FEBS Letters
Volume 583, Issue 19 , Pages 3158-3164, 6 October 2009

Ce-wts-1 plays important roles in Caenorhabditis elegans development

Edited by Tamas Dalmay

College of Life Science, Peking University, Beijing 100871, PR China

Received 14 July 2009; received in revised form 2 September 2009; accepted 2 September 2009. published online 07 September 2009.

Article Outline

Abstract 

The Hippo–Warts pathway defines a novel signaling cascade involved in organ size control and tumor suppression. However, the developmental function of this pathway is less understood. Here we report that the Caenorhabditis elegans homolog of Warts, Ce-wts-1, plays important roles during worm development. The null allele of Ce-wts-1 causes L1 lethality. Partial loss of Ce-wts-1 function by RNAi reveals that Ce-wts-1 is involved in many developmental processes such as larval development, growth rate regulation, gut granule formation, pharynx development, dauer formation, lifespan and body length control. Genetic analyses show that Ce-wts-1 functions synergistically with the TGF-β Sma/Mab pathway to regulate body length. In addition, CE-WTS-1::GFP is enriched near the inner cell membrane, implying its possible membrane-related function.

Keywords: Warts family, Ce-wts-1, TGF-β, Development, Membrane, Caenorhabditis elegans

 

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1. Introduction 

Cell proliferation, differentiation and programmed cell death are essential processes during animal development. Any aberration in these processes may lead to tumorigenesis. Mutations of two classes of genes, protooncogenes and tumor suppressor genes, can lead to cancer development [1].

Recently, the Salvador–Hippo–Warts cascade was identified as a new tumor suppressor network [2]. The signal from extracellular milieu is received by transmembrane proteins, such as Fat. Then it is relayed by cytoplasmic proteins, including Expanded, Merlin, Hippo, Salvador, Warts (Wts; also called Lats in mammals), and Mats. Finally, it leads to the phosphorylation and inhibition of Yorkie, a transcriptional coactivator that positively regulates cell proliferation and survival. A lack of these tumor suppressors leads to overgrowth in a variety of tissues in Drosophila melanogaster, while a gain-of-function leads to reduced proliferation and ectopic apoptosis [3]. The importance of this pathway is emphasized by its simultaneous and unisonous control of cell proliferation and apoptosis, and its evolutionary conservation. The increasing evidence also indicates that the deregulation of this pathway occurs in human tumors.

To understand how inactivation of tumor suppressors leads to tumorigenesis, it is common to decipher the pathways in animal developmental processes. The developmental roles of the Hippo–Warts pathway are poorly documented, especially in C. elegans [2]. In this report, we explored the function of Warts, a core component of the Hippo/Warts pathway, in worm development.

We identified the C. elegans homolog of warts, Ce-wts-1. Null allele of Ce-wts-1 leads to L1 lethality. Partial depletion of Ce-wts-1 exhibits many defects during development. Genetic analyses show that Ce-wts-1 genetically interacts with the TGF-β pathway and other small mutations. The expression pattern of Ce-WTS-1::GFP reveals that Ce-WTS-1 is mainly expressed intracellularly near the membrane.

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2. Materials and methods 

2.1. Strains 

The nematode C. elegans was maintained as described by Brenner [4]. Worms were grown at 20°C unless otherwise noted. The following alleles were used in this work: LGI: rnt-1(ok351), mef-2(gv2), Ce-wts-1(ok753); LGII: sma-6(wk7), eat-2(ad465), eat-3(ad426); LGIII: sma-2(e502), sma-3(e491), daf-7(e1372); LGIV: sma-4(e729), tax-6(p675), eat-1(ad427), pha-3(ad607); LGV: dbl-1(wk70), sma-1(ru18); LGX: kin-29(oy38), pha-2(ad472), sma-5(n678) [5]. The transgenic markers are: juIs76 (Punc-25::gfp) for D-type neurons, wIs51 (scm-1::gfp), jcIs1 (ajm-1::gfp) for seam cells, fwEX1(pRF4, Ce-wts-1::gfp).

2.2. Construction of Ce-wts-1::gfp reporter 

The Ce-wts-1::gfp reporter contains the promoter, coding sequence and 3’UTR (T20F10, nt 17884–27380). gfp was inserted at the C-terminus of Ce-wts-1. The reporter DNA was coinjected with pRF4 (rol-6) and at least two transgenic lines were analyzed.

2.3. RNAi and microinjection 

Single-stranded RNA was transcribed from the T7 and SP6-flanked PCR templates. The PCR template used for synthesizing RNA is: Ce-wts-1 (cDNA, nt 1645–2620). The single-stranded RNAs were annealed and injected into N2 and different mutants. Eggs laid between 24 and 48h after microinjection were collected for further analyses.

2.4. Body length measurement 

L4 hermaphrodites grown at 20°C were transferred to fresh NGM plates. One day later, animals were mounted on 5% agarose pad and photographed under 10× or 20× objectives with Zeiss AxioCam. Only animals with split vulva, mature oocytes and few embryos (⩽2) were used for body length measurement by Image J [5].

2.5. Measurement of seam cell size 

The ajm-1::gfp that specifically marked the seam cell membrane was used to measure the seam cell area [6], [7]. Late L3 larvae were picked and seam cells were photographed under 100× objective. Three consecutive cells ideal for measurement were selected per worm and measured by Zeiss AxioVision.

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3. Results 

3.1. Ce-wts-1 encodes the warts homolog in C. elegans 

Searching C. elegans database revealed a worm homolog of warts, T20F10.1, which we named Ce-wts-1. Similar to Warts in fly and mammals, Ce-wts-1 encodes a kinase of the Nuclear Dbf-2-related (NDR) family [8], [9]. The predicted Ce-WTS-1 product contains 908 amino acids, with a characteristic Serine/threonine kinase domain between aa502 and aa807. The Ce-WTS-1 kinase domain is 54% identical to those of fly Warts and human LATS kinase domains. ok753 allele of Ce-wts-1 (provided by C. elegans Gene Knockout Consortium), contains a frame-shift deletion from aa544 to aa657, and causes L1 lethality (data not shown). We injected a 10kb genomic DNA which contains 2kb promoter, 6kb coding region and 2kb 3′UTR of Ce-wts-1 into heterozygous ok753 worms, and found that this Ce-wts-1 genomic DNA could completely rescue ok753−/− lethality, indicating that the L1 lethal arrest of ok753−/− is due to Ce-wts-1 mutation (data not shown). Since ok753 removes most of the kinase domain, which has been demonstrated to be crucial for Warts function [10], we believe that ok753 may be a null allele of Ce-wts-1. We also noted that Ce-wts-1 genomic DNA lacking the first 1kb intron failed to rescue ok753−/−, suggesting that the first intron may contain positive gene regulatory elements.

3.2. Loss of Ce-wts-1 function has pleiotropic effects in C. elegans development 

Because ok753−/− animals arrest at L1 stage, we could not explore the developmental role of Ce-wts-1 beyond L1 stage. Alternatively, we used RNAi to create the partial loss of Ce-wts-1 function. We synthesized Ce-wts-1 double-stranded RNA (dsRNA) and microinjected it into worms. We checked the specificity of Ce-wts-1(RNAi) by injecting it into fwEX1(pRF4, Ce-wts-1::gfp) worms. The results showed that the expression of Ce-wts-1::gfp was largely eliminated by Ce-wts-1(RNAi), confirming that the dsRNA is indeed targeting Ce-wts-1 gene (Fig. 1I and J). We found Ce-wts-1(RNAi) caused many developmental defects, including larval lethality, constitutive dauer and longer lifespan, growth retardation, less gut granules, distorted pharynx, and small body size.

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  • Fig. 1. 

    Ce-wts-1(RNAi) causes pleiotropic defects in worm development. (A) Depletion of Ce-wts-1 activity lead to larval lethality and increased constitutive dauer formation (Daf-c). (B) Ce-wts-1(RNAi) worms grew much slower than N2. (C and D) Knockdown of Ce-wts-1 caused decreased gut granules (arrows, pictures were taken at the same exposure time). (E and F) Pharynxes were distorted in Ce-wts-1(RNAi) worms (broken line). (G and H) Ce-wts-1(RNAi) worms were smaller than wild-type. (I and J) Pharynx GFP was largely eliminated by Ce-wts-1(RNAi) (J), compared with worms before RNAi (I).

3.2.1. Larval lethality 

As shown in Fig. 1A, 47% (n=241) of RNAi-treated worms die at different larval stages, and 53% of them can grow to the adult stage.

3.2.2. Growth retardation 

Decrease of Ce-wts-1 activity leads to slow growth (Fig. 1B). It took nearly 95h for severely affected worms to grow to the young adult stage at 20°C. The rest worms took about 73h to enter the young adult stage, while N2 worms needed about 55h. Interestingly, Lats1−/− knockout mice also display decreased growth rate [11], suggesting the function of Ce-wts-1 in regulating animal growth may be conserved.

3.2.3. Constitutive dauer formation and long lifespan 

Data from the Ruvkun lab showed that inactivation of Ce-wts-1 by RNAi feeding could make worms live longer [12]. Our experiments confirmed this observation (data not shown). Study from the Kenyon lab showed that a weak allele of daf-2 lives longer, while a strong allele becomes dauer worm at L2 stage [13]. We thus examined the role of Ce-wts-1 in dauer formation. In C. elegans, the TGF-β dauer pathway inhibits entry into dauer stage in wild-type worms, and DAF-7 is its ligand. daf-7(e1372) is a temperature-sensitive mutation. At 15°C, only a small fraction of daf-7 mutants get into dauer stage. At 25°C, 100% become dauer worms. When Ce-wts-1 dsRNA was injected into daf-7 worms at 15°C, the percentage of dauer worm increased from 13% (n=50) to 79% (n=153) (Fig. 1A), suggesting a genetic interaction between the Hippo–Warts pathway and the TGF-β dauer pathway.

3.2.4. Less gut granules 

Gut granules can be visualized by its autofluorescence under microscopic DAPI channel. We found that autofluorescence from gut granules drastically diminished in Ce-wts-1(RNAi) worms (Fig. 1C and D).

3.2.5. Distorted pharynx 

We found that in the slower-growing RNAi worms, the pharynxes were distorted to some extent (Fig. 1E and F). This phenotype is also found in other small mutants.

3.2.6. Small body size 

In D. melanogaster, loss of warts function leads to tissue overgrowth [9]. However, Lats1−/− knockout mice exhibit small size/decreased weight, although there are hyperplastic changes in the pituitary [11]. In C. elegans, knockdown of Ce-wts-1 also displayed the small body size phenotype (Fig. 1G and H). At young stage, the body length of RNAi-treated worms was 29% shorter than that of wild-type animals (n=37) (Table 1). We checked whether the small body size phenotype was the consequence of less cells. We injected Ce-wts-1 dsRNA into juIs76 and wIs51, which specifically marked D-type neurons and hypodermal seam cells [14]. We found in Ce-wts-1(RNAi) worms, numbers of D-type neurons and hypodermal seam cells (19.8±0.8, 16.0±0.8, n=15) were similar to those of wild-type worms (18.9±0.3, 16.0±0.4, n=15). We then measured seam cell size using jcIs1(ajm-1::gfp) that labels the seam cell membrane [6], [7]. In late L3 Ce-wts-1(RNAi) worms, the average area of seam cells was 83.4±17.6μm2 (n=52), significantly smaller than that of wild-type worms (117.4±11.3μm2, n=39; P<0.001). These results imply that smaller cell size may be responsible for the small size phenotype in Ce-wts-1(RNAi) worms.

Table 1. Genetic interactions between Ce-wts-1 and other Small mutations.
GenotypeBody length (mm)a (means±S.D.)Relative value of the mean body lengthProteinReferences
N20.96±0.04100
Ce-wts-l(RNAi)0.68±0.0771bSerine/threonine kinase
dbl-1(wk70)0.59±0.0462Ligand of TGF-β signaling[15]
dbl-1; Ce-wts-l(RNAi)0.43±0.0445b
sma-2(e502)0.54±0.0457R-Smad[16], [18]
sma-2; Ce-wts-l(RNAi)0.43±0.0444b
sma-3(e491)0.60±0.0562R-Smad[16], [18]
sma-3; Ce-wts-1(RNAi)0.39±0.0241b
sma-4(e729)0.52±0.0261Co-Smad[16], [18]
sma-4; Ce-wts-1(RNAi)0.48±0.0150b
sma-6(wk7)0.59±0.0361Type I receptor of TGF-β signaling[17]
sma-6; Ce-wts-1(RNAi)0.44±0.0346b
lon-1(e185)1.03±0.04107With homology to CRISP protein[29]
lon-1; Ce-wts-1(RNAi)0.96±0.07100
lon-2(e678)1.01±0.05105Glypican protein of HSPG[30]
lon-2; Ce-wts-1(RNAi)0.75±0.0378b
sma-1(ru18)0.67±0.0670βH-spectrin[19]
sma-1; Ce-wts-1(RNAi) Embryo lethal
sma-5(n678)0.50±0.0352Serine/threonine kinase[27]
sma-5; Ce-wts-1(RNAi) L1 lethal
kin-29(oy38)0.62±0.0565Serine/threonine kinase[28]
kin-29; Ce-wts-1(RNAi) L1 lethal
eat-1(ad427)0.80±0.0483α-Actinin associated LIM protein[5]
eat-1; Ce-wts-1(RNAi) L1 lethal
eat-3(ad426)0.76±0.0679Dynamin-like GTP binding protein[25]
eat-3; Ce-wts-1(RNAi) L1 lethal
pha-2(ad472)0.73±0.0576Homeodomain transcription factor[26]
pha-2; Ce-wts-1(RNAi) L1 lethal

aBody length was only measured for young adult worms with split vulva, mature oocytes and ⩽2 embryos. At least 15 worms were measured per genotype.

bThese body length data differ significantly from those of wild-type and the parental single mutants in unpaired t-test (P<0.005).

3.3. Ce-wts-1 synergistically interacts with small genes 

To explore how Ce-wts-1 interacts with signaling pathways during development, we choose the small phenotype of Ce-wts-1(RNAi) worms to do genetic analyses. In C. elegans, four pathways have been reported in regulating body length [5]. These pathways are: a TGF-β Sma/Mab pathway including dbl-1, sma-2, sma-3, sma-4 and sma-6 [15], [16], [17], [18]; a spectrin pathway including sma-1, spc-1 and unc-70 [19], [20], [21]; a calcineurin pathway including tax-6 and cnb-1 [22], [23]; a feeding defective pathway including eat-1, eat-2, eat-3, pha-2, pha-3, etc. [5], [24], [25], [26]. In addition, there are still some small genes that have not been assigned to these pathways, like sma-5, kin-29, etc. [27], [28]. To explore whether Ce-wts-1 also genetically interacts with these mutations, we injected Ce-wts-1 dsRNA into dbl-1(wk79), sma-2(e502), sma-3(e491), sma-4(e729), sma-6(wk7); sma-1(ru18); tax-6(p675); eat-1(ad427), eat-2(ad465), eat-3(ad426), pha-2(ad472), pha-3(ad607); sma-5(n678), rnt-1(ok351), kin-29(oy38), tph-1(mg280).

Inactivation of TGF-β Sma/Mab pathway leads to worms with 60–70% of body length of wild-type (Table 1). However, when Ce-wts-1 dsRNA was injected into TGF-β Sma/Mab pathway mutants, the double mutants’ body length was only 41–50% of wild-type (Table 1). In addition, double mutants grow much slower, usually taking 6–7days to enter adulthood. The brood size of double mutants also drastically decreased (data not shown). According to previous studies [15], wk70 is a null allele of dbl-1. Double mutations of dbl-1 and sma-2/sma-3/sma-4 did not enhance the Small phenotype of single mutants. Therefore, there is strong redundancy between Ce-wts-1 and TGF-β Sma/Mab pathway in regulating body length. Additionally, lon-1, which was negatively regulated by TGF-β Sma/Mab pathway [29], could suppress Ce-wts-1 Small phenotype, which indicated lon-1 was downstream of Ce-wts-1 (Table 1). Moreover, the Long phenotype of lon-2, an upstream regulator of TGF-β Sma/Mab pathway [30], was suppressed by Ce-wts-1(RNAi) (Table 1). Based on these data, we conclude that Ce-wts-1 functions redundantly with TGF-β Sma/Mab pathway, sharing the same upstream regulator and downstream target.

We also found that Ce-wts-1; sma-1 was totally embryonically lethal; Ce-wts-1; sma-5, Ce-wts-1; kin-29, Ce-wts-1; eat-1, Ce-wts-1; eat-3, Ce-wts-1; pha-2 were 100% L1 lethal, while other double mutants did not exhibit obvious synthetic phenotypes (Table 1). The synthetic larval lethality of these double mutants implies Ce-wts-1 synergistically interacts with sma-5, kin-29, sma-1, eat-1, eat-3, pha-2 to regulate worm early development.

3.4. Ce-WTS-1 is expressed near the cell membrane 

To determine the temporal and spatial expression patterns of Ce-wts-1, we constructed Ce-wts-1::gfp fusion gene and microinjected it into wild-type animals. The fusion gene contains a full length genomic Ce-wts-1 DNA, with gfp ligated in frame to its C-terminus. This fusion reporter could fully rescue ok753−/− lethal phenotype, suggesting it may reveal the endogenous expression pattern of Ce-WTS-1.

The expression of Ce-wts-1 begins at the comma stage (Fig. 2A and B) and proceeds during the entire larval and adult stages. Ce-WTS-1::GFP was detectable in many tissues, including pharynx, gut, vulval, spermathecal, and seam cells (Fig. 2C–I). The subcellular localization of Ce-WTS-1 appears to be close to the membrane or membrane-associated, i.e., in gut apical membrane, vulval cell membrane, spermathecal cell membrane and seam cell membrane, by comparing Ce-WTS-1 expression patterns with NHX-2::GFP expression in gut [31], AJM-1::GFP expression in vulval, spermathecal and seam cells [32]. However, the ‘DAS’ transmembrane domain prediction program predicted no transmembrane domain in CE-WTS-1 (data not shown). In addition, neither fly Warts or human LATS1 contains transmembrane domains [33]. Therefore, Ce-WTS-1 is likely accumulated intracellularly near the cell membrane and may interact with membrane or membrane associated proteins.

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  • Fig. 2. 

    The expression of Ce-wts-1 was enriched beneath the cell membrane. (A and B) The onset of Ce-wts-1 expression was detected at comma stage. (C–I) Obvious membrane localization of Ce-wts-1 was evident at apical membrane of intestine cells (arrows, D), vulval cell membrane (arrows, F), cell membrane of inflated spermatheca (arrows, H), and hypodermal seam cell membrane (arrows, I). The corresponding DIC pictures were on the left (C, E and G).

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4. Discussion 

In this report, we have identified a C. elegans homologue of the tumor suppressor warts. Ce-WTS-1 has the characteristic Serine/threonine kinase domain of the Nuclear Dbf-2-related (NDR) family. Like human Lats1 [11], loss of Ce-wts-1 function leads to larval lethality, slow growth rate and small animal size/decreased weight. However, we have not observed obvious tissue over-growth or hyperplastic cells in Ce-wts-1(RNAi) worms, which is different from warts’ role in inhibiting tissue growth in D. melanogaster. Further efforts are required to verify whether Ce-wts-1 inhibits tissue over-growth.

It is known that cytoplasmic Warts inhibits Yorkie translocation into nucleus by phosphorylating Yorkie. Recently, several studies in human cultured cells indicated that plasma membrane anchoring is important for Lats1 kinase activity [34], [35]. They showed that when LATS1 was co-expressed with membrane-bound hMOB1, an interacting protein of LATS1, its kinase activity greatly increased, compared with non-membrane-bound hMOB1. However, it is still unclear whether Warts is physically membrane-attached. In our study, we constructed a Ce-wts-1::gfp fusion gene and found Ce-WTS-1 is mainly localized intracellularly near the cell membrane in multiple tissues of worm. Interestingly, the subcellular location of T10H10.1, the C. elegans homologue of Warts interacting protein, Salvador, is also expressed at the membrane region of spermathecal cells (unpublished data). Therefore, the expression pattern revealed by Ce-WTS-1::GFP may provide an in vivo evidence for membrane anchoring property of Warts and further investigation is required to address how membrane association is related to the physiological functions of Warts.

In C. elegans, there are at least two TGF-β-like signaling pathways: the TGF-β dauer pathway and the TGF-β Sma/Mab pathway. Through double mutant analyses, we found that Ce-wts-1 genetically interacted with both TGF-β pathways in C. elegans. Ce-wts-1 synergistically interacted with daf-7 to inhibit worms entering into dauer stage and with the Sma/Mab pathway components to regulate worm body length. In cancer biology, the TGF-β pathway is a well-known tumor suppressor pathway and the Hippo–Warts pathway is a novel tumor suppressor pathway. Considering the evolutionary conservation of these pathways, it is tempting to speculate that both the Hippo–Warts pathway and the TGF-β pathway may coordinate to inhibit the tumorigenesis.

Body size is determined by cell number and cell size. In Ce-wts-1(RNAi) worms, we did not observe obvious changes of cell number in D-type neurons and hypodermal seam cells. However, the size of seam cells in Ce-wts-1(RNAi) worms is obviously smaller than that of wild-type, implying that the small body size of Ce-wts-1(RNAi) animals may be due to decreased cell size. Studies on cell size control in mouse and fly revealed that insulin/IGF-mTOR-S6K/eIF4E phosphorylation cascade positively regulates protein synthesis and cell size [36]. The Xu T. lab found that there was a 25% decrease of growth hormone level in Lats1−/− knockout mice [11]. Therefore, it is likely that Ce-wts-1 might use similar insulin/IGF-mTOR-S6K/eIF4E pathway to regulate protein synthesis and body size. Indeed, the Ohshima Y. lab recently showed there was a drastic decrease of total protein contents in sma-1, sma-2, sma-4, sma-5 and sma-6 [37]. Thus, total protein contents and insulin signaling should be measured in Ce-wts-1(RNAi) worms in future experiments to explore how Ce-wts-1 regulates body size.

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Acknowledgements 

We thank Drs. Mei Ding, Dong Liu, Lei Liu, Xun Huang for comments and language editing on the manuscript. Some strains used in this work were received from the Caenorhabditis elegans Genetics Center, which is supported by a grant from the National Institutes of Health. This work was supported by a grant from the Ministry of Science and Technology of China to Q. Fan (2007CB946900, 2007CB946904).

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PII: S0014-5793(09)00686-3

doi:10.1016/j.febslet.2009.09.002

FEBS Letters
Volume 583, Issue 19 , Pages 3158-3164, 6 October 2009

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