An RNAi screen of Rab GTPase genes in C. elegans reveals that somatic cells of the reproductive system depend on rab-1 for morphogenesis but not stem cell niche maintenance

Abstract

Membrane trafficking is a crucial function of all cells and is regulated at multiple levels from vesicle formation, packaging, and localization to fusion, exocytosis, and endocytosis. Rab GTPase proteins are core regulators of eukaryotic membrane trafficking, but developmental roles of specific Rab GTPases are less well characterized, potentially because of their essentiality for basic cellular function. C. elegans gonad development entails the coordination of cell growth, proliferation, and migration-processes in which membrane trafficking is known to be required. Here we take an organ-focused approach to Rab GTPase function in vivo to assess the roles of Rab genes in reproductive system development. We performed a whole-body RNAi screen of the entire Rab family in C. elegans to uncover Rabs essential for gonad development. Notable gonad defects resulted from RNAi knockdown of rab-1, the key regulator of ER-Golgi trafficking. We then examined the effects of tissue-specific RNAi knockdown of rab-1 in somatic reproductive system and germline cells. We interrogated the dual functions of the distal tip cell (DTC) as both a leader cell of gonad organogenesis and the germline stem cell niche. We find that rab-1 functions cell-autonomously and non-cell-autonomously to regulate both somatic gonad and germline development. Gonad migration, elongation, and gamete differentiation-but surprisingly not germline stem niche function-are highly sensitive to rab-1 RNAi.

Keywords: C. elegans; RNAi; Rab GTPase; gamete differentiation; germline; gonad; stem cell.

Figure 1.. Whole-body rab-1 knockdown leads to large-scale defects in the gonad.

(A) Cartoon showing position of two gonad arms inside of worm body (above) and representative gonad shapes of control (lower left) and rab-1 RNAi gonads (center and right). Vulva shown with V, and location where proximal gonad fails to form in RNAi samples shown with asterisks. (B-D) Representative images of germ cells (yellow; mex-5p::H2B::mCherry::nos-2 3′UTR) (B’-D’) and somatic gonad DTC and sheath cells (magenta; GFP::INX-9) (B’’-D’’). Adults that escaped severe embryonic and larval defects after hatching on rab-1 RNAi (C-C’’, D-D’’) have fewer germ cells and smaller gonads in 4/16 samples compared to adults hatched on L4440 empty RNAi vector (B-B’’), and display turning and gonad growth defects (C-C”) as well as catastrophic gonad defects (D-D”). (E-G) Representative images of strain expressing markers for the DTC (cyan; lag-2p::mNeonGreen::PLC^δPH) (E’-G’) and gonadal sheath cells (magenta; mKate::INX-8) (E’’-G’’). Adults that escaped severe embryonic and larval defects after hatching on rab-1 RNAi (F-F’’,G-G’’) have DTC and sheath cells in the correct relative positions, but with aberrant morphology in 6/12 samples compared to L4440 treated controls (E-E’’). Phenotypes range from growth defects (F-F”) to catastrophic gonad defects (G-G”). (H-I) Single z-slices of images shown in C and G. Arrows show intracellular membrane protein bubbles and asterisks show encapsulation of germ cells by somatic gonad. In B and E, only the distal portion of the gonad is captured in the field of view (see yellow box in A). Gonads outlined in dashed lines. Scale bars = 20 μm. (J) Graph showing percentage lethality and percentage growth defects in larvae exposed to rab-1 RNAi from the L1 stage. Error bars represent standard error of the mean (SEM).

Figure 2.. C. elegans with germline-specific rab-1 RNAi knockdown are smaller, but develop largely normal gonads that produce defective embryos.

(A, D-E) Representative DIC images of young adult MAH23 worms treated with L4440 control or rab-1 RNAi for 48 (A,D) or 72 hours (E). (A, scale bar = 100 μm; D-E, scale bar = 20 μm). (B-C) MAH23 worms treated with rab-1 RNAi (n=24) RNAi have decreased body length (B) and width (C) compared to L4440 treated controls (n=15). (D) A majority (n = 19/21) of rab-1 RNAi treated worms (middle) developed gonads normally with 2/21 having second turn migration defects (bottom). (E) rab-1 RNAi treated (bottom) worms have defective embryogenesis compared to L4440 (top) treated controls. Statistical significance was calculated by unpaired, two-tail Student’s t-tests, error bars represent ±SEM, and **** denotes p-value < 0.0001. Yellow arrows in E denote uterine contents: embryos in the case of L4440 control and abnormal masses in the case of rab-1 RNAi. Gonads outlined in dashed lines (D-E).

Figure 3.. Somatic gonad-specific RNAi knockdown of rab-1 causes gonad growth and migration, vulva, and body size defects by early adulthood.

Representative images of young adult rde-1(ne219) mutants rescued with a lag2p::mNG::PLC^δPH::F2A::rde-1 transgene restoring RNAi function and driving membrane-localized fluorescence protein mNeonGreen in cells that express the lag-2 promoter on empty L4440 vector control (A) and rab-1 RNAi (B) for 48 hours after L1 arrest. Fluorescence merged with a single DIC z-slice (left), fluorescence alone (right, 8 μm maximum intensity projection displayed on a log scale to show DTC and dimmer vulval precursor cells, arrows). Scale bars = 20 μm. Gonads outlined in dashed lines, control uterine lumen outlined in solid black line. Unbroken basement membrane in rab-1 RNAi treated animal bracketed in yellow. (C) Tissue-specific rab-1 RNAi treated worms (n=17) have delayed vulva formation compared to controls (n=16). For controls, 1/16 had not yet completed vulva formation, but 17/17 rab-1 RNAi treated worms had a delay. (D) Tissue-specific rab-1 RNAi treated worms (n=50) have defective DTC migration compared to controls (n=31). One gonad arm scored per worm. Growth of both gonad arms was typically affected, but the deeper DTC under the gut was difficult to score for orientation of turning. All control DTCs completed migration. For rab-1 RNAi, 17/50 had no turn, 7/50 had just a first turn, 9/50 had a misdirected second turn, 4/50 had no extension after the second turn, and 13/50 completed migration. Error bars show Standard Error of the sample proportion in C and D (see Methods). (E-F) Representative 20x brightfield images of young adult control (left) and rab-1 RNAi (right) worms; scale bars = 100 μm). (G-H) Worms treated with rab-1 RNAi (n=20) have decreased body length (G) and width (H) compared to controls (n=20). Mean ± S.E.M graphed in G and H. Statistical significance was calculated by unpaired, two-tail Welch’s t-tests. Length t(21.82)=10.03, p<0.0001, width t(29.39)=5.120, p<0.0001.

Figure 4.. Reproductive system defects persist after prolonged rab-1 RNAi knockdown in specific cells.

Representative images (with gonads outlined in dashed black or white lines) of reproductive-age animals with tissue-specific RNAi in cells expressing a lag2p::mNG::PLC^δPH::F2A::rde-1 transgene restoring RNAi function and driving membrane-localized fluorescence protein mNeonGreen on empty L4440 vector control (A) and rab-1 RNAi (B-C) for 72 hours after L1 arrest. Fluorescence merged with a single DIC z-slice (left), fluorescence alone (middle, maximum intensity projection through slices with mNeonGreen signal). A’ and B’ show insets indicated by yellow boxes. Scale bars = 20 μm. Gonads outlined in black or white dashed lines. Dashed yellow lines in A’ and B’ indicates length of DTC as measured for (D). Arrows (A-C) indicate site of expected vulval formation, showing normal vulva (A), protruding vulva (B) and vulvaless (C) phenotypes. Arrowhead in A indicates embryonic mNG expression. (D-F) Quantification of rab-1 tissue-specific RNAi treated worm defects in DTC growth (D), vulva formation (E), and DTC migration (F) compared to controls. Both gonad arms in the same worm were scored if both were visible. (D) rab-1 RNAi treated worms (n=21) have shorter DTCs than controls (n=18). Welch’s t-test t(36.11)=5.368, p<0.0001, error bars show S.E.M. (E) While 6/6 control samples had normal vulvas, only 1/17 rab-1 RNAi samples had normal vulva, 5/17 had a missing vulva, 10/17 had a protruding vulva, and 1/17 ruptured through its protruding vulva on the slide. Error bars show standard error of the sample proportion (see Methods). (F) While 18/18 control samples had complete DTC migration, only 5/25 rab-1 RNAi samples completed migration, 10/25 failed to make any turns or elongate, 4/25 made the second turn in the wrong direction, and 6/25 made both turns and then failed to extend. Error bars in E and F show standard error of the sample proportion (see Methods).

Figure 5.. RNAi knockdown of rab-1 in specific somatic gonad cells causes germline proliferation and differentiation defects.

Representative images (with gonads outlined in dashed white lines) of DAPI-stained reproductive-age adult rde-1(ne219) mutants rescued with a lag2p::mNG::PLC^δPH::F2A::rde-1 transgene restoring RNAi function in cells that express the lag-2 promoter on empty L4440 vector control (A-A’) and the range of defects observed after tissue-specific rab-1 RNAi (B-C, F-F’) for 72 hours after L1 arrest. A’ and B’ show insets indicated by yellow boxes (A-B). Dividing germ cells, yellow ovals. Dashed green line shows length of progenitor zone as measured for (D). Gametes, when present, are circled in purple (oocytes) and blue (sperm). Embryos circled in orange. Arrows indicate position of expected vulva formation (C and F). Scale bars = 20 μm. (D) Tissue-specific rab-1 RNAi treated worms (n=14) shorter progenitor zones than control (n=12), Welch’s t-test, t(23.52)=4.106, p=0.0004, error bars show median with interquartile range. (E) Gamete (left) and embryo (right) defects observed for tissue-specific rab-1 RNAi (n=18) and control (n=12). Error bars show standard error of the sample proportion (see Methods). All 12 controls had both gametes and embryos. For rab-1 tissue-specific RNAi, 15/18 had gamete defects (12/18 lacked gametes, 2/18 had only endomitotic oocytes, 1/18 had only sperm), 3/18 had both gametes, and 2/18 had embryos. (F-F’) A DAPI stained reproductive age animal treated with tissue specific rab-1 RNAi with failure of gametogenesis, showing most proximal germ cells in pachytene. (G-H’) Whole-body rab-1 RNAi treated worms expressing a somatic gonad marker in cyan (GFP::INX-9) and a marker of germ cell histones (mex-5p::H2B::mCherry::nos-2 3′UTR) shifted onto rab-1 RNAi plates at the L2 stage and imaged ~48 hours later (3 days post L1 arrest). (G) rab-1 RNAi treated sample with sperm showing meiotic progression past pachytene. (n=4/9 RNAi treated worms) (H-H’) rab-1 RNAi treated sample showing failure to progress through meiosis, with proximal most germ cells showing pachytene morphology (n=5/9). Yellow arrowheads indicate germ cell nuclei with pachytene morphology.