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Genes to Cells (2004) 9, 773-779. doi:10.1111/j.1365-2443.2004.00771.x
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cGMP and a germ-line signal control body size in C. elegans through cGMP-dependent protein kinase EGL-4

Yoshiya Nakano, Yasuko Nagamatsu and Yasumi Ohshima*

Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Mechanisms involved in the control of body size are largely unknown. In the nematode C. elegans, several small body size mutants were isolated, and the responsible genes were reported to encode putative components of a TGFß signalling pathway. Recently, mutants in the egl-4 gene encoding cGMP-dependent protein kinases were found to have a larger body size, and it was suggested that EGL-4 down-regulates the TGFß/DBL-1 pathway. We show that a permeable cGMP analogue 8-Br-cGMP significantly reduces body size of the wild-type but not that of an egl-4 mutant, indicating that cGMP controls body size through EGL-4. Laser ablation of germ-line cells revealed that a germ-line signal and EGL-4 function in the same pathway. Targeted expression of EGL-4 indicates that EGL-4 can function in hypodermis, neurones and intestine both cell-autonomously and cell-nonautonomously to control organ and body size. We propose a signal cascade for the control of body size that involves a germ-line signal, cGMP, G-kinase EGL-4 and DBL-1/TGFß pathway. It is interesting that two important pathways involving cGMP and TGFß, respectively, are related. Also, the results suggest a novel mechanism for the control of organ and body size in which hypodermis plays a key role


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Mechanisms involved in the control of body size are largely unknown (Conlon & Raff 1999). In C. elegans, many small body size mutants were isolated, and several genes responsible for their phenotypes have been reported to encode putative components of a TGFß (transforming growth factor ß) signalling pathway. It includes DBL-1 ligand, DAF-4 and SMA-6 receptors, SMA-2, SMA-3 and SMA-4 Smad transcription factors (Estevetz et al. 1993; Savage et al. 1996; Krishna et al. 1999; Suzuki et al. 1999; Morita et al. 1999).

In C. elegans, the body size is too small to measure the weight. The only quantitative data showing the small body size of the small mutants in the reports cited above was the body length. We recently designed a system to measure diameters and volume as well as length of a C. elegans worm. By using this system, we found that the volume of the small mutants was really smaller, and that lon (long) mutants were not larger in volume although longer. Also, we isolated and characterized many larger body size mutants using this system (Hirose et al. 2003). We and others showed that egl-4 mutants have a larger body size (Daniels et al. 2000; Fujiwara et al. 2002; Hirose et al. 2003). The egl-4 gene was shown to be involved in the control of sensory behaviours and dauer larva formation (Daniels et al. 2000), olfactory adaptation (L’Etoile et al. 2002) and regulation of behavioural state (Fujiwara et al. 2002) as well. It was also shown that the egl-4 gene encodes cyclic GMP-dependent protein kinases (Fujiwara et al. 2002; L’Etoile et al. 2002; Hirose et al. 2003). We also developed a method to analyse morphology and volume of major organs using a confocal microscope: cell size in the major organs is increased in the egl-4 mutants while cell numbers are not. Genetic interaction studies strongly suggest that the DBL-1/TGFß pathway functions downstream of EGL-4 for body size control since the body sizes of double mutants carrying egl-4 and sma-6 or dbl-1 mutations are close to those of the single small mutants (Hirose et al. 2003). In contrast, three small mutants in the DBL-1 pathway have much smaller cell size and indistinguishable cell numbers in major organs (Nagamatsu & Ohshima 2004). Based on the analyses of nuclear DNA contents and total protein contents, we proposed that decreased cell size and body size of the small mutants in the DBL-1 pathway are related to decreased levels of protein expression while increased cell size in egl-4 large mutants is related to increase in fluid content (Nagamatsu & Ohshima 2004).

In the present report, we examined factors acting upstream of EGL-4, and site and mode of action of EGL-4, in the control of body size and organ size. The results are of interest in that two important signals, cGMP and a germ-line signal, are related to the TGFß signal pathway via EGL-4, and provide novel insights into the mechanisms controlling body size.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Effect of 8-Br-cGMP on body size

Since the egl-4 gene is involved in body size regulation and encodes cyclic GMP-dependent protein kinases (Fujiwara et al. 2002; Hirose et al. 2003), it is assumed that cyclic GMP functions to control body size in the pathway involving EGL-4. To confirm this, we investigated the effect of 8-bromoguanosine 3', 5'-cyclic monophosphate (8-Br-cGMP), a permeable cGMP analogue (Birnby et al. 2000), on the body size. If 8-Br-cGMP penetrates into the cells of a worm and activates EGL-4 kinase, the worm will be smaller. The wild-type and egl-4 animals were grown on agar plates containing 8-Br-cGMP, and the body volume of an adult 100 h after hatch was measured (Fig. 1). The wild-type animals grown with 1 mM or 5 mM 8-Br-cGMP were significantly smaller than control. After culture for two more days in a separate experiment, the results were similar (data not shown). In the presence of 8-Br-cGMP, no significant delay in growth was observed with the wild-type and most animals became adults that were fertile by 100 h after hatch. In contrast, body size of egl-4 adults was not reduced at all in the presence of 8-Br-cGMP 100 h after hatch (Fig. 1) or 2 days later (data not shown). Addition of 5 mM 8-Br-cGMP did not change the average number of body wall muscle cells 100 h after hatch, or the number of intestinal nuclei in L4 larvae (data not shown), suggesting that cells are smaller in the presence of 8-Br-cGMP, as in small mutants of the DBL-1/TGFß pathway (Nagamatsu & Ohshima 2004).



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  		Figure 1  Effect of 8-Br cGMP on body size of the wild-type and an egl-4 mutant. Volumes of adult worms grown on NGM agar plates containing an indicated concentration of 8-Br-cGMP for 100 h after hatch are shown. Error bars indicate standard deviations. The figure is based on two independent experiments. Total numbers of animals examined are as follows: for N2 54 (0 mM), 49 (1 mM) and 47 (5 mM), and for egl-4(ks61) 47 (0 mM), 42 (1 mM) and 45 (5 mM). The data in the presence of the drug are significantly different from that in the absence for N2 (P < < 0.01), and not significantly different for egl-4 (ks61) (P > 0.1) in t-tests.

 
We also investigated the effect of 8-Br-cAMP, a permeable cAMP analogue. In the presence of 1 or 5 mM 8-Br-cAMP, both wild-type and egl-4 animals stopped growth at an early larval stage, suggesting that cAMP has quite different effects and that the effects of the cGMP analogue described above are specific. These results demonstrate that cGMP controls body size through EGL-4.

A germ-line signal functions in the same pathway as does EGL-4

When the two ancestry cells of the germ-line in a wild-type animal were ablated with laser microbeam at the L1 larval stage, the resulting adult had a longer lifespan (Hsin & Kenyon 1999) and a larger body size as well (Patel et al. 2002). egl-4 mutants show qualitatively similar phenotypes in body size and lifespan (Hirose et al. 2003). The similarity in the phenotypes between egl-4 mutants and germ-line ablated wild-type animals may mean that a signal from germ-line functions in the same pathway as does EGL-4. To examine this possibility, we measured the body size of wild-type and egl-4 animals grown to adults after germ-line ablation (Fig. 2). We confirmed that germ-line ablated wild-type worms became significantly larger than non-operated animals. However, in egl-4 mutants, the volume of an adult worm without germ-line cells was close to that of a non-operated worm. Germ-line ablation in wild-type animals did not change the average number of body wall muscle cells or intestinal nuclei (data not shown), which supports that germ-line ablated wild-type animals are similar to egl-4 animals analysed previously (Hirose et al. 2003). In the presence of 8-Br-cGMP, germ-line ablated wild-type animals became significantly larger than non-operated animals, although to a lesser extent than in the absence (Fig. 2).



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  		Figure 2  Germ-line ablation increases body size of the wild-type but not an egl-4 mutant. Germ-line (–) worms were subjected to laser surgery of Z2, Z3 germ line precursor cells at L1 larval stage as described in Experimental procedures. Worms were cultured on NGM agar plates with (+) or without (–) 5 mM 8-Br-cGMP until measurement of body volume 6 days after hatch. Error bars indicate standard deviations. The numbers of examined animals are, in order from the top, 60, 42, 37 and 20 for N2, 40 and 42 for egl-4 (ks61). An asterisk indicates significant difference from the value for non-operated controls in t-tests (P < 0.01).

 
Site of action of EGL-4

The egl-4 gene expression was seen in neurones, hypodermis and intestine under promoter (a) and in body wall muscles under promoter (b) by using a GFP reporter construct (Hirose et al. 2003). When an egl-4 cDNA was expressed in an egl-4 mutant under promoter (a), the phenotypes of the mutant were rescued, but they were not rescued by expression under promoter (b), suggesting that expression in either neurones, hypodermis or intestine is critical for the functions of EGL-4 (Hirose et al. 2003). To determine the site of action of EGL-4 in the control of body size, an egl-4 cDNA was expressed under a cell or organ specific promoter as well as under the egl-4 promoter (a), and effects on body size and organ size were examined (Fig. 3). For examination of organ size, hypodermis and intestine were chosen since they are the two largest organs in C. elegans, and also they are the organs in which size difference is the greatest between the wild-type and egl-4 large mutants or small DBL-1 pathway mutants (Hirose et al. 2003; Nagamatsu & Ohshima 2004). The total body size and the size of hypodermis and intestine in an egl-4 (ks61) mutant are larger by about 70% than those of the wild-type N2 (Fig. 3) as shown previously (Hirose et al. 2003). When a full size egl-4 cDNA was expressed under its own promoter (promoter a), body and organ sizes were reduced to levels around those of the wild-type (Fig. 3). When the same egl-4 cDNA was expressed under a tax-2 promoter that drives expression in about 10 pairs of head sensory neurones (Coburn & Bargmann 1996), or under a col-19 promoter specific for adult hypodermis (Liu et al. 1995), the body size was reduced to a level close to that of the wild-type (108% or 97% of the wild-type value). In these cases, organ size was also reduced to a wild-type level (101–117%) except for incomplete reduction of hypodermal volume under expression with the tax-2 promoter (136% of the wild-type level). In contrast, when the cDNA was expressed under a dss-1 promoter specific for intestine (Hirose et al. 2003), body size of the egl-4 mutant was reduced only to a limited extent (to 157%). In this case, intestinal size was reduced to an almost wild-type level (111%) while hypodermal size was reduced to a much less extent (140%).



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  		Figure 3  Effect of targeted expression of egl-4 a1 cDNA on the body or organ volume in the egl-4 (ks61) mutant. Relative body or organ volume is shown by a bar and in percentage of that of the wild-type N2. Notations for expression site shown in the parentheses are N for neurones, SN for sensory neurones, H for hypodermis and I for intestine. An asterisk indicates significant difference from the value for N2 in t-tests (P < 0.01). All the data for egl-4 transgenic lines in which egl-4 cDNA was expressed are significantly different from the value for an egl-4 line without cDNA expression (P < 0.01) except for the body volume of animals in which dss-1 promoter was used for cDNA expression (157% of the N2 value). Numbers of worms examined are 21–39 in each transgenic line. Each of the body size results is the average of the result for a col-19p::gfp line and that for a dss-1p::gfp line.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
It is remarkable that such a clear effect of 8-Br-cGMP on body size was observed (Fig. 1). The results presented in Fig. 3 indicate that expression of the egl-4 gene in hypodermis or head sensory neurones is important for control of the whole body size. Importance of hypodermal expression of sma-6 or sma-3 genes in body length control was reported previously (Yoshida et al. 2001; Wang et al. 2002). Since col-19 gene is expressed only in the adult hypodermis (Liu et al. 1995), our results indicate that expression of egl-4 in adult hypodermis is sufficient to keep normal body size in the wild-type. As for neurones, AWC-specific expression of egl-4 was reported to be important in olfactory adaptation (L’Etoile et al. 2002), and egl-4 expression in sensory neurones under a tax-4 promoter was reported to recover the normal body length in an egl-4 mutant (Fujiwara et al. 2002).

Expression of the egl-4 gene in hypodermis or intestine can keep normal size of its own organ, indicating organ-autonomous and probably cell-autonomous function of the egl-4 gene. Also, cell-nonautonomous effects of the egl-4 gene expression were shown: effect of the expression in sensory neurones on intestinal size (reduced to 101%) and hypodermal expression on intestinal size (reduced to 117%) are strong, while those of expression in sensory neurones on hypodermal size (reduced to 136%) or intestinal expression on hypodermal size (reduced to 140%) are less strong. These results indicate that the egl-4 gene can act both organ-autonomously and organ-nonautonomously. It is likely that the level of the organ-nonautonomous effect depends on the numbers of cells and spatial relationship of expressing and target organs which are specific for each pair. Also, level of the driving power of each promoter and expression timing may also contribute to the organ-nonautonomous effect.

Based on the results presented here and previously, we propose a model for the EGL-4 signal pathway in body size control as shown in Fig. 4. Laser ablation of germ-line cells did not induce gigantism of the egl-4 mutant at all (Fig. 2). This is not likely to be because such big mutants as egl-4 cannot become still larger since daf-16 (mgDf50); egl-4 (ks61) double mutant is significantly larger than the egl-4 (ks61) single mutant (Hirose et al. 2003), and since the big daf-16 (mu86) mutant becomes even larger when germ-line cells are ablated (Patel et al. 2002). These results indicate that EGL-4 and a germ-line signal function in the same pathway. The results showing that both the germ-line signal and EGL-4 depend on DAF-16 for lifespan control and that both of them do not depend on DAF-16 for body size control (Hsin & Kenyon 1999; Patel. 2002; Hirose et al. 2003) support this. This conclusion is also supported by the similarity in the phenotypes of body size and lifespan between egl-4 mutant animals (Hirose et al. 2003) and germ-line ablated wild-type animals, and by the results shown in this report that both animals have indistinguishable cell and nuclear numbers. Since the body sizes of the double mutants carrying egl-4 and dbl-1 or sma-6 mutations are close to those of the dbl-1 or sma-6 small mutants of the DBL-1/TGFß pathway, this pathway functions downstream of EGL-4 (Fujiwara et al. 2002; Hirose et al. 2003). Therefore, DBL-1/TGFß pathway also functions in the same pathway as does the germ-line signal. For the epistasis between the germ-line signal and EGL-4, we propose that the germ-line signal functions upstream of EGL-4 since the germ-line signal seems to be conserved and fundamental among animals (see below), and since brood size of an egl-4 null hermaphrodite is only slightly decreased (Hirose et al. 2003) while laser ablation completely abolishes germ-line. Comparison of adult body size between germ-line ablated and control animals in the presence of 8-Br-cGMP (Fig. 2) suggest that cGMP and the germ-line signal independently control EGL-4.



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  		Figure 4  A model for the EGL-4 signal pathway controlling body size in C. elegans. Arrows indicate stimulation of activity and T-bars indicate inhibition.

 
Among the factors postulated in this model, it is certain that one or some of guanylate cyclases are involved to produce cGMP. It is possible that a phosphodiesterase specific to cGMP, or to both cAMP and cGMP, is also involved by reducing a cGMP level. The C. elegans genome has about 40 candidate guanylate cyclase genes and six candidate cyclic nucleotide phosphodiesterase genes based on Worm Base (http://www.wormbase.org/).

Estrogen is a likely candidate for the signal from germ-line that controls (represses) body size in mammals (Sharpe 1998; Patel et al. 2002). In C. elegans, although the nature of the germ-line signal postulated in Fig. 4 is unknown, it could be an oestrogen-like steroid as based on the analogy from mammals. If so, a steroid hormone receptor may act between the germ-line signal and a guanylate cyclase in the model of Fig. 4. There are about 100 candidate genes for nuclear hormone receptors and many for steroid metabolism in the C. elegans genome. Alternatively, another kind of factor like fibroblast growth factor (FGF)-like EGL-17 (Burdine et al. 1997) might be the germ-line signal.

Since organ-nonautonomous control of organ size by EGL-4 was observed (Fig. 3), a diffusible factor probably functions downstream of EGL-4. A plausible candidate of this diffusible factor is DBL-1 ligand of the DBL-1/TGFß pathway. The dbl-1 gene was reported to be expressed predominantly in neurones (Suzuki et al. 1999) or in neurones and body wall muscles (Morita et al. 1999). If this is the case, DBL-1 cannot be the organ-nonautonomous effector of EGL-4 shown in the experiments for Fig. 3, although it could be the native effector of the wild-type neurones. An unknown factor controlled by EGL-4 may be the downstream diffusible factor. Alternatively, the dbl-1 gene may be expressed at a low, but effective level in other organs such as hypodermis and intestine. As for SMA-6 putative receptor and SMA-2, 3 and 4 putative transcription factors, their genes are more widely expressed (Krishna et al. 1999; Yoshida et al. 2001; Wang et al. 2002) as for the egl-4 gene (Hirose et al. 2003), allowing them to be candidate factors downstream of EGL-4 such as substrates.

Apart from the requirement for a diffusible factor, at least in the control of organ size by sensory neurones, our results suggest an alternative, interesting possibility of organ-nonautonomous control mechanism of organ size by EGL-4. In this mechanism, hypodermal size is controlled by expression of egl-4 in itself or in neurones, and the hypodermal size indirectly controls the size of other organs such as intestine and muscle by controlling the size of the cuticle that is synthesized by hypodermis (White 1988). Thus, the role of hypodermis in body size control could be important in nematodes, and it may be similar to the role of the major tissue for bone formation in mammals, chondrocytes or the cartilage growth plate (Kronenberg 2003). It is noted that oestrogen, bone morphogenetic proteins (BMPs) of the TGFß family and fibroblast growth factors are known to control this endochondral bone formation (Bord et al. 2001; Kronenberg 2003). Also, guanylate cyclases and cGMP-dependent protein kinases are expressed in chondrocytes (Ruth 1999) and the latter was shown to have a role in bone formation in mice (Pfeifer et al. 1996; Ruth 1999). All these findings support similarity between hypodermis in nematodes and chondrocytes in mammals in body size control through formation of skeleton, and may suggest control of a mammalian BMP by a cGMP-dependent protein kinase pathway.

In conclusion, our results reveal a signal cascade for the control of body size that involves a germ-line signal, cGMP, G-kinase EGL-4 and DBL-1/TGFß pathway. The effect of cGMP on body size is striking, and it is interesting that two important pathways involving cGMP and a TGFß, respectively, are related. Also, the results suggest a novel mechanism for the control of organ and body size in which hypodermis plays a key role.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Strains and culture of C. elegans

Bristol strains of C. elegans were used in this work including the wild-type strain N2 (Brenner 1974) obtained from Caenorhabditis Genetics Center, and a large body size mutant FK229 egl-4 (ks61) isolated in our laboratory (Hirose et al. 2003).

C. elegans worms were handled essentially as described (Brenner 1974; Sulston & Hodgkjn 1988). A few or several adult worms were picked and cultured on 6 cm NGM agar plates seeded with E. coli strain OP50 at 20 °C. Plates for examination of the effect of 8-Br-cGMP were made as follows. A 100 µL of distilled water, 20 mM or 100 mM 8-Br-cGMP (Sigma, sodium salt monohydrate) was spread on each plate made of 2 mL NGM agar in a 3.5 cm Petri dish and allowed to diffuse into the agar so that the final concentration was 0 mM, 1 mM or 5 mM. A 20 µL of concentrated overnight culture of E. coli OP50 was spread on each plate for culture.

In this study, worms 4 days after becoming adults, which are termed 4 day (old) adults, or worms 100 h after hatch were analysed for body or organ size, as indicated. To obtain 4-day-old adults, 20 or more worms at the young adult stage were picked up and cultured. After 2 days, the worms were picked on to new plates to remove the progeny, and cultured for 2 more days.

Measurement of body size

Total body volume, body length and diameters of a worm were measured as described (Hirose et al. 2003); only body volume is shown here.

Germ-line ablation

New born L1 larvae were mounted on a 5% agar pad in S-medium containing10% polyvinylpyrrolidone (PVP) and covered with a cover slip. Germ-line precursor cells (Z2 and Z3) were irradiated with 440 nm laser microbeam through a 7-amino-4-methyl-coumarin solution using VSL-337 Nitrogen-laser (Laser Science, Inc.), MicroPoint Laser Ablation System and Zeiss Axioskop microscope as described (Bargmann & Horvitz 1991; Avery & Horvitz 1987). The worms which were mounted on the pad but were not irradiated were used as the untreated control. After laser ablation, the worms were transferred on to NGM plates and grown for measurement of body size after 6 or 7-days.

cDNA expression and measurement of organ size

Constructions for the fusions of egl-4 a1 cDNA (Hirose et al. 2003) and a promoter of tax-2 (Coburn & Bargmann 1996), col-19 (Liu et al. 1995) or dss-1 (Hirose et al. 2003) for expression in chemosensory neurones, hypodermis or intestine were done as follows. col-19p::gfp, dss-1::gfp (Hirose et al. 2003) and tax-2p::gfp (Coburn & Bargmann 1996) were digested with PstI/BamHI to obtain each promoter and ligated into pPD49.26 (A. Fire). pPD49.26/Ppkga/PKGa (Hirose et al. 2003) was digested to obtain egl-4 a1 cDNA, which was ligated into col-19p, dss-1p or tax-2p::pPD49.26 to obtain col-19p, dss-1p or tax-2p::PKGa. The following pairs of constructs were injected to egl-4 (ks61) to get the transgenic strains shown in the parentheses for measurement of the organ volume.

Also, transgenic lines without egl-4 cDNA but with col-19p::gfp or dss-1p::gfp in N2 background (FK336 or FK290) and in egl-4 (ks61) background (FK338 or FK299) were prepared as controls. The concentrations of the constructs at injection were 20 µg/mL for an egl-4 cDNA construct, except for FK346 egl-4; Ex[col-19p::egl-4, dss-1p::gfp] in which 10 µg/mL was used, 10 µg/mL for col-19p::gfp and 50 µg/mL for dss-1::gfp, and the total DNA concentration was made 100 µg/mL with pBluescript SK+ plasmid when necessary. Germ-line transformation was done as described (Mello et al. 1991).

Volumes of hypodermis or intestine of transgenic worms expressing GFP specifically in the organ were measured with a confocal microscope as described (Hirose et al. 2003). Hypodermal volume was measured using transgenic lines carrying col-19p::gfp described above (FK336, 338, 351, 349, 345 and 354 for the data from top to bottom in Fig. 3, Hypodermis panel). Intestinal volume was measured with transgenic lines carrying dss-1p::gfp (FK290, 299, 357, 355, 346 and 348 from top to bottom in Fig. 3, Intestine panel). Body volume was measured as described by Hirose et al. (2003) using both transgenic lines carrying either col-19p::gfp or dss-1p::gfp and with or without the corresponding promoter/egl-4 cDNA construct, and the average of the results obtained for each is shown in Fig. 3, Body panel.


   Acknowledgements
 
Some nematode strains were obtained from the Caenorhabditis Genetics Center, which is funded by a grant from the National Institute of Health for Research Resources. We thank A. Fire for vectors, A.E. Rougvie for pJA1 carrying col-19 promoter, C.I. Bargmann for tax-2 promoter, K. Ishii and T. Tani for dss-1::gfp, T. Hirose for information, M. Fujiwara, M. Koga and other members of our laboratory for discussion, M. Fujiwara for critical reading of the manuscript, and M. Ohara for language assistance. This work was supported by The Japan Society for the Promotion of Science (Research for the Future 97L00401) to Y.O.


   Footnotes
 
Communicated by: Masayuki Yamamoto

* Correspondence: E-mail: yohshscb{at}mbox.nc.kyushu-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 17 May 2004
Accepted: 28 June 2004




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