquired using a ×40 objective lens. Error bars, s.e.m. Scale bars, 20 μm

Fig. 3

Mitochondrial transcription acts in parallel with signaling pathways converging on the germline. Hoechst 33342 staining of D1 adult animals in control conditions and upon rpom-1 silencing. a, b Inhibition of mitochondrial transcription causes pachytene arrest in otherwise wild-type animals. In contrast, treatment of daf-2/ IGFR (c, d) and daf-1/ TGFR (e, f) homozygous mutants with rpom-1(RNAi) at 20 °C produces dwarf gonads and augments their reported defects. Mutants with attenuated protein synthesis rates, such as rsks-1/ S6K (g, h) and ife-5/eIF4E (i, j) are indistinguishable from their control counterparts upon treatment with rpom-1(RNAi). k, l Glp-1/ Notch loss of function produces germline-less animals at restrictive temperatures. The red dashed lines indicate the border between the mitotic region and the transition zone. m Egg laying measurements in wild-type, daf-2(e1370), rsks-1(ok1255) and ife-5(ok1934) mutants. n Quantification of the number of germ nuclei reaching diakinesis in wild-type, daf-2(e1370), daf-1(m40), rsks-1(ok1255) and ife-5(ok1934) genetic backgrounds. (n > 40; ***P < 0.001, unpaired t-test). Error bars, s.e.m. Images were acquired using a ×40 objective lens. Scale bars 20 μm

Fig. 4

Rpom-1 expression is compartmentalized. a RPOM-1 abundance (in green) is low in the distal arm and the mitotic region of the gonads, becomes evident at the onset of the pachytene region (arrowhead) and substantially increases close to the turn and in the proximal arm, where the oocytes mature. LAG-2::myr::tdTomato (in red) marks the distal tip cell (white star) and its membrane projections. b A reporter strain overexpressing RPOM-1 under the control of its endogenous promoter and 3′UTR reveals higher expression in the oocytes and lower in the syncytium. Ds; distal, pr; proximal, −1 denotes the most proximal oocyte. Images were acquired using ×40 and ×63 objective lenses. Scale bars, 20 μm

Fig. 5

Transition from globular to tubular mitochondria is a prerequisite for germline homeostasis. a Confocal image of an adult C. elegans gonad. In the distal arm, mitochondria have a globular shape (arrow), which gradually switches to a more elongated/ tubular one in the oocytes of the proximal arm (arrowhead). b The turn of the gonad, shown in magnification, is the site where the shape alteration occurs. There, both globular (arrow) and tubular (arrowhead) mitochondria can be observed. c DAPI staining of wild-type and fzo-1(tm1133)/Mitofusin homozygous mutants. The red dashed line marks the gonad turn. d Day 1 adult fzo-1(tm1133)/Mitofusin mutant animals, bearing defects in mitochondrial fusion, produce significantly fewer germ nuclei in diplotene as well as nuclei in diakinesis compared to their control counterparts. (n > 40; ***P < 0.001, unpaired t-test). e Quantification of mitochondrial length in the proximal and distal arm of the gonads. f Animals with perturbed mitochondrial dynamics (fusion-fission), such as fzo-1, drp-1 and eat-3 mutants become sterile when exposed to a mild heat stress (25 °C). One-way ANOVA was used for the estimation of statistical significance (n > 40; ***P < 0.001). Error bars, s.e.m. Images were acquired using X40 and X63 objective lenses. Ds; distal, pr; proximal, −1 denotes the proximal-most oocyte. Scale bars, 20 μm

Fig. 6

Sperm-derived signals promote mitochondrial maturation. a Mitochondria in the proximal gonad arm are tubular under control conditions (arrowheads). b Knockdown of fzo-1 leads to mitochondrial network fragmentation and globular mitochondria in the proximal gonad arm (arrows). c Inhibition of MPK-1/MAPK signaling via mpk-1(RNAi) results in a failure of mitochondria to elongate proximally (arrows). d Mitochondria are exclusively globular in the proximal arm of gld-1(RNAi)-treated animals (arrows). e GSA-1 inhibition results in failure of oocyte maturation as well as mitochondrial elongation. f Quantification of mitochondrial length in proximal gonad arm oocytes upon the respective RNAi treatments (n = 40; ***P < 0.001, one-way ANOVA was used for multiple comparisons). g DIOC6(3) staining reveals that mitochondria polarize in the course of germ cell differentiation. Inhibition of GOA-1, a negative regulator of sperm signaling, boosts mitochondrial potential in the proximal gonad arm. Treatment with gsa-1(RNAi) results in a failure of mitochondria to polarize proximally. h Quantification of the DIOC6(3) fluorescence per oocyte of the proximal gonad arm. The dashed lines surround the germline syncytium. (n = 40; ***P < 0.001, unpaired t-test). Error bars, s.e.m. Images were acquired using a X40 objective lens. Scale bar, 20 μm

Fig. 7

Mitochondria functionally mature during germ nuclei differentiation. a The ATP/ADP sensor Perceval was overexpressed in the C. elegans germline under the control of pie-1 promoter, to achieve germline-specific expression. Perceval emission increases upon ATP binding. Fluorescence could be mainly detected in the oocytes, indicating increased ATP production in the proximal arm. b DIOC6(3) mitochondrial dye preferentially stains energized mitochondria in the proximal gonad arm. c TMRE staining reveals increased electrochemical potential in the oocytes of the proximal arm. d Mitochondrial ROS production increases as the germ nuclei mature and give rise to oocytes. Arrowheads highlight tubular mitochondria in the proximal gonad arm. Sp; spermatheca, ds; distal, pr; proximal, −1 denotes the most proximal oocyte. Images were acquired using ×40 and ×63 objective lenses. Scale bars, 20 μm

Fig. 8

Mammalian stem cell differentiation upon LIF removal is accompanied by an increase in POLRMT expression. Elevated POLRMT expression is observed in cells with increased cytoplasmic OCT-4 abundance (arrowheads). In contrast, adjacent areas with increased nuclear OCT-4 abundance (stars) display lower POLRMT expression. Images were acquired using a ×40 objective lens. Scale bars, 20 μm

Fig. 9

Intact mitochondrial bioenergetics safeguards germline homeostasis. Expression of rpom-1 (the nematode orthologue of POLRMT) increases progressively as the germ nuclei mature and mitochondria acquire an elongated, tubular shape. The boost in mitochondrial metabolic activity is manifested by enhanced ATP and ROS production, as well as increased electrochemical potential in the proximal gonad arm. Mitochondrial maturation is under the control of MPK-1/MAPK and MSP signaling pathways