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Dev Biol. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Dev Biol. 2016 June 1; 414(1): 58–71. doi:10.1016/j.ydbio.2016.03.028.
CACN-1 is required in the Caenorhabditis elegans somatic gonad for proper oocyte development Alyssa D. Cecchetelli, Julie Hugunin, Hiba Tannoury, and Erin J. Cram* Department of Biology, Northeastern University, Boston, MA 02115, United States
Abstract Author Manuscript Author Manuscript
CACN-1/Cactin is a conserved protein identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode Caenorhabditis elegans. In addition to possessing distal tip cells that migrate past their correct stopping point, animals depleted of cacn-1 are sterile. In this study, we show that CACN-1 is needed in the soma for proper germ line development and maturation. When CACN-1 is depleted, sheath cells are absent and/or abnormal. When sheath cells are absent, hermaphrodites produce sperm, but do not switch appropriately to oocyte production. When sheath cells are abnormal, some oocytes develop but are not successfully ovulated and undergo endomitotic reduplication (Emo). Our previous proteomic studies show that CACN-1 interacts with a network of splicing factors. Here, these interactors were screened using RNAi. Depletion of many of these factors led to missing or abnormal sheath cells and germ line defects, particularly absent and/or Emo oocytes. These results suggest CACN-1 is part of a protein network that influences somatic gonad development and function through alternative splicing or post-transcriptional gene regulation.
Keywords CACN-1; Somatic gonad; Sperm-oocyte switch; Spliceosome; Post-transcriptional regulation; C.
elegans
1. Introduction
Author Manuscript
Cell-cell interactions and signaling are crucial for proper development and differentiation. Germ line development, differentiation, and maturation across species is regulated through soma-germ line interactions (Lehmann, 2012; Hanna and Hennebold, 2014; Killian and Hubbard, 2005; McCarter et al., 1997; Eppig, 1991). Somatic cells provide a niche for proper germ line differentiation and development (Jemc, 2011). Defects in development of the soma and disruption of somatically expressed genes can cause reproductive defects including ovarian failure, adeno-carcinomas, granulosa tumors and infertility (Matzuk and Lamb, 2008; Singh and Schimenti, 2015; Pangas et al., 2008; Wu et al., 2007). However, the molecular mechanisms that control somatic gonadal cell specification and development are not well understood.
*
Correspondence to: Biology Department, Northeastern University, 360 Huntington Avenue, 134 Mugar Hall, Boston, MA, 02115. United States. ; Email: [email protected] (E.J. Cram)..
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The reproductive system of the nematode Caenorhabditis elegans is an ideal model for the study of somatic gonad development and soma-germ line interactions. The C. elegans reproductive system consists of two symmetrical U-shaped gonad arms connected to a shared uterus. Each somatic gonad arm is enveloped by an outer basal lamina and consists of the distal tip cell (DTC), 5 pairs of gonadal sheath cells, the spermatheca, and the spermatheca-uterine valve (sp-ut) (McCarter et al., 1997; Hubbard and Greenstein, 2000; Strome, 1986). The distal mitotic germ cell pool is the source of meiotic cells, which move proximally as they undergo gametogenesis. Sperm are produced in the last stage of larval development and stored in the spermatheca. The hermaphrodites then undergo the spermooycte switch and produce oocytes throughout adulthood. Oocytes progress through meiosis as they move proximally, arresting at diakinesis of meiosis I in the proximal arm. Upon receiving cues from sperm and sheath cells, the most proximal oocyte adjacent to the spermatheca (the −1 oocyte) matures, is ovulated into the spermatheca, fertilized, and finally expelled into the uterus (Hubbard and Greenstein, 2005). Germ cells and somatic gonadal cells arise from Z2/Z3 and Z1/Z4 precursor cells respectively (Lints and Hall, 2013). These cells and their daughters are not spatially segregated until the L2/L3 molt when the gonad begins to establish organization and structure (Kimble and Hirsh, 1979). During early L3, guided by the distal tip cells (DTC), the two gonad arms begin to elongate away from the midpoint of the animal. Germ cells proliferate throughout the arm. Midway through L3 the most proximal germ cells begin to enter meiosis (Killian and Hubbard, 2005; Hansen et al., 2004; Kimble and White, 1981). The epithelial sheath cells that surround the gonad play key roles in germ line patterning during development (Killian and Hubbard, 2005).
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Sheath cells originate as five lateral pairs adjacent to the proximal germ line. As the animal develops, sheath cells migrate and expand, forming a single layer of cells covering most of the gonad arm (Hall et al., 1999; Hirsh et al., 1976). The distal sheath cells (Sh1-2) promote germ line proliferation and exit from meiotic pachytene (Killian and Hubbard, 2005; McCarter et al., 1997), whereas the 3 pairs of proximal sheath cells (Sh 3-5) are important for oocyte maturation and for the smooth-muscle-like contractions that push oocytes into the spermatheca for fertilization (Strome, 1986; Miller et al., 2003, 2001; McCarter et al., 1999). Defects in sheath cells, including mutations in sheath cell expressed genes like ceh-18, a POU class homeoprotein (Rose et al., 1997), pro-1, a protein involved in rRNA processing (Voutev et al., 2006; Killian and Hubbard, 2004), and mup-2, a troponin homolog (Myers et al., 1996), or ablation of sheath cell precursor cells (Sh15) (Killian and Hubbard, 2005; McCarter et al., 1997) produce an array of germ line phenotypes including tumors (Killian and Hubbard, 2005; McCarter et al., 1997; Killian and Hubbard, 2004), sperm filled proximal gonad arms that lack oocytes (Killian and Hubbard, 2005) and endo-mitotically duplicating oocytes (Killian and Hubbard, 2005; McCarter et al., 1997; Rose et al., 1997; Myers et al., 1996) (the Emo phenotype (Iwasaki et al., 1996)). Even though it is clear that sheath cell development has a crucial role in proper germ line formation, little is known about the molecular mechanisms that control sheath cell specification, development and patterning in C. elegans. For example, the protein LIN-9 functions in an RB-related pathway, and is necessary for the development of the correct number of somatic sheath cells, however the mechanism of this regulation remains unknown (Beitel et al., 2000). Dev Biol. Author manuscript; available in PMC 2017 June 01.
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We identified CACN-1, a protein of unknown function, conserved from yeast to humans, in a screen for regulators of DTC migration (Cram et al., 2006; Tannoury et al., 2010). CACN-1 is the homolog of Cactin, which functions with the Rel/NFKB pathway to control dorsal-ventral patterning in Drosophila (Lin et al., 2000), in innate immunity in Litopenaeus vannamei (Zhang et al., 2014), and with TRIM39 to negatively regulate the NFKB pathway in human cell lines (Suzuki et al., 2015). CACN-1 lacks enzymatic activity but contains a nuclear localization signal at its N-terminus, two coiled-coiled domains and a conserved Cterminus (Schultz et al., 2000; Kosugi et al., 2009). Recently, a functional role for CACN-1 has begun to emerge. Our proteomics work suggests CACN-1 is part of a novel network containing many spliceosomal components (Doherty et al., 2014). This result is consistent with the observation that both human and Arabidopsis thaliania (Baldwin et al., 2013) CACTIN proteins co-purify with spliceosomal proteins (Jurica et al., 2002; Bessonov et al., 2008; Ilagan et al., 2009; Ashton-Beaucage et al., 2014), and that in Schizosaccharomyces pombe, Cay1/cactin promotes proper splicing and protein stability of the telomeric protein Rap1 (Lorenzi et al., 2015).
Author Manuscript
The spliceosome is responsible for the removal of introns and ligation of exons (Zahler, 2012) and for alternative splicing, which allows for variety in mRNA products and is a mechanism to regulate gene expression (Wollerton et al., 2001). The spliceosome consists of over 100 different factors including small ribonuclear-protein particles (snRNPs), accessory proteins and an array of RNA binding proteins (RBPs) (Zahler, 2012; Chen and Cheng, 2012). In the C. elegans genome 2562 annotated genes, or 13% of the genome, are alternatively spliced (Zahler, 2005). In addition, regulation of gene expression in the C. elegans germ line is primarily translational, most commonly through binding of RBPs to the 3′ UTR of target messages (Merritt et al., 2008). Even though splicing and posttranscriptional gene regulation play an important role in C. elegans biology, many C. elegans splicing factors and RBPs remain uncharacterized.
Author Manuscript
CACN-1 has been previously shown to cause germ line over-proliferation and issues in germ cell differentiation when depleted solely in the germ line of C. elegans (Kerins et al., 2010). However, CACN-1's role in the somatic gonad and the mechanism by which it exerts these germ line effects remains largely unknown. In this study, we demonstrate that CACN-1 is necessary primarily in the soma for the presence and proper morphology of the gonadal sheath cells that regulate the sperm-oocyte switch as well as germ line maturation and ovulation in C. elegans. Depletion of a set of previously identified CACN-1 interacting proteins (Doherty et al., 2014) similarly results in sheath cell and germ line defects. The results of this study reveal that CACN-1 and its network of interacting proteins are important for proper development of the gonad and suggest that CACN-1 may be involved in premRNA splicing or post-transcriptional regulation to mediate these decisions.
2. Materials and methods 2.1. Nematode strains Nematodes were grown on nematode growth media (NGM) (0.107 M NaCl, 0.25% wt/vol Peptone (Fischer Science Education), 1.7% wt/vol BD Bacto-Agar (Fisher Scientific), 0.5% Nyastatin (Sigma), 0.1 mM CaCl2, 0.1 mM MgSO4, 0.5% wt/vol Cholesterol, 2.5 mM Dev Biol. Author manuscript; available in PMC 2017 June 01.
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KPO4), seeded with Escherichia coli OP50 using standard techniques (Myers et al., 1996). Nematodes were cultured at 23°C unless specified otherwise. The strains used in this study are as follows: N2 (wild type reference strain from Bristol), NL2550 ppw-1(pk2505) and NL2098 rrf-1(pk1417) and the GFP expression lines DG1575 lim-7::GFP and OD27 AIR-2::GFP. 2.2. RNA interference
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Starved dauer nematodes were allowed to recover for 48 h on NGM plates newly seeded with OP50. This procedure produces young gravid adults for egg collection. Eggs were released using an alkaline hypochlorite solution as described in Hope (1999), and washed 3 × with filter sterilized M9 buffer (22 mM KH2PO4, 42 mM NaHPO4, 86 mM NaCl, and 1 mM MgSO4) (‘egg prep’). Clean eggs were then transferred to NGM previously seeded with HT115(DE3) bacteria that express dsRNA for RNAi. Strains utilized in each RNAi experiment are indicated. The RNAi feeding protocol was performed essentially as described in Timmons et al. (2001). HT115(DE3) bacteria transformed with the dsRNA construct of interest were grown over night at 37°C in Luria broth (LB) supplemented with 40 μg/ml ampicillin. The following day, 150 μl of the culture was seeded on NGM agar supplemented with 25 μg/ml carbenicillin and isopropylthio-β-galactoside (IPTG) and incubated at room temperature for 24–72 h to induce dsRNA expression. Eggs collected from the alkaline lysis procedure were transferred onto these plates and incubated at 23°C for the times specified.
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The cacn-1 ORF RNAi clone is a full-length cDNA matching Wormbase (WS2000) predictions (Open biosystems; Huntsville, AL, USA). All CACN-1 interactor RNAi clones are described in Doherty et al. (2014). Empty pPD129.36 vector (“empty RNAi”) was used as a negative control in all RNAi experiments. 2.3. RNA in situ hybridization
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RNA in situ hybridization was performed in accordance with Lee and Schedl (2006). Nematodes subjected to both cacn-1 and control empty RNAi were collected at adulthood (54 h post ‘egg prep’). Gonad arms were extracted in 1 × Phosphate Buffered Saline (PBS) and fixed in 3% paraformaldehyde/0.25% glutaraldehyde/0.1 M K2HPO4 (pH 7.2). Samples were washed with in 1 × Phosphate Buffered Saline and 0.1% Tween 20 (PBST) and stored in cold methanol overnight, then washed with PBST and digested with Proteinase K, 50 μg/ml in PBST, for 30 min. Gonad arms were then washed with PBST, fixed with 3% paraformalde-hyde/0.25% glutaraldehyde/0. 1 M K2HPO4 (pH 7.2) for 15 min, and washed with PBST containing 2 mg/ml glycine. Gonad arms were then hybridized in a 1:2 ratio of RNA cacn-1 DIG labeled probe to hybridization buffer (5 × sodium chloride sodium citrate solution, 50% formamide, 100 μg/ml salmon sperm DNA, 50 μg/ml heparin sodium salt and 0.1% Tween 20), in a 48°C water bath for 24 h. Anti-cacn-1 probes were generated using 580 base pairs of the cacn-1 gene. PCR was run with DIG labeled nucleotides (Roche Diagnostics Corporation, Indianapolis, IN, USA). Sense and anti-sense probes were created using asymmetric one-way PCR. Probes were then diluted with sterile water and dehydrated with 1 M NaCl overnight at −20°C. The reaction Dev Biol. Author manuscript; available in PMC 2017 June 01.
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was centrifuged and washed with ethanol. The remaining DNA probe was then re-suspended in hybridization buffer and stored at −20°C.
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After gonad arms were hybridized with anti-cacn-1 DIG labeled probes they were washed several times with hybridization buffer and PBST both with and without 0.5 mg/ml BSA (New England Biolabs, Ipswich, MA, USA). Probe was detected using a diluted 1:1000 alkaline-phosphatase-conjugated anti-DIG (Roche Diagnostics Corporation, Indianapolis, IN, USA) in PBST with BSA overnight at 4°C. Gonad arms were then washed with PBST both with and without BSA as well as a staining solution containing 100 mM NaCl, 5 mM MgCl2, 100 mM Tris (pH 9.5), 0.1% Tween 20, and 1 mM levamisole. Gonad arms were stained with staining solution containing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NDT) dissolved tablet (Sigma Aldrich, St. Louis, MO, USA) and 100 ng/ml DAPI in the dark for approximately four hours at room temperature, washed with PBST, re-suspended in 100 ng/ml DAPI, and washed a final time in PBS. Excess liquid was removed and gonad arms were mounted onto slides with 2% agar pads and observed using the DIC filter of the fluorescent microscope for cacn-1 staining within the gonad arms of all the treated animals. 2.4. DAPI and Texas Red-X phalloidin staining
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N2 nematodes were stained with 4′,6-diamidino-2-phenylindolea (DAPI), a nuclear stain, to observe oocytes and sperm within gonad arms. Young adults were collected with 1 × Phosphate Buffered Saline and 0.1% Tween 20 (PBST) into a 15 ml glass conical tube and allowed to settle by gravity. The animals were then washed three times with PBST to remove excess bacteria and fixed with cold methanol for 20 min. After fixation, animals were washed once more with PBST. The whole animal was then stained with 100 ng/ml of DAPI (Sigma-Aldrich, St. Louis, MO) in PBS for at least 20 min. After washing with PBS, excess liquid was removed and animals were mounted on slides coated with 2% agarose in H2O. Gonad arm content and phenotypes were observed for nuclear staining using a Nikon 80i epifluorescence microscope equipped with a DAPI fluorescent filter.
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To stain gonad arms with Texas Red-X phalloidin, an actin stain, animals were washed with PBS and transferred to a watch glass. Using a 25 gauge syringe (BD PrecisionGlide Needle (Becton Dickinson, Franklin Lakes, NJ, USA)), animals were sliced at the level of the pharynx or vulva, causing gonad arms to extrude from the body. The dissected gonad arms were fixed in 4% formaldehyde and then washed several times with PBST. After washing, gonad arms were stained with Texas Red-X phalloidin (Molecular Probes, Invitrogen, Life Technologies, Grand Island, NY, USA) at a final concentration of 0.4 units/ml, and placed in the dark at room temperature for 2 h or 4°C overnight. Gonad arms were than washed with PBST and stained with 100 ng/ml DAPI in PBS for 20 min, washed in PBS and left to settle. Extra liquid was removed and gonad arms were transferred onto slides coated with 2% agarose in H2O for observation as described above. 2.5. Fluorescence microscopy and analysis To analyze gonad arm morphology, partially synchronized populations of animals were mounted in a ~5 μl drop of M9 and 0.08 M sodium azide (or 0.01% tetramisole and 0.1%
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Tricaine) onto a slide with a 2% agarose in H2O pad. Animals were observed using a 20 × or 60 × oil-immersion objective with a Nikon Eclipse 80i epiflourescence microscope equipped with a Spot RT3 CCD camera (Diagnostic instruments; Sterling Heights, MI, USA). Images were captured using Spot advanced version 4.6 software (Diagnostic Instruments, Sterling Heights, MI, USA). All Differential Interference Contrast (DIC) pictures were taken at 37.44 ms exposure. Fluorescent images were taken using either a GFP, DAPI or RFP filter as appropriate. Animals were scored for the presence and absence of sperm, oocytes and/or embryos in both the proximal and distal gonad arm of each animal. Cells were identified as sperm or oocytes based on nuclear staining and morphology. Animals stained with Texas Red-X phalloidin were scored for the presence and absence of sheath cells based on the visible actin filaments. Due to variability in the dissection and phalloidin staining procedure, as well as the variation in the orientation of the dissected gonads, images from this experiment were scored blindly for the presence of actin in the proximal gonad. For all experiments a Fishers exact t-test (two dimensional χ2 analysis) using GraphPad Prism statistical software was used to compare the percent of normal gonad arms between empty and all other RNAi treatments.
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3. Results 3.1. CACN-1 is necessary for the production of normal oocytes
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Depletion of CACN-1 via feeding RNAi leads to sterility in wild type animals. Sterility can be caused by abnormal germ line development or maturation, as well as by defects in the somatic gonad (Hubbard and Greenstein, 2000). To investigate the cause of sterility, we used DIC microscopy and DAPI, a nuclear stain, to observe gonad arms and germ line development of wildtype and cacn-1 RNAi treated animals. Gonad arms in wildtype (N2) young adults at 65 h contained aligned oocytes in the proximal gonad, adjacent to the spermfilled spermatheca, with developing embryos in the uterus (Fig. 1). Animals treated with cacn-1 RNAi exhibited proximal gonad arms that either contained sperm, but lacked oocytes (44%) or contained abnormal oocytes in addition to sperm (51%; n=208) (Fig. 1). Occasionally, we observed gonads comprised entirely of undifferentiated germ cells (1%; n=200). C. elegans germ cells, sperm and oocytes exhibit characteristic DNA morphologies (Hansen and Schedl, 2013; Greenstein, 2005). To visualize the DNA in these cells, gonad arms were stained with DAPI, a nuclear stain. N2 gonad arms contained normally stained germ line cells, oocytes, and sperm. Consistent with the DIC imaging, DAPI staining of CACN-1 depleted animals revealed proximal gonad arms that contained sperm, but lacked oocytes (43%), or for those that contained oocytes, large, dense areas of nuclear staining indicative of endomitotically duplicating oocytes (Emo) (Iwasaki et al., 1996) (57%; n=134) (Fig. 1D). In C. elegans, when ovulation fails, oocytes typically go through multiple rounds of nuclear envelope breakdown and replication becoming highly polyploidy (Iwasaki et al., 1996). Disruption of many different genes can result in the Emo phenotype, including genes involved in oocyte maturation and maintenance (Killian and Hubbard, 2004; Iwasaki et al., 1996), as well as somatic gonad structure and development (McCarter et al., 1997; Myers et al., 1996; Wissmann et al., 1999; Ono and Ono, 2004; Greenstein et al., 1994).
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To determine whether animals that lacked oocytes were simply developmentally delayed in the sperm to oocyte switch, animals were reared on cacn-1 RNAi and observed over a time course throughout early adulthood (54–72 h). All timepoints had a similar penetrance of proximal gonad arms that contained sperm, but lacked oocytes (42%; n=740) with most of the remainder of the proximal gonads containing abnormal oocytes in addition to sperm (55%; n=740) (Fig. 1B). These data suggest that the absence of oocytes is not the result of a delay in oocyte production, but instead represents a failure to switch to oocyte production. Therefore, CACN-1 may regulate the sperm to oocyte switch. 3.2. cacn-1 is expressed in the somatic gonad
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A transgenic construct, expressing CACN-1::GFP under the control of 913 bp of upstream sequence, is expressed in the pharynx, intestine, vulva and somatic gonad (Tannoury et al., 2010). A mini-gene construct, containing this sequence upstream of cacn-1 cDNA, is sufficient to rescue larval development and fertility in cacn-1(tm3042) mutant animals (Tannoury et al., 2010). However, expression of GFP was not observed in the germ line, likely due to silencing of the multi-copy extrachromosomal array. To observe the endogenous cacn-1 mRNA expression pattern in the gonad, animals were reared on cacn-1 and empty control RNAi for 54 h, dissected, and subjected to RNA in situ hybridization. As expected, cacn-1 treated animals had substantially smaller gonad arms than wild type (Tannoury et al., 2010). In control animals, cacn-1 mRNA was expressed from the distal gonad arm through to the proximal arm, the region where oocyte maturation occurs (Fig. 2A and D). No staining was observed when CACN-1 was depleted via RNAi, confirming not only the specificity of our CACN-1 probe, but also that feeding RNAi efficiently depletes CACN-1 expression (Fig. 2A and D). To determine if cacn-1 mRNA is expressed in the somatic gonadal sheath cells and/or the underlying germ line, RNA in situ hybridization was repeated on animals resistant to RNAi in the soma, NL2098 rrf-1(pk1417) (Sijen et al., 2001), or in the germ line NL2550 ppw-1(pk2505) (Grishok, 2005). Both rrf-1(pk1417) and ppw-1(pk2505) animals depleted of cacn-1 exhibited reduced staining compared to control animals (p
Dev Biol. Author manuscript; available in PMC 2017 June 01. Published in final edited form as: Dev Biol. 2016 June 1; 414(1): 58–71. doi:10.1016/j.ydbio.2016.03.028.
CACN-1 is required in the Caenorhabditis elegans somatic gonad for proper oocyte development Alyssa D. Cecchetelli, Julie Hugunin, Hiba Tannoury, and Erin J. Cram* Department of Biology, Northeastern University, Boston, MA 02115, United States
Abstract Author Manuscript Author Manuscript
CACN-1/Cactin is a conserved protein identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode Caenorhabditis elegans. In addition to possessing distal tip cells that migrate past their correct stopping point, animals depleted of cacn-1 are sterile. In this study, we show that CACN-1 is needed in the soma for proper germ line development and maturation. When CACN-1 is depleted, sheath cells are absent and/or abnormal. When sheath cells are absent, hermaphrodites produce sperm, but do not switch appropriately to oocyte production. When sheath cells are abnormal, some oocytes develop but are not successfully ovulated and undergo endomitotic reduplication (Emo). Our previous proteomic studies show that CACN-1 interacts with a network of splicing factors. Here, these interactors were screened using RNAi. Depletion of many of these factors led to missing or abnormal sheath cells and germ line defects, particularly absent and/or Emo oocytes. These results suggest CACN-1 is part of a protein network that influences somatic gonad development and function through alternative splicing or post-transcriptional gene regulation.
Keywords CACN-1; Somatic gonad; Sperm-oocyte switch; Spliceosome; Post-transcriptional regulation; C.
elegans
1. Introduction
Author Manuscript
Cell-cell interactions and signaling are crucial for proper development and differentiation. Germ line development, differentiation, and maturation across species is regulated through soma-germ line interactions (Lehmann, 2012; Hanna and Hennebold, 2014; Killian and Hubbard, 2005; McCarter et al., 1997; Eppig, 1991). Somatic cells provide a niche for proper germ line differentiation and development (Jemc, 2011). Defects in development of the soma and disruption of somatically expressed genes can cause reproductive defects including ovarian failure, adeno-carcinomas, granulosa tumors and infertility (Matzuk and Lamb, 2008; Singh and Schimenti, 2015; Pangas et al., 2008; Wu et al., 2007). However, the molecular mechanisms that control somatic gonadal cell specification and development are not well understood.
*
Correspondence to: Biology Department, Northeastern University, 360 Huntington Avenue, 134 Mugar Hall, Boston, MA, 02115. United States. ; Email: [email protected] (E.J. Cram)..
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The reproductive system of the nematode Caenorhabditis elegans is an ideal model for the study of somatic gonad development and soma-germ line interactions. The C. elegans reproductive system consists of two symmetrical U-shaped gonad arms connected to a shared uterus. Each somatic gonad arm is enveloped by an outer basal lamina and consists of the distal tip cell (DTC), 5 pairs of gonadal sheath cells, the spermatheca, and the spermatheca-uterine valve (sp-ut) (McCarter et al., 1997; Hubbard and Greenstein, 2000; Strome, 1986). The distal mitotic germ cell pool is the source of meiotic cells, which move proximally as they undergo gametogenesis. Sperm are produced in the last stage of larval development and stored in the spermatheca. The hermaphrodites then undergo the spermooycte switch and produce oocytes throughout adulthood. Oocytes progress through meiosis as they move proximally, arresting at diakinesis of meiosis I in the proximal arm. Upon receiving cues from sperm and sheath cells, the most proximal oocyte adjacent to the spermatheca (the −1 oocyte) matures, is ovulated into the spermatheca, fertilized, and finally expelled into the uterus (Hubbard and Greenstein, 2005). Germ cells and somatic gonadal cells arise from Z2/Z3 and Z1/Z4 precursor cells respectively (Lints and Hall, 2013). These cells and their daughters are not spatially segregated until the L2/L3 molt when the gonad begins to establish organization and structure (Kimble and Hirsh, 1979). During early L3, guided by the distal tip cells (DTC), the two gonad arms begin to elongate away from the midpoint of the animal. Germ cells proliferate throughout the arm. Midway through L3 the most proximal germ cells begin to enter meiosis (Killian and Hubbard, 2005; Hansen et al., 2004; Kimble and White, 1981). The epithelial sheath cells that surround the gonad play key roles in germ line patterning during development (Killian and Hubbard, 2005).
Author Manuscript Author Manuscript
Sheath cells originate as five lateral pairs adjacent to the proximal germ line. As the animal develops, sheath cells migrate and expand, forming a single layer of cells covering most of the gonad arm (Hall et al., 1999; Hirsh et al., 1976). The distal sheath cells (Sh1-2) promote germ line proliferation and exit from meiotic pachytene (Killian and Hubbard, 2005; McCarter et al., 1997), whereas the 3 pairs of proximal sheath cells (Sh 3-5) are important for oocyte maturation and for the smooth-muscle-like contractions that push oocytes into the spermatheca for fertilization (Strome, 1986; Miller et al., 2003, 2001; McCarter et al., 1999). Defects in sheath cells, including mutations in sheath cell expressed genes like ceh-18, a POU class homeoprotein (Rose et al., 1997), pro-1, a protein involved in rRNA processing (Voutev et al., 2006; Killian and Hubbard, 2004), and mup-2, a troponin homolog (Myers et al., 1996), or ablation of sheath cell precursor cells (Sh15) (Killian and Hubbard, 2005; McCarter et al., 1997) produce an array of germ line phenotypes including tumors (Killian and Hubbard, 2005; McCarter et al., 1997; Killian and Hubbard, 2004), sperm filled proximal gonad arms that lack oocytes (Killian and Hubbard, 2005) and endo-mitotically duplicating oocytes (Killian and Hubbard, 2005; McCarter et al., 1997; Rose et al., 1997; Myers et al., 1996) (the Emo phenotype (Iwasaki et al., 1996)). Even though it is clear that sheath cell development has a crucial role in proper germ line formation, little is known about the molecular mechanisms that control sheath cell specification, development and patterning in C. elegans. For example, the protein LIN-9 functions in an RB-related pathway, and is necessary for the development of the correct number of somatic sheath cells, however the mechanism of this regulation remains unknown (Beitel et al., 2000). Dev Biol. Author manuscript; available in PMC 2017 June 01.
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We identified CACN-1, a protein of unknown function, conserved from yeast to humans, in a screen for regulators of DTC migration (Cram et al., 2006; Tannoury et al., 2010). CACN-1 is the homolog of Cactin, which functions with the Rel/NFKB pathway to control dorsal-ventral patterning in Drosophila (Lin et al., 2000), in innate immunity in Litopenaeus vannamei (Zhang et al., 2014), and with TRIM39 to negatively regulate the NFKB pathway in human cell lines (Suzuki et al., 2015). CACN-1 lacks enzymatic activity but contains a nuclear localization signal at its N-terminus, two coiled-coiled domains and a conserved Cterminus (Schultz et al., 2000; Kosugi et al., 2009). Recently, a functional role for CACN-1 has begun to emerge. Our proteomics work suggests CACN-1 is part of a novel network containing many spliceosomal components (Doherty et al., 2014). This result is consistent with the observation that both human and Arabidopsis thaliania (Baldwin et al., 2013) CACTIN proteins co-purify with spliceosomal proteins (Jurica et al., 2002; Bessonov et al., 2008; Ilagan et al., 2009; Ashton-Beaucage et al., 2014), and that in Schizosaccharomyces pombe, Cay1/cactin promotes proper splicing and protein stability of the telomeric protein Rap1 (Lorenzi et al., 2015).
Author Manuscript
The spliceosome is responsible for the removal of introns and ligation of exons (Zahler, 2012) and for alternative splicing, which allows for variety in mRNA products and is a mechanism to regulate gene expression (Wollerton et al., 2001). The spliceosome consists of over 100 different factors including small ribonuclear-protein particles (snRNPs), accessory proteins and an array of RNA binding proteins (RBPs) (Zahler, 2012; Chen and Cheng, 2012). In the C. elegans genome 2562 annotated genes, or 13% of the genome, are alternatively spliced (Zahler, 2005). In addition, regulation of gene expression in the C. elegans germ line is primarily translational, most commonly through binding of RBPs to the 3′ UTR of target messages (Merritt et al., 2008). Even though splicing and posttranscriptional gene regulation play an important role in C. elegans biology, many C. elegans splicing factors and RBPs remain uncharacterized.
Author Manuscript
CACN-1 has been previously shown to cause germ line over-proliferation and issues in germ cell differentiation when depleted solely in the germ line of C. elegans (Kerins et al., 2010). However, CACN-1's role in the somatic gonad and the mechanism by which it exerts these germ line effects remains largely unknown. In this study, we demonstrate that CACN-1 is necessary primarily in the soma for the presence and proper morphology of the gonadal sheath cells that regulate the sperm-oocyte switch as well as germ line maturation and ovulation in C. elegans. Depletion of a set of previously identified CACN-1 interacting proteins (Doherty et al., 2014) similarly results in sheath cell and germ line defects. The results of this study reveal that CACN-1 and its network of interacting proteins are important for proper development of the gonad and suggest that CACN-1 may be involved in premRNA splicing or post-transcriptional regulation to mediate these decisions.
2. Materials and methods 2.1. Nematode strains Nematodes were grown on nematode growth media (NGM) (0.107 M NaCl, 0.25% wt/vol Peptone (Fischer Science Education), 1.7% wt/vol BD Bacto-Agar (Fisher Scientific), 0.5% Nyastatin (Sigma), 0.1 mM CaCl2, 0.1 mM MgSO4, 0.5% wt/vol Cholesterol, 2.5 mM Dev Biol. Author manuscript; available in PMC 2017 June 01.
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KPO4), seeded with Escherichia coli OP50 using standard techniques (Myers et al., 1996). Nematodes were cultured at 23°C unless specified otherwise. The strains used in this study are as follows: N2 (wild type reference strain from Bristol), NL2550 ppw-1(pk2505) and NL2098 rrf-1(pk1417) and the GFP expression lines DG1575 lim-7::GFP and OD27 AIR-2::GFP. 2.2. RNA interference
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Starved dauer nematodes were allowed to recover for 48 h on NGM plates newly seeded with OP50. This procedure produces young gravid adults for egg collection. Eggs were released using an alkaline hypochlorite solution as described in Hope (1999), and washed 3 × with filter sterilized M9 buffer (22 mM KH2PO4, 42 mM NaHPO4, 86 mM NaCl, and 1 mM MgSO4) (‘egg prep’). Clean eggs were then transferred to NGM previously seeded with HT115(DE3) bacteria that express dsRNA for RNAi. Strains utilized in each RNAi experiment are indicated. The RNAi feeding protocol was performed essentially as described in Timmons et al. (2001). HT115(DE3) bacteria transformed with the dsRNA construct of interest were grown over night at 37°C in Luria broth (LB) supplemented with 40 μg/ml ampicillin. The following day, 150 μl of the culture was seeded on NGM agar supplemented with 25 μg/ml carbenicillin and isopropylthio-β-galactoside (IPTG) and incubated at room temperature for 24–72 h to induce dsRNA expression. Eggs collected from the alkaline lysis procedure were transferred onto these plates and incubated at 23°C for the times specified.
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The cacn-1 ORF RNAi clone is a full-length cDNA matching Wormbase (WS2000) predictions (Open biosystems; Huntsville, AL, USA). All CACN-1 interactor RNAi clones are described in Doherty et al. (2014). Empty pPD129.36 vector (“empty RNAi”) was used as a negative control in all RNAi experiments. 2.3. RNA in situ hybridization
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RNA in situ hybridization was performed in accordance with Lee and Schedl (2006). Nematodes subjected to both cacn-1 and control empty RNAi were collected at adulthood (54 h post ‘egg prep’). Gonad arms were extracted in 1 × Phosphate Buffered Saline (PBS) and fixed in 3% paraformaldehyde/0.25% glutaraldehyde/0.1 M K2HPO4 (pH 7.2). Samples were washed with in 1 × Phosphate Buffered Saline and 0.1% Tween 20 (PBST) and stored in cold methanol overnight, then washed with PBST and digested with Proteinase K, 50 μg/ml in PBST, for 30 min. Gonad arms were then washed with PBST, fixed with 3% paraformalde-hyde/0.25% glutaraldehyde/0. 1 M K2HPO4 (pH 7.2) for 15 min, and washed with PBST containing 2 mg/ml glycine. Gonad arms were then hybridized in a 1:2 ratio of RNA cacn-1 DIG labeled probe to hybridization buffer (5 × sodium chloride sodium citrate solution, 50% formamide, 100 μg/ml salmon sperm DNA, 50 μg/ml heparin sodium salt and 0.1% Tween 20), in a 48°C water bath for 24 h. Anti-cacn-1 probes were generated using 580 base pairs of the cacn-1 gene. PCR was run with DIG labeled nucleotides (Roche Diagnostics Corporation, Indianapolis, IN, USA). Sense and anti-sense probes were created using asymmetric one-way PCR. Probes were then diluted with sterile water and dehydrated with 1 M NaCl overnight at −20°C. The reaction Dev Biol. Author manuscript; available in PMC 2017 June 01.
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was centrifuged and washed with ethanol. The remaining DNA probe was then re-suspended in hybridization buffer and stored at −20°C.
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After gonad arms were hybridized with anti-cacn-1 DIG labeled probes they were washed several times with hybridization buffer and PBST both with and without 0.5 mg/ml BSA (New England Biolabs, Ipswich, MA, USA). Probe was detected using a diluted 1:1000 alkaline-phosphatase-conjugated anti-DIG (Roche Diagnostics Corporation, Indianapolis, IN, USA) in PBST with BSA overnight at 4°C. Gonad arms were then washed with PBST both with and without BSA as well as a staining solution containing 100 mM NaCl, 5 mM MgCl2, 100 mM Tris (pH 9.5), 0.1% Tween 20, and 1 mM levamisole. Gonad arms were stained with staining solution containing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NDT) dissolved tablet (Sigma Aldrich, St. Louis, MO, USA) and 100 ng/ml DAPI in the dark for approximately four hours at room temperature, washed with PBST, re-suspended in 100 ng/ml DAPI, and washed a final time in PBS. Excess liquid was removed and gonad arms were mounted onto slides with 2% agar pads and observed using the DIC filter of the fluorescent microscope for cacn-1 staining within the gonad arms of all the treated animals. 2.4. DAPI and Texas Red-X phalloidin staining
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N2 nematodes were stained with 4′,6-diamidino-2-phenylindolea (DAPI), a nuclear stain, to observe oocytes and sperm within gonad arms. Young adults were collected with 1 × Phosphate Buffered Saline and 0.1% Tween 20 (PBST) into a 15 ml glass conical tube and allowed to settle by gravity. The animals were then washed three times with PBST to remove excess bacteria and fixed with cold methanol for 20 min. After fixation, animals were washed once more with PBST. The whole animal was then stained with 100 ng/ml of DAPI (Sigma-Aldrich, St. Louis, MO) in PBS for at least 20 min. After washing with PBS, excess liquid was removed and animals were mounted on slides coated with 2% agarose in H2O. Gonad arm content and phenotypes were observed for nuclear staining using a Nikon 80i epifluorescence microscope equipped with a DAPI fluorescent filter.
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To stain gonad arms with Texas Red-X phalloidin, an actin stain, animals were washed with PBS and transferred to a watch glass. Using a 25 gauge syringe (BD PrecisionGlide Needle (Becton Dickinson, Franklin Lakes, NJ, USA)), animals were sliced at the level of the pharynx or vulva, causing gonad arms to extrude from the body. The dissected gonad arms were fixed in 4% formaldehyde and then washed several times with PBST. After washing, gonad arms were stained with Texas Red-X phalloidin (Molecular Probes, Invitrogen, Life Technologies, Grand Island, NY, USA) at a final concentration of 0.4 units/ml, and placed in the dark at room temperature for 2 h or 4°C overnight. Gonad arms were than washed with PBST and stained with 100 ng/ml DAPI in PBS for 20 min, washed in PBS and left to settle. Extra liquid was removed and gonad arms were transferred onto slides coated with 2% agarose in H2O for observation as described above. 2.5. Fluorescence microscopy and analysis To analyze gonad arm morphology, partially synchronized populations of animals were mounted in a ~5 μl drop of M9 and 0.08 M sodium azide (or 0.01% tetramisole and 0.1%
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Tricaine) onto a slide with a 2% agarose in H2O pad. Animals were observed using a 20 × or 60 × oil-immersion objective with a Nikon Eclipse 80i epiflourescence microscope equipped with a Spot RT3 CCD camera (Diagnostic instruments; Sterling Heights, MI, USA). Images were captured using Spot advanced version 4.6 software (Diagnostic Instruments, Sterling Heights, MI, USA). All Differential Interference Contrast (DIC) pictures were taken at 37.44 ms exposure. Fluorescent images were taken using either a GFP, DAPI or RFP filter as appropriate. Animals were scored for the presence and absence of sperm, oocytes and/or embryos in both the proximal and distal gonad arm of each animal. Cells were identified as sperm or oocytes based on nuclear staining and morphology. Animals stained with Texas Red-X phalloidin were scored for the presence and absence of sheath cells based on the visible actin filaments. Due to variability in the dissection and phalloidin staining procedure, as well as the variation in the orientation of the dissected gonads, images from this experiment were scored blindly for the presence of actin in the proximal gonad. For all experiments a Fishers exact t-test (two dimensional χ2 analysis) using GraphPad Prism statistical software was used to compare the percent of normal gonad arms between empty and all other RNAi treatments.
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3. Results 3.1. CACN-1 is necessary for the production of normal oocytes
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Depletion of CACN-1 via feeding RNAi leads to sterility in wild type animals. Sterility can be caused by abnormal germ line development or maturation, as well as by defects in the somatic gonad (Hubbard and Greenstein, 2000). To investigate the cause of sterility, we used DIC microscopy and DAPI, a nuclear stain, to observe gonad arms and germ line development of wildtype and cacn-1 RNAi treated animals. Gonad arms in wildtype (N2) young adults at 65 h contained aligned oocytes in the proximal gonad, adjacent to the spermfilled spermatheca, with developing embryos in the uterus (Fig. 1). Animals treated with cacn-1 RNAi exhibited proximal gonad arms that either contained sperm, but lacked oocytes (44%) or contained abnormal oocytes in addition to sperm (51%; n=208) (Fig. 1). Occasionally, we observed gonads comprised entirely of undifferentiated germ cells (1%; n=200). C. elegans germ cells, sperm and oocytes exhibit characteristic DNA morphologies (Hansen and Schedl, 2013; Greenstein, 2005). To visualize the DNA in these cells, gonad arms were stained with DAPI, a nuclear stain. N2 gonad arms contained normally stained germ line cells, oocytes, and sperm. Consistent with the DIC imaging, DAPI staining of CACN-1 depleted animals revealed proximal gonad arms that contained sperm, but lacked oocytes (43%), or for those that contained oocytes, large, dense areas of nuclear staining indicative of endomitotically duplicating oocytes (Emo) (Iwasaki et al., 1996) (57%; n=134) (Fig. 1D). In C. elegans, when ovulation fails, oocytes typically go through multiple rounds of nuclear envelope breakdown and replication becoming highly polyploidy (Iwasaki et al., 1996). Disruption of many different genes can result in the Emo phenotype, including genes involved in oocyte maturation and maintenance (Killian and Hubbard, 2004; Iwasaki et al., 1996), as well as somatic gonad structure and development (McCarter et al., 1997; Myers et al., 1996; Wissmann et al., 1999; Ono and Ono, 2004; Greenstein et al., 1994).
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To determine whether animals that lacked oocytes were simply developmentally delayed in the sperm to oocyte switch, animals were reared on cacn-1 RNAi and observed over a time course throughout early adulthood (54–72 h). All timepoints had a similar penetrance of proximal gonad arms that contained sperm, but lacked oocytes (42%; n=740) with most of the remainder of the proximal gonads containing abnormal oocytes in addition to sperm (55%; n=740) (Fig. 1B). These data suggest that the absence of oocytes is not the result of a delay in oocyte production, but instead represents a failure to switch to oocyte production. Therefore, CACN-1 may regulate the sperm to oocyte switch. 3.2. cacn-1 is expressed in the somatic gonad
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A transgenic construct, expressing CACN-1::GFP under the control of 913 bp of upstream sequence, is expressed in the pharynx, intestine, vulva and somatic gonad (Tannoury et al., 2010). A mini-gene construct, containing this sequence upstream of cacn-1 cDNA, is sufficient to rescue larval development and fertility in cacn-1(tm3042) mutant animals (Tannoury et al., 2010). However, expression of GFP was not observed in the germ line, likely due to silencing of the multi-copy extrachromosomal array. To observe the endogenous cacn-1 mRNA expression pattern in the gonad, animals were reared on cacn-1 and empty control RNAi for 54 h, dissected, and subjected to RNA in situ hybridization. As expected, cacn-1 treated animals had substantially smaller gonad arms than wild type (Tannoury et al., 2010). In control animals, cacn-1 mRNA was expressed from the distal gonad arm through to the proximal arm, the region where oocyte maturation occurs (Fig. 2A and D). No staining was observed when CACN-1 was depleted via RNAi, confirming not only the specificity of our CACN-1 probe, but also that feeding RNAi efficiently depletes CACN-1 expression (Fig. 2A and D). To determine if cacn-1 mRNA is expressed in the somatic gonadal sheath cells and/or the underlying germ line, RNA in situ hybridization was repeated on animals resistant to RNAi in the soma, NL2098 rrf-1(pk1417) (Sijen et al., 2001), or in the germ line NL2550 ppw-1(pk2505) (Grishok, 2005). Both rrf-1(pk1417) and ppw-1(pk2505) animals depleted of cacn-1 exhibited reduced staining compared to control animals (p
