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Genes to Cells (2007) 12, 407-420. doi:10.1111/j.1365-2443.2007.01061.x
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The adaptor-like protein ROG-1 is required for activation of the Ras-MAP kinase pathway and meiotic cell cycle progression in Caenorhabditis elegans

 Yosuke Matsubara1, Ichiro Kawasaki4, Seiichi Urushiyama2, Tomoharu Yasuda1,b, Masaki Shirakata1, Yuichi Iino4, Hiroshi Shibuya2,3 and Yuji Yamanashi1,3,*

1 Department of Cell Regulation, and
2 Department of Molecular Cell Biology, Medical Research Institute, and 3 School of Biomedical Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
4 Molecular Genetics Research Laboratory, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The Ras-MAP kinase pathway regulates varieties of fundamental cellular events. In Caenorhabditis elegans, this pathway is required for oocyte development; however, the nature of its up-stream regulators has remained elusive. Here, we identified a C. elegans gene, rog-1, which encodes the only protein having the IRS-type phosphotyrosine-binding (PTB) domain in the worms. ROG-1 has no obvious domain structure aside from the PTB domain, suggesting that it could serve as an adaptor down-stream of protein-tyrosine kinases (PTKs). RNA interference (RNAi)-mediated down-regulation of rog-1 mRNA significantly decreased brood size. rog-1(tm1031) truncation mutants showed a severe disruption in progression of developing oocytes from pachytene to diakinesis, as was seen in worms carrying a loss-of-function mutation in the let-60 Ras or mpk-1 MAP kinase gene. Furthermore, let-60 Ras-regulated activation of MPK-1 in the gonad is undetectable in rog-1(tm1031) mutants. Conversely, a gain-of-function mutation in the let-60 Ras gene rescues the brood size reduction and germ cell abnormality in rog-1(tm1031) worms. Consistently, rog-1 is preferentially expressed in the germ cells and its expression in the gonad is essential for oocyte development. Thus, ROG-1 is a key positive regulator of the Ras-MAP kinase pathway that permits germ cells to exit from pachytene.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The Ras-MAP kinase pathway plays an essential role in controlling cell fate by regulating cellular responses to signals from microenvironments (Schlessinger 2000). Several routes have been established that lead to the activation of MAP kinase, including one initiated by the activation of receptor-type protein-tyrosine kinases (PTKs), in which intermediary proteins relay the signal, resulting in the activation of Ras. Activated Ras promotes the sequential activation/phosphorylation of MEK kinase, MEK and MAP kinase. Once the MAP kinase is activated, it is competent to phosphorylate a variety of substrates, including transcription factors, cytoskeletal proteins and protein kinases, and is involved in the control of many fundamental cellular processes such as cell proliferation, survival, apoptosis, differentiation, motility and metabolism (Qi & Elion 2005). For example, it has been established that the tight regulation of MAP kinase activity serves to control the meiotic cell cycle progression of oocytes in Xenopus and mammals (Kishimoto 2003; Fan & Sun 2004). In these animals, fully grown oocytes are arrested at the diplotene/diakinesis stage, the germinal vesicle (GV) stage, and MAP kinase activity is crucial for hormone-induced meiotic resumption of the GV stage oocytes (Gotoh et al. 1995; Su et al. 2002).

In Caenorhabditis elegans, a MAP kinase MPK-1 is activated upon the maturation of oocytes as well as in early meiotic prophase I of the pachytene stage in oocyte development (Miller et al. 2001; Page et al. 2001). In the hermaphrodite, the gonad consists of two U-shaped tubes that are each connected at their proximal terminals to a uterus. Mitotic germ cells are located in the distal-most region of the gonad arm, which forms a syncytium. As the cells move away from the distal tip, they enter meiotic prophase I in the transition zone and progress to pachytene. Then, upon transition through the loop region of the gonad, they exit pachytene and enter diakinesis, where they become fully enclosed with a plasma membrane. That loss-of-function mutations in the genes of the Ras-MAP kinase pathway, including let-60 Ras, lin-45 Raf, mek-2 MEK, mpk-1/sur-1 MAP kinase and ksr-2, which encodes a scaffold protein, cause pachytene arrest of germ cells demonstrates an essential role for this pathway in the exit from pachytene and/or entry into diakinesis (Church et al. 1995; Ohmachi et al. 2002). The Ras-MAP kinase pathway is also essential for vulval induction and sex myoblast migration, where receptor PTKs LET-23/epidermal growth factor receptor (EGFR) and EGL-15/fibroblast growth factor receptor (FGFR) regulate this pathway via adaptors, respectively. In contrast, up-stream regulators of Ras in oocyte development are largely unknown.

In general, activation of receptor PTKs induces autophosphorylation of the cytoplasmic region and subsequent recruitment of signaling molecules having the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domain, which specifically targets a short peptide having a phosphotyrosine. For example, an activated and thus autophosphorylated FGFR recruits and phosphorylates an adaptor protein, SNT-1/FRS2, in a manner dependent on interaction of the SNT-1 PTB domain and its target motif (Asn-Pro-Xaa-Tyr) in FGFR. Phosphorylated SNT-1 then recruits the Grb2/Sos complex, a potent activator of Ras, via the SH2 domain of Grb2 (Ong et al. 2000). Following a database search, we found several uncharacterized genes encoding proteins having a PTB domain in the C. elegans genome. The PTB domain can be further classified into the Shc-type or IRS-type based on primary structure and target specificity (Wolf et al. 1995). Interestingly, we found only a single gene that encodes an adaptor-like protein having the IRS-type PTB domain in the C. elegans genome. Given that IRS-, SNT- and Dok-family adaptors, which have this type of PTB domain in common, play essential roles in a wide range of signaling situations in higher eukaryotes, we set out to investigate the physiological role of this gene, named rog-1.

Here, we report that a mutation in rog-1 prevents oocyte development at the pachytene stage of meiosis and causes sterile and embryonic lethal phenotypes. We also provide evidence that rog-1 functions up-stream of let-60 Ras and is required for the activation of MAP kinase in the gonad.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Isolation and identification of rog-1

Aiming to obtain an insight into functions of a protein having the PTB domain, we investigated the complete C. elegans genomic sequence via WormBase and found F54D12.6 as a unique gene that would encode a protein containing the IRS-type of PTB domain, which was previously described as a hypothetical adaptor involved in PTK and/or Ras-mediated signaling (Borland et al. 2001; Huang & Stern 2005). In order to define the entire coding region of the gene, we performed the standard plaque hybridization screening of a cDNA library, which had been prepared from worms at mixed stages, with a cDNA probe containing the sequence corresponding to the PTB domain. Also, the 5' end of the cDNA was determined by reverse transcription and polymerase chain reaction (RT-PCR) using SL1 or SL2, a trans-spliced leader sequence, as a primer. Together, we cloned a cDNA encompassing the entire coding region, which spans six exons and encodes a protein of 323 amino acids (Fig. 1A–C). This novel gene appeared to use consensus splice sites and its cDNA was successfully amplified when we used either SL1 or SL2 spliced leader sequences as a forward primer. Of note is that coding sequence only in exons 1–3, but not in the others, had been predicted for F54D12.6 (Fig. 1B). Thus, we experimentally characterized this novel gene as described below, and named it rog-1 (Ras-activating factor in development of the germ-line) based on its function.


Figure 1
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  		Figure 1  Characterisation of the rog-1 gene and ROG-1 protein. (A) Nucleotide sequences of six exons and intervening introns of the rog-1 gene. Nucleotides in the rog-1 exons and introns are demonstrated in uppercase and lowercase, and coding and non-coding sequences in the exons are in bold and italic, respectively. Nucleotides of F54D12.6 and F54D12.5 previously reported in WormBase are boxed, and their predicted coding sequences are shaded in gray. (B) A schematic diagram of the genomic structure of the rog-1 locus. Boxes represent exons, with the coding region filled and the 3' untranslated region left open. Exons are numbered so that the first exon includes the initiation codon. Regions of F54D12.6 and F54D12.5 previously reported in WormBase and of the large deletion in the rog-1(tm1031) allele, where a thymine is concurrently inserted, are indicated. (C) Schematic diagrams of the predicted wild-type ROG-1 protein and tm1031 mutant, which has four additional amino acids at the C-terminus generated by the mutation concurrently with the truncation. The IRS-type PTB domain and the positions of the SH2 target motifs encompassing Tyr-142, Tyr-169 and Tyr-277 are indicated (Y). (D) Alignment of the amino acid sequences of IRS-type PTB domains. Amino acids that are identical or similar in the ROG-1 PTB domain (37-128 amino acids) and others are shaded in black and gray, respectively. The asterisks show arginines characteristic of the IRS-type PTB domain, which are shown to coordinate a phosphotyrosine in the PTB target motifs. The position of the large deletion found in ROG-1(tm1031) and the amino acid numbers of the first and last residues of each panel are indicated. ROG-1(tm1031) lacks the core region of the PTB domain. (E) Phylogenic relationships between the PTB domains. Phylogenic relationships were calculated based on amino acid sequences of the PTB domains of indicated human adaptor molecules and ROG-1. The ROG-1 PTB domain is distantly related to the Dab or Shc-type PTB domain. The scale bar represents evolutionary distance (estimated numbers of amino acid substitutions per site).

 
The rog-1 gene is on LGII, in the region between the lin-42 and sup-9 loci, and encodes a protein (ROG-1) containing the IRS-type PTB domain from amino acid 37-128 (Fig. 1C), which is most homologous (36%–47% identity) to the PTB domains of SNT or Dok-family adaptors among Homo sapiens proteins (Fig. 1D,E). That ROG-1 does not have any other functional domains predictable from the primary sequence suggests a role as an adaptor protein. In general, mammalian Dok and SNT adaptors interact with PTKs via the PTB domain and recruit SH2-containing molecules upon tyrosine phosphorylation of the SH2 target motifs in the C-terminal region. In this context, it should be noted that ROG-1 also has three SH2 target motifs of the form Tyr-Xaa-Xaa-Pro or Tyr-Xaa-Xaa-Gln (Fig. 1C, marked with Y).

rog-1 is essential for development of germ cells

To investigate the physiological role of ROG-1, we used a reverse genetic approach. First, we employed RNA interference (RNAi), which has been shown to efficiently interfere with the expression of a specific gene in many model animals, including C. elegans (Fire et al. 1998). The 461-nucleotide fragment of the rog-1 cDNA, including the PTB domain sequence, was subcloned into the L4440 double promoter vector to generate a plasmid (L4440-rog-1) for feeding RNAi (Timmons & Fire 1998). This cDNA fragment did not show significant sequence similarity to any other C. elegans gene in a BLAST search, assuring that its RNAi effect was specific to rog-1. Down-regulation of the expression of rog-1 via RNAi significantly decreased brood size and increased embryonic lethality as compared to the controls (Fig. 2A,B), suggesting a role for rog-1 in oogenesis and embryogenesis. To further examine the function of rog-1, we obtained a deletion mutant generated with the trimethylpsoralen (TMP) and UV irradiation method by the National Bioresource Project for C. elegans in Japan (Gengyo-Ando & Mitani 2000). The rog-1(tm1031) mutant allele has a 516-bp deletion in the second exon and intron, where a thymine of unknown origin is inserted (Fig. 1B), resulting in the generation of a premature STOP codon 262 nucleotides down-stream of the start codon in the rog-1 mRNA. Thus, we concluded that the rog-1(tm1031) allele encodes a truncated protein comprising only 83 amino acids of ROG-1 fused with an additional 4 amino acids generated by the mutation. Given that the rog-1(tm1031) mutant lacks a major portion of the PTB domain, including an arginine predicted to coordinate a phosphotyrosine (Fig. 1C,D) (Eck et al. 1996), ROG-1(tm1031) appears to lack the PTB-dependent functions at least. rog-1(tm1031) homozygous hermaphrodites showed the same defects as rog-1(RNAi) worms, but to a greater extent. Namely, the rog-1 mutants showed greater reductions in brood size and increases in rates of embryonic lethality among the progeny than rog-1(RNAi) worms (Fig. 2). Autofluorescent gut granules of these dead embryos were observed with UV illumination; however, these granules did not show any gut structure. Also, we failed to find any dead embryo that appeared to be at or after the comma stage of development (data not shown), suggesting that the developmental arrest during embryogenesis occurred after the initiation of gut differentiation but before completion of its morphogenesis. In addition, the low rate of lethality among embryos laid by a heterozygous mother indicates a maternal effect as shown in Fig. 2D. Because the mutant phenotypes are essentially the same as those found in the RNAi-treated worms and the mutation that causes a large truncation of ROG-1 results in a completely recessive phenotype, tm1031 appears to be a loss-of-function mutation.


Figure 2
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  		Figure 2  rog-1 is essential for production and viability of embryos. Brood size (A, C) and embryonic lethality (B, D) in rog-1(RNAi) worms (A, B) or rog-1(tm1031) mutants (C, D) were evaluated as described in Experimental procedures. (A, B) HT115(DE3) bacteria having the expression plasmid for rog-1 dsRNA (rog-1 RNAi) or the control vector L4440 (Control) were grown overnight at 37 °C in LB and incubated on NGM agar plates supplemented with 50 µg/mL ampicillin and 1 mM IPTG overnight at 37 °C to allow generation of the dsRNA. Then, L4 hermaphrodites to be tested were cloned on the plates and fed at 20 °C. (C, D) Wild-type N2 (+/+) L4 hermaphrodites and heterozygous (+/–) or homozygous (–/–) L4 hermaphrodites of rog-1(tm1031) mutants were cloned and fed on NGM plates seeded with E. coli strain OP50. Worms were transferred to new plates every day for 3 days, and the phenotypes of their progeny were scored. Error bars represent SE calculated from experiments with 8–14 mothers that laid 150–3700 embryos. The mother's genotype was confirmed by PCR using specific primers for rog-1 as described in Experimental procedures.

 
Assuming that a defect in oogenesis or fertilization caused the severe decrease of brood size in rog-1(tm1031) homozygous hermaphrodites, we morphologically examined the homozygous mother's gonads. In the wild-type controls, DNA staining highlighted thread-like chromosomes in pachytene and condensed bivalents in diakinesis (Fig. 3C,G,H). In contrast, whereas the gonads of rog-1(tm1031) adult hermaphrodites had similar mitotic and transition zone nuclei at the distal end (Fig. 3A–F), the rest of the organ was greatly perturbed and had very few cells with the morphological characteristics of diakinesis, such as bivalent chromosomes, a granular cytoplasm and an enlarged nucleus as seen in wild-type animals (Fig. 3G–J). However, there were many developing germ cell nuclei that contained thread-like chromosomes characteristic of the pachytene stage in rog-1(tm1031) gonads as well as in N2 gonads (Fig. 3C,F). These results indicate that the progression from pachytene to diakinesis is severely disrupted in rog-1(tm1031) mutant gonads. Also, large aggregates of granular material accumulated within the syncytial core and clumps of pachytene stage nuclei were observed in the gonads of rog-1(tm1031) adults (Fig. 3D,E). Because these phenotypes are similar to those of mutants having a mutation in the genes essential for the Ras-MAP kinase pathway (Church et al. 1995), rog-1 appears to be required for the Ras-dependent development of oocytes. On the other hand, the rog-1(tm1031) mutants appeared to develop normally aside from the gonads; in particular, they did not show any abnormality in development of the vulva and male spicule and also lacked rod-like L1 lethality (data not shown). As these results were obtained from progeny of rog-1(tm1031) homozygous parents, maternal rescue cannot explain these phenotypes. Thus, rog-1 is dispensable for the Ras-dependent organogenesis. It is of note that rog-1(tm1031) homozygous males could produce outcross progeny normally, indicating that the rog-1 defect is confined to the female germ-line. Taken together, our findings demonstrate an essential role for the rog-1 gene in the transition of developing oocytes from pachytene to diakinesis and in embryogenesis.


Figure 3
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  		Figure 3  rog-1 is essential for the transition of developing oocytes from pachytene to diakinesis. Differential interference contrast images of the gonads dissected from adult hermaphrodites (DIC) and fluorescence micrographs of DNA visualized with Hoechst 33342 (DNA) are shown. (A–F) rog-1(tm1031) mutant hermaphrodites have many developing germ cells at the pachytene stage. Wild-type N2 worms (A–C) and rog-1(tm1031) mutants (D–F) were cultured at 20 °C. Arrowheads and arrows indicate aggregates of granular material and clumps of pachytene nuclei, respectively (D, E), both of which are hardly detectable in the wild-type worms (A, B). There are many developing germ cell nuclei containing thread-like pachytene-stage chromosomes in wild-type and rog-1(tm1031) worms (C, F). (G–R) Gain-of-function mutations of let-60 rescue the germ-line pachytene arrest phenotype of rog-1(tm1031) hermaphrodites. Wild-type N2 worms (G, H) and rog-1(tm1031) mutants (I, J) were cultured at 20 °C, whereas the mutants of let-60(n1046) (K, L), let-60(ga89ts) (M, N), rog-1(tm1031), let-60(n1046) (O, P) and rog-1(tm1031); let-60(ga89ts) (Q, R) were cultured at the restrictive temperature (25 °C). Arrowheads indicate diakinesis-stage nuclei, which are hardly detectable in rog-1(tm1031) mutant gonads (J). Dorsal is up and anterior is to the left. The distal end of each gonad is indicated with an asterisk. The scale bar represents 20 µm.

 
rog-1 expression in the gonad is essential for oocyte development

Since rog-1 is required for the development of germ cells, it is likely that this gene is expressed and functions in the gonad. To evaluate levels of rog-1 mRNA in the germ cells, we employed glp-4(bn2ts) mutants, because these worms grown at 25 °C, but not 16 °C, virtually lack germ cells (Beanan & Strome 1992). An RT-PCR-based analysis of the gld-1 gene, which shows a germ-line-restricted expression (Jones & Schedl 1995), demonstrated negligible expression in glp-4(bn2ts) mutants grown at 25 °C, but not 16 °C, confirming the very small numbers of germ cells in glp-4(bn2ts) mutants at the non-permissive temperature (25 °C) (Fig. 4A). Similarly, when the adult glp-4(bn2ts) mutants were cultured at 25 °C, the rog-1 mRNA was undetectable. In contrast, the mRNA expression of a ubiquitously expressed gene, act-1 (Files et al. 1983), was decreased but detectable at the non-permissive temperature in the mutant worms. These results indicate that the rog-1 gene is preferentially expressed in the germ cells, implying a role in these cells.


Figure 4
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  		Figure 4  rog-1 plays an essential role in the germ cells. (A) Total RNA was isolated from mixed-stage glp-4(bn2ts) mutant worms grown either at a permissive temperature (16 °C; lanes 1, 3, 5) or at a restrictive temperature (25 °C; lanes 2, 4, 6); the worms grown at 25 °C, but not 16 °C, virtually lack germ cells. RT-PCR was performed with a specific pair of primers for gld-1, rog-1 or act-1 and amplified DNA fragments were separated by electrophoresis on a 2% agarose gel and visualized with ethidium bromide. gld-1 is a germ-cell specific marker, whereas act-1 is expressed ubiquitously. (B, C) Wild-type N2 or rrf-1(pk1417) mutant worms were microinjected with rog-1 dsRNA (rog-1 RNAi) or M9 buffer (Control) and brood size (B) or embryonic lethality (C) was examined as in Experimental procedures. In rrf-1(pk1417) mutants, RNAi is effective only in the germ-line, not in the soma. The administration of rog-1 RNAi resulted in the same low brood sizes and high embryonic lethality in rrf-1(pk1417) mutants as in N2, indicating that rog-1 plays an essential role in the germ-line. Error bars represent SE calculated from experiments with 10–17 mothers that laid 70–2600 embryos.

 
Because the rrf-1 gene function is required for RNAi in the soma, but not in the gonad (Sijen et al. 2001), mRNAs that are expressed in the germ cells are exclusively targeted in the rrf-1(pk1417) mutants. Thus, we employed rrf-1(pk1417) to examine whether the expression of the rog-1 gene in the germ cells is required for oogenesis. Double-stranded RNA (dsRNA) directed against the rog-1 transcript was prepared from the plasmid L4440-rog-1 in vitro to be injected into the rrf-1(pk1417) mutants or the wild-type controls. Injection of the rog-1 dsRNA into N2 worms resulted in the same defects as the feeding RNAi but to a greater extent (Fig. 4B,C, in comparison with Fig. 2A,B). Also, the rrf-1(pk1417) mutants showed nearly the same responses to the injection of the rog-1 dsRNA as N2 worms, indicating that rog-1 expression in the gonad is essential for oocyte development and subsequent embryogenesis. As ROG-1 appears to be a cytoplasmic adaptor protein, it probably plays an essential role in the gonad syncytium. This assumption is consistent with the maternal-effect embryonic lethality described above (Fig. 2D).

rog-1 is required for activation of MPK-1 MAP kinase in the gonad

It has been established that the transition of developing oocytes from pachytene to diakinesis is controlled by the Ras-MAP kinase pathway in C. elegans (Sternberg & Han 1998). LET-60, MEK-2 and MPK-1/SUR-1 are the nematode counterparts of the mammalian Ras, MEK and Erk MAP kinase, respectively, and many loss-of-function mutations of these genes cause a pachytene arrest (Church et al. 1995). Like other species, C. elegans LET-341 SOS-1, a potent activator of LET-60 Ras, is necessary for several Ras-dependent developmental processes, including the transition from pachytene to diakinesis (Chang et al. 2000). However, the nature of an up-stream regulator of SOS-1/Ras-MAP kinase signaling in this particular event has been an open question. Given that the rog-1(tm1031) hermaphrodite also showed a pachytene arrest phenotype (Fig. 3I,J), ROG-1 is likely involved in the signaling mediated by the Ras-MAP kinase pathway. Because adaptors having a PTB domain usually interact with PTKs and regulate down-stream pathways, including the Ras-MAP kinase pathway, we hypothesized that ROG-1 might be an up-stream regulator of the pathway in oocyte development. To test this, we performed immunostaining experiments to visualize the phosphorylation, and thereby activation, of MPK-1 MAP kinase, which plays an essential role during the transition of developing oocytes from pachytene to diakinesis (Page et al. 2001). It is of note that MPK-1 is also activated in a few oocytes in the proximal region of the gonad in response to a signal from the sperm (Miller et al. 2001). Immunostaining of the dissected wild-type N2 gonads with monoclonal antibodies to the di-phosphorylated, activated form of MPK-1 successfully revealed these activations (Fig. 5A,B). In contrast, MPK-1 was not detectably activated in the gonads of rog-1(tm1031) homozygotes (Fig. 5C,D), indicating that ROG-1 is essential for the activation of MPK-1 at least in germ cells during pachytene. However, the role of ROG-1 in MPK-1's activation in oocytes in the proximal region is yet unclear because the pachytene arrest phenotype of the rog-1(tm1031) mutant hinders the production of fully developed oocytes.


Figure 5
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  		Figure 5  Gain-of-function mutations of let-60 rescue the germ-line MPK-1 inactivation phenotype of rog-1(tm1031) hermaphrodites. Gonads were dissected from young adult hermaphrodites within 1 day of the L4/adult molt, and stained with antibodies to the activated (di-phosphorylated) form of MPK-1 (pMPK-1; left panels). DNAs were counterstained with DAPI (DNA; right panels). Wild-type N2 (A, B), rog-1(tm1031) (C, D) were grown at 20 °C, whereas let-60(n1046) (E, F), let-60(ga89ts) (G, H), rog-1(tm1031), let-60(n1046) (I, J) and rog-1(tm1031); let-60(ga89ts) (K, L) were grown at the restrictive temperature (25 °C). The activation of MPK-1 is apparent in the pachytene zone (P) and in a few oocytes at the proximal end of the gonads except for the rog-1(tm1031) single mutant (C). Arrowheads indicate diakinesis-stage nuclei, of which there are very few in the gonad of the rog-1(tm1031) single mutant (D). The distal end of each gonad is indicated with an asterisk. The scale bar represents 50 µm.

 
rog-1 acts up-stream of let-60 Ras

Because rog-1 is required for the activation of MPK-1 in the gonad, we next examined whether it plays a role up-stream of let-60 Ras by interfering with the expression of rog-1 in various let-60 mutants carrying a reduction-of-function allele, n2021, or a gain-of-function allele, n1046 or ga89ts (Ferguson & Horvitz 1985; Eisenmann & Kim 1997). As mentioned above, N2 worms subjected to the rog-1 feeding RNAi (rog-1(RNAi)) or rog-1(tm1031) worms had a partially or fully decreased brood size, respectively (Fig. 6A). Administration of the rog-1 feeding RNAi to let-60(n2021), a reduction-of-function mutant, further decreased brood size as compared to that of rog-1(RNAi) alone or let-60(n2021) alone, indicating that both rog-1 and let-60 function as a positive regulator of oocyte development. However, the rog-1(tm1031); let-60(n1046 or ga89ts) double mutants showed comparable brood sizes to the single let-60(n1046 or ga89ts) mutants, indicating that each gain-of-function mutation of let-60 Ras was epistatic to the loss-of-function mutation of rog-1 in the pathway. With regard to embryonic lethality, essentially the same conclusion was drawn (Fig. 6B). The rog-1(RNAi) and rog-1(tm1031) mutants had moderately and extremely high rates of embryonic lethality, respectively, whereas N2 had a very low rate. Administration of the rog-1 RNAi to let-60(n2021) significantly increased the level of embryonic lethality; however, the rog-1(tm1031); let-60(n1046 or ga89ts) double mutants and the single let-60(n1046 or ga89ts) mutants showed the same low rates of embryonic lethality. These results together indicate that rog-1 acts up-stream of let-60 Ras in the same or a closely related pathway.


Figure 6
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  		Figure 6  rog-1 acts up-stream of let-60 Ras. Wild-type N2, rog-1(RNAi), let-60 reduction-of-function mutant n2021 and rog-1(RNAi); let-60(n2021) were grown at 20 °C, whereas let-60 gain-of-function mutants, n1046 and ga89ts, or rog-1(tm1031) worms with or without the let-60 mutation (n1046 or ga89ts) were grown at the restrictive temperature (25 °C). Note that let-60(n2021) was subjected to the control feeding RNAi with empty L4440 vector. Brood size (A) or embryonic lethality (B) was evaluated as described in Experimental procedures. Error bars represent SE calculated from experiments with 8–10 mothers that laid 70–1900 embryos.

 
Further, we examined the dissected gonads of let-60(n1046 or ga89ts) mutants and rog-1(tm1031); let-60(n1046 or ga89ts) double mutants (Fig. 3K–R). Although both let-60 gain-of-function mutants had abnormal vulvae as well as reduced brood sizes, the germ cell morphology in their gonads, including characteristic chromosome condensation patterns, were apparently normal, indicating a practically unperturbed transition from pachytene to diakinesis (Fig. 3K–N). As expected, worms carrying both the rog-1(tm1031) and let-60(n1046 or ga89ts) mutations showed an almost indistinguishable development of oocytes from that in let-60(n1046 or ga89ts) single mutants (Fig. 3O–R), confirming that let-60 Ras is epistatic to rog-1 in the pathway. In addition, we examined the MPK-1 phosphorylation pattern in the gonads of rog-1(tm1031); let-60(n1046 or ga89ts) double mutants and found that it was almost indistinguishable from that in the gonads of let-60(n1046 or ga89ts) single mutants (Fig. 5E–L). Taken together, our results demonstrate that ROG-1 is a key positive regulator of LET-60 Ras in the development of oocyte in C. elegans.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we have identified the gene rog-1, which encodes an adaptor-like protein that has an IRS-type PTB domain. Several lines of evidence have also demonstrated that rog-1 acts up-stream of let-60 Ras to activate MPK-1 MAP kinase in the gonad and play an essential role in the progression of developing oocytes from pachytene to diakinesis. Given that worms carrying a loss-of-function mutation in the let-60 Ras, mek-2 MEK or mpk-1 MAP kinase gene show the pachytene exit defect, we concluded that the adaptor-like protein ROG-1 is a positive regulator of the Ras-MAP kinase pathway. However, whereas mutations in these genes also caused abnormal development of the vulva and male spicule in addition to death in the first larval stage with a fluid-filled appearance, rog-1(tm1031) loss-of-function mutants did not show these phenotypes. In addition, rog-1(tm1031) homozygous males could produce normal numbers of outcross progeny, suggesting an intact spermatogenesis. Thus, the ROG-1-mediated regulatory mechanism of the Ras-MAP kinase pathway is probably restricted in the female germ-line. Consistent with this notion, rog-1 is preferentially expressed in this tissue (Fig. 4A), and germ-line-specific down-regulation of this expression caused the same defects as were observed in rog-1(tm1031) mutants (Fig. 4B,C).

The PTB domain was originally identified as a module that specifically binds to a short peptide of the form Asn-Pro-Xaa-Tyr in a manner dependent on tyrosine phosphorylation of the target motif. There are several proteins having the PTB domain in C. elegans, including CED-6, FEH-1 and LIN-10, which are known to play essential roles in the engulfment of apoptotic cells, pharyngeal pumping and vulval induction, respectively (Liu & Hengartner 1998; Zambrano et al. 2002; Glodowski et al. 2005). Moreover, mutations that disrupt the PTB domain of ARH, FE65 and CCM2(MGC4607) are associated with inherited hypercholesteremia, Alzheimer's disease and cerebral cavernous malformation, respectively, suggesting the biological importance of proteins carrying the PTB domain (Mammarella et al. 2000; Garcia et al. 2001; Hu et al. 2002). In general, this domain is classified into two distinct subgroups: the longer Shc-type and the shorter IRS-type (Wolf et al. 1995). The IRS-type PTB domain coordinates the phosphotyrosine residue through two core arginines (Fig. 1D), which are considered to interact with a phosphorylated tyrosine, but not with a non-phosphorylated tyrosine, in the target motifs. Thus, this type of PTB domain implies an involvement in PTK-mediated signal transduction. However, not a few PTB domains bind to non-phosphorylated targets and may be segregated as the Dab-type from the phosphotyrosine-specific Shc-type of PTB domains (Uhlik et al. 2005). As mentioned earlier, ROG-1 has the IRS-type PTB domain, where the two arginines thought to coordinate a phosphotyrosine are conserved, suggesting a role in PTK-mediated signaling.

Spatiotemporal control of the Ras-MAP kinase pathway by up-stream regulators, including PTKs and G-protein-coupled receptors, is an important factor determining the specificity of cellular responses. In C. elegans, the regulatory mechanism of the Ras-MAP kinase pathway in vulval induction has been precisely elucidated (Sternberg & Han 1998). In hermaphrodites, vulval induction is initiated upon activation of a receptor PTK, LET-23/EGFR, in the vulval precursor cells by its extracellular ligand LIN-3. Then, activated, and thus autophosphorylated, LET-23 recruits the adaptor protein SEM-5/Grb2 that is complexed with SOS-1, a potent activator of Ras (Chang et al. 2000), to activate the Ras-MAP kinase pathway. Mutations that disable or activate these molecules are known to induce a vulvaless or multivulva phenotype, respectively. In contrast, up-stream regulators of the Ras-MAP kinase pathway essential for the oocyte to progress through the pachytene stage of meiosis have been unknown apart from SOS-1, even though a stimulating signal is postulated to be provided by somatic gonadal sheath cells that are in direct contact with germ cells in pachytene (McCarter et al. 1997). Given that ROG-1 is essential for activation of the Ras-MAP kinase pathway in the gonad and has the IRS-type PTB domain, the ROG-1-Ras-MAP kinase pathway may be regulated by receptor PTKs having the PTB target motif: Asn-Pro-Xaa-Tyr or the like (Uhlik et al. 2005). Following a database search, we found DAF-2 insulin receptor, EGL-15 FGFR and VER-3 VEGFR to be receptor PTKs that have the PTB target motif. Since daf-2 and egl-15, but not ver-3, transcripts are detectable in the gonad (results from NEXTDB by Y. Kohara, Tokyo, Japan. Available at: <http://nematode.lab.nig.ac.jp/>), ROG-1 may interact with these receptor PTKs via the PTB domain. The mammalian adaptor SNT-1/2 also has a PTB domain, which shows high homology to the ROG-1 counterpart (Fig. 1D,E), and binds to the PTB target motif of receptor PTKs, FGFR and NTRK1. Then, SNT-1/2 is tyrosine phosphorylated to recruit Grb2 and SHP2 via the SH2-binding motifs, and activates the Ras-MAP kinase pathway. This signaling event is, for example, essential for oocyte maturation in Xenopus laevis regulated by FGFR (Mood et al. 2002). Because ROG-1 has multiple SH2 target motifs (Fig. 1C), it may similarly provide a molecular platform necessary for signal transduction from an as yet unidentified PTK to the Ras-MAP kinase pathway in the gonad.

Since Ras is attached to the cellular membrane via lipidation, its regulators must be recruited to the membrane/juxtamembrane compartment for interaction to occur. Although the PTB domain of ROG-1 shows the highest amino acid identity (38%–47%) to that of SNT1/2 and of Dok-4/5/6, it apparently lacks a module for interacting with membranes. SNT1/2 has the N-terminal motif needed for myristoylation, which facilitates localization to the membrane; and Dok-4/5/6 has the PH domain known to interact with specific types of lipids in the membrane compartment (Grimm et al. 2001; Crowder et al. 2004). However, binding to receptor PTKs via the PTB domain by itself would give rise to a juxtamembrane-based localization of ROG-1. Indeed, the adaptor Shc, which lacks a PH domain and lipid-modification site, directly binds to receptor PTKs to be phosphorylated and activate Ras. Moreover, the PTB domains are similar to the PH domains in three-dimensional structure, and indeed the PTB domains of Shc, IRS-1, Dab1/2, X11, Numb, Talin and several other proteins have been shown to interact with membrane lipids, raising the possibility that ROG-1 is recruited to the membrane/juxtamembrane compartment upon receptor PTK-mediated signaling. Given that SNT1/2 and Dok-4/5/6 are considered to positively regulate the Ras-MAP kinase pathway down-stream of PTKs and that ROG-1 is unique in having the IRS-type PTB domain in C. elegans (Grimm et al. 2001; Gotoh et al. 2004), we speculate that SNT1/2, Dok-4/5/6 and ROG-1 may have evolved from a common ancestor of adaptor proteins. Although the precise mechanisms underlying the positive regulatory function of ROG-1 remain unclear, their elucidation would contribute to a comprehensive understanding of how oocytes develop in C. elegans.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains and alleles

The maintenance and genetic manipulation of C. elegans were carried out as described (Brenner 1974). C. elegans strain N2 variety Bristol was used as the wild-type. Worms were grown at 20 °C on nematode growth medium (NGM) agar plates spread with E. coli OP50 unless otherwise noted. Mutations used in this study were LGI, glp-4(bn2ts) and rrf-1(pk1417); LGII, rog-1(tm1031); and LGIV, let-60(ga89ts), let-60(n1046) and let-60(n2021). The analysis of rog-1(tm1031) mutant phenotypes was carried out after eight rounds of backcrosses of the original isolate to N2. Genotyping of rog-1(tm1031) was performed with PCR using the two sets of rog-1 specific primers: rog-1-type-Fw1, 5'-CTGCCGTACCGAAAACCGAT-3'; rog-1-type-Rv1, 5'-CTGACAGGTGTTCTGTGTAT-3'; rog-1-type-Fw2, 5'-CTGAAATATCTCATCAGCTTTT-3'; rog-1-type-Rv2, 5'-GTAGTCCGTCTGACAGGTGT-3'.

Cloning of full-length rog-1 cDNA

Based on the nucleotide sequence of F54D12.6 (Chromosome II: 1374991-1373695; WormBase), forward (rog-1-ex1-Fw, 5'-CGATTCTGGGCAATCGACGTGG-3'; 1374963-1374942) and reverse (rog-1-ex3-Rv, 5'-TACGGATTGCTGTCGCTGGTACCGG-3'; 1373839-1373863) primers were designed to amplify a partial rog-1 cDNA fragment by PCR, which was performed with C. elegans mixed stage cDNAs generated from total RNAs. The amplified rog-1 cDNA fragment was used as a probe for screening a Nematode embryo lambda cDNA library (Stratagene) according to standard plaque hybridization procedures, with the aim of obtaining a full-length rog-1 cDNA. Because cDNA corresponding to the 5' portion of the rog-1 mRNA had not been obtained, we amplified it by standard RT-PCR using SL1 or SL2, a trans-spliced leader sequence, as the 5' primer, together with the rog-1-ex3-Rv primer. The complete sequence data for rog-1 are available from GENBANK/EMBL/DDBJ under accession number AB259784.

Evaluation of brood size and embryonic lethality

Brood size and embryonic lethality were evaluated by placing L4 hermaphrodites on to individual plates, and transferring the worms to a new plate every day for 3 days to enumerate non-hatched embryos at day 1 and adult animals at day 3 on each plate. Brood size is the sum of non-hatched and hatched progeny per mother hermaphrodite. Embryonic lethality represents the ratio of the number of non-hatched embryos to brood size.

Feeding RNAi

A cDNA fragment covering the ROG-1 PTB domain was amplified with the rog-1-ex1-Fw and rog-1-ex3-Rv primers and inserted appropriately into the L4440 double promoter vector to generate a rog-1 dsRNA expression plasmid (L4440-rog-1). E. coli strain HT115(DE3) was transformed with L4440-rog-1 or empty L4440 vector, grown in Luria broth (LB) overnight at 37 °C, and, for the expression of dsRNA, incubated overnight at 37 °C on NGM agar plates supplemented with 50 µg/mL ampicillin and 1 mM isopropyl thiogalactoside (IPTG). L4 hermaphrodites were transferred to these plates and fed at 20 °C for the subsequent evaluation of brood size and embryonic lethality as described above.

Injection RNAi

Sense and anti-sense strands of RNAs were transcribed in vitro from the plasmid L4440-rog-1 using RiboMAX Large Scale RNA Production Systems—T7 (Promega). A mixture of these RNAs was heated at 65 °C for 5 min and then slowly allowed to cool to generate dsRNAs, which were microinjected into L4 hermaphrodites according to the standard protocol (Mello et al. 1991).

DNA staining with Hoechst 33342

DNAs in the extruded gonads were visualized with Hoechst 33342 in Fig. 3 as follows. Worms were cut open in 10 µL of 100 µg/mL Hoechst 33342 in M9 buffer on a poly L-lysine-treated slide. Dyes were allowed to penetrate into worm tissues for 5 min, and the specimens were covered with a coverslip, sealed with nail polish and observed with Nomarski and fluorescence microscopy.

RT-PCR with glp-4(bn2ts) mutants

For the temperature shift experiments in Fig. 4A, glp-4(bn2ts) embryos were prepared by NaOCl/NaOH digestion of the gravid hermaphrodites raised at 16 °C. Then, the embryos were allowed to develop at 25 °C or 16 °C to adulthood. Adult hermaphrodites were lyzed in 500 µg/mL of proteinase K for 1 h at 55 °C and RNAs were extracted with phenol/chloroform, precipitated with ethanol and resuspended in nuclease-free water. Residual DNAs were digested by treatment with RNase-free DNase I. Oligo (dT)12-18-primed reverse transcription was performed using SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's directions. Subsequent PCR was performed with cDNAs generated from approximately 5 µg of each RNA sample and the following primer sets, which were designed to amplify the rog-1, gld-1 and act-1 cDNAs, respectively:

rog-1-Fw, 5'-CGGGATCCACCATGCGGGCAATTATTTCGGT-3';

rog-1-Rv, 5'-CCGCTCGAGGGGTATAATGCCG-3';

gld-1-Fw, 5'-TGGGAGCATCTCGAAGACGATCTGCACGTTCTTGTGC-3';

gld-1-Rv, 5'-GAAAGAGGTGTTGTTGACTGAAGAAGCCGAGGGACTTG-3';

act-1-Fw, 5'-CGTGGTTACTCTTTCACCACCACCGCTG-3';

act-1-Rv, 5'-CATTTAGAAGCACTTGCGGTGAACGATGG-3'.

Immunostaining of activated MPK-1

For immunostaining of the phosphorylated, and thereby activated, form of MPK-1, a monoclonal antibody (Sigma, M8159) specific to di-phosphorylated ERK-1/2 was used. This antibody has been shown to react with the di-phosphorylated form of the MAP kinases of various species, including C. elegans MPK-1 (Miller et al. 2001; Page et al. 2001). Young adult worms were dissected in 10 µL of 0.2 mM levamisole in PBS on a poly L-lysine-treated slide glass, and extruded gonads were fixed in 3.7% formaldehyde, followed by immersion in liquid nitrogen for 5 min with a coverslip, which was removed immediately after the treatment. The frozen gonads were dehydrated at –20 °C in methanol for 15 min and acetone for 15 min and dried under air. Then, the gonads on the slide glasses were incubated in 100 µL of 1.5% ovalbumin/1.5% bovine serum albumin in PBS (OVA/BSA) at room temperature for 30 min. The OVA/BSA-treated gonads were further treated with a diphospho-specific anti-ERK monoclonal antibody (1 : 200) in 100 µL of OVA/BSA at 4 °C for 12–18 h. After a wash with PBS at room temperature for 30 min twice, the primary antibody-treated gonads were incubated in 100 µL of OVA/BSA at room temperature for 30 min and then treated with 100 µL of Alexa 594-conjugated goat antibodies to mouse IgG (Invitrogen, A-11005) 1 : 500 in PBS at room temperature for 6 h. After another wash with PBS at room temperature for 30 min, the gonads were counterstained with 0.1 µg/mL of 4',6-diamidino-2-phenylindole (DAPI) in PBS, washed again and mounted in Elvanol mounting fluid. Finally, a coverslip was placed on the fluorescence-labeled gonads on the slide glass and sealed with nail polish, and the images were observed under a fluorescence microscope.


   Acknowledgements
 
We thank Hidehito Kuroyanagi, Yoshishige Kimura, Yoshiki Andachi, and Yuji Kohara for discussions. Some of the nematode strains used in this study were provided by the National Bioresource Project for C. elegans in Japan and the Caenorhabditis Genetics Center. This work was supported by Grants-in-Aid for Scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from the Uehara Foundation.


   Footnotes
 
Communicated by: Tadashi Yamamoto

aPresent address: Molecular Neuroscience Unit, Okinawa Institute of Science and Technology, 12-2 Suzaki, Uruma, Okinawa 904-2234, Japan. Back

bPresent address: Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. Back

* Correspondence: E-mail: yamanashi.creg{at}mri.tmd.ac.jp


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Received: 14 November 2006
Accepted: 18 December 2006





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