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Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The size of an animal is basically determined by the number and the size of its component cells. In mammals, differences in cell number are much more important than those in individual cell size for determining interspecies difterences in the mass (Conlon & Raff 1999). In invertebrates, cell size variation also seems important (Böhni et al. 1999; Flemming et al. 2000).
As to the factors known to control body size or growth in various animals, many are involved in insulin/insulin-Iike growth factor signalling or TGFß signalling, suggesting that they are major and conserved signal pathways controlling body size (Böhni et al. 1999; Patterson & Padgett 2000; Massague et al. 2000). In Caenorhabditis elegans, many small body size mutants have been identified. Several genes responsible for their phenotypes such as sma-2, 3, 4 (Smad transcription factors), daf-4 and sma-6 (receptors) and dbl-1lcet-1 (ligand) encode components of a TGFß signalling pathway (Savage et al. 1996; Estevez et al. 1993; Krishna et al. 1999; Suzuki et al. 1999; Morita et al. 1999; Savage-Dunn et al. 2000).
In C. elegans, the body size is too small for measuring the weight. The only quantitative data showing the small body size of the small mutants was the body length in the reports cited above. 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 mutants were not larger in volume although longer. Also, we could isolate and characterize many larger body size mutants (Hirose et al. 2003). In that paper, we showed that egl-4 mutants have a much larger body size and that the egl-4 gene encodes cyclic GMP-dependent protein kinases. We also developed a method to analyse morphology and volume of major organs using a confocal microscope. It was shown by this method that cell size in the major organs is increased in the egl-4 mutants while cell numbers are not. Genetic interaction studies suggest that the DBL-1/TGFß pathway functions downstream of EGL-4 for body size control (Hirose et al. 2003).
To better understand the mechanisms of body size control in C. elegans, we have precisely analysed small mutants in the DBL-1 pathway concerning body size, organ size and cell numbers, as had been done for the egl-4 mutants. We have also analysed nuclear DNA contents and total protein contents of the small mutants and an egl-4 Iarge mutant. Our findings provide novel and interesting insights into the mechanisms controlling body size.
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Results |
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Since DBL-1/TGFß pathway functions downstream of EGL-4 in body size control, it is important to examine morphology of the small mutants in the DBL-1 pathway to elucidate mechanisms governing body size not only by this pathway but also by EGL-4. As the first step, we precisely determined the volume of major organs as well as body volume of sma-2, sma-4 and sma-6 mutants in the DBL-1 pathway, as we had previously done with egl-4 mutants (see Experimental procedures and Hirose et al. 2003 for methods). The results are presented in Fig. 1. sma-1 (e30) mutant (McKeown et al. 1998) was also analysed as an example of small mutants not related to the DBL-1 pathway. Total body volume of sma-2, sma-4 and sma-6 mutants was approximately, or less than, half of that of the wild-type in 4 day old adults (the average volumes were 1.7–2.3 nL for the mutants in contrast to 4.1 nL for the wild-type), while that of sma-1 was slightly decreased. Volumes of major organs such as hypodermis, intestine and muscle of the three mutants were reduced to about half or less, approximately in proportion to the reduction in total body volumes (for the wild-type, the average volume of hypodermis, intestine and muscle was 1.15 nL, 0.89 nL and 0.73 nL, respectively, which represented 28%, 21% and 18% of the total body volume). Among the three organs, intestine showed the highest reduction in volume.
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Reduction in organ volume could be due to decreased cell number, cell volume or both. To determine which is the case, we examined cell numbers or nuclear numbers in hypodermis, intestine and muscle in the wild-type, the four small mutants described above, a lon-1 mutant and an egl-4 mutant (Fig. 2). The lon-1 mutant was examined in this study mainly for analysis of chromosomal ploidy as will be described later. An adult hermaphrodite of the wild-type has 188 hypodermal nuclei, 165 (88%) of which reside in a large syncytium called hyp7 surrounding most of the body or in two seam syncytia (White 1988). For hypodermis therefore nuclear numbers are more significant than cell numbers for comparison. Most of the nuclei in hyp7 or seam syncytia have distinct nuclear morphology and are easy to identify, while nuclei in other hypodermal cells are more difficult to discern. Thus, we measured the number of these distinct hypodermal nuclei near the upper side of an adult animal, which should comprise most of the 165/2 or 83 nuclei/side of an adult animal. For all the mutants examined, these hypodermal nuclear numbers did not differ significantly from that of the wild-type (Fig. 2, open bars).
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Ninety-five body wall muscle cells, which can be specifically identified with polarized light microscopy (Waterston et al. 1980), comprise most of the muscle cells expressing the muscle reporter myo-3p::gfp. Therefore, we counted body wall muscle cells (Fig. 2, grey bars). Their numbers were invariant for all the strains examined and very close to the predicted value of 47 or 48 per side (White 1988). All these results indicate that cell size in hypodermis, intestine and body wall muscle is much decreased in accordance with the total organ volumes in the three small mutants of the DBL-1 pathway. Results for the organ size and cell numbers of intestine and body wall muscle in the wild-type and the egl-4 mutant had been reported previously (Hirose et al. 2003).
Nuclear DNA contents are not much changed in the small mutants and in an egl-4 mutant
To elucidate the mechanisms of cell size decrease in the small DBL-1 pathway mutants described above and those of cell size increase in egl-4 mutants, we have analysed chromosomal ploidies and total protein contents in these strains. Chromosomal ploidy is a well known, universal control factor for cell size (Conlon & Raff 1999). For analysis of chromosomal ploidy, we examined nuclei of worms stained with 4',6-diamidino-2-phenylindole (DAPl) as described in Experimental procedures. As organs, we chose hypodermis and intestine for the following reasons. First, they are the largest organs in C. elegans (see above). Second, a large volume increase of about 70% was observed in these organs of egl-4 mutants (Hirose et al. 2003). Third, the intestine showed the largest reduction in the total volume and cell volume in the small mutants (Fig. 1). Lastly, all the intestinal nuclei in the wild-type become 32C by the adult stage (Hedgecock & White 1985). Similarly, most of the 133 nuclei of the large hyp7 syncytium that are postembryonic are 4C at the L4 stage (Hedgecock & White 1985). Flemming et al. (2000) found that the average ploidies of hyp7 nuclei of the wild-type in the adult stage was 10.7 implying that they also contain 8C and 16C nuclei. Since this polyploidization is developmentally regulated, it could be sensitive to a mutation affecting body size.
The results are presented as chromosomal ploidy in Fig. 3, which were calculated by assuming that neuronal nuclei are diploid. This assumption is reasonable since wild-type neuronal nuclei are diploid (Hedgecock & White 1985) and the average fluorescent intensities of DAPI stained neuronal nuclei are similar for the wild-type and the mutants examined here (except for sma-1).
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Since nuclear DNA contents are not much changed in the small or large mutants, we were interested to know if the level of gene expression is altered in these mutants. The total protein content of a worm should be a good measure of the level of general gene expression. The protein contents are much decreased in all the small mutants (to 29–47% of the wild-type value) and increased slightly (by 19%) in the egl-4 mutant (Fig. 4).
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In egl-4 mutants, organ volume and cell volume in the intestine and hypodermis were increased by about 70% (Hirose et al. 2003). In contrast, the increase in nuclear DNA contents is probably slight (23% increase in intestine and 13% decrease in hypodermis, Fig. 3), with a small increase in total protein content (19%, Fig. 4). Therefore, efficiency of general protein expression in the egl-4 mutant is likely to be similar with that of the wild-type. A plausible explanation for most of the cell size increase in the egl-4 mutant is increase of water or fluid content. If so, cellular and body volume of the egl-4 mutant could be more sensitive to high osmolarity in the medium, due to altered osmotic regulation, than that of the wild-type. C. elegans animals are usually cultured on agar plates containing 0.05 M NaCl. We examined the effect on body volume of the wild-type and the egl-4 mutant of higher NaCl concentrations in the plates (Fig. 5). While the body volume of the wild-type decreased slightly (at 0.5 M NaCl, 13% decrease from the value at the normal concentration), volume decrease of the egl-4 mutant was more significant (26%).
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Discussion |
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The volumes of the three major organs in the small mutants (Fig. 1) had not been reported previously. They were much decreased as expected, with the reduction in intestinal volumes being the largest. Concerning the volumes of the three organs analysed, that of the hypodermis seems to show highest correlation with the total body volume, which is reasonable since the hypodermis is the largest. It is noted here that hypodermal expression of sma-6 or sma-3 is critical for their control of body length (Yoshida et al. 2001; Wang et al. 2002).
The numbers of cells or nuclei in the three major organs of the mutants examined in Fig. 2 did not change significantly from those of the wild-type N2, except for marginally significant decrease in the intestinal nuclear number of sma-2. Average hypodermal nuclear numbers of N2 and sma-2 (e502) were reported previously to be 76.3 and 73.6 by Flemming et al. (2000), which are similar to those shown here (80.7 and 73.9). Other results shown in Fig. 2 for the small mutants and lon-1, and those on hypodermis of egl-4 are novel. Suzuki et al. (1999) and Wang et al. (2002) reported no change in the numbers of hypodermal seam cell nuclei in dbl-1 (ev580) and dbl-1 (wk70) mutants, respectively, but gave no numerical data. Our quantitative data establish decrease in organ size and cell size without significant changes in cell numbers in the small mutants. This is an important step toward elucidation of the molecular mechanisms of body size control by the DBL-1 pathway.
The results on chromosomal ploidies (Fig. 3) needs some discussion. Flemming et al. (2000) reported reduction in hypodermal DNA content in sma-2 (e502) to 74% of the wild-type value. Our results show a reduction to 70% in the same sma-2 mutant, which is in good agreement with their data. Flemming et al. (2000) and Morita et al. (2002) reported average hypodermal chromosomal ploidy of 10.7 and 12.47, respectively, in the adults of N2, and the latter also reported that of 14.36 for lon-1 (e185). These values significantly differ from our values of 7.2 for N2 and 7.7 for lon-1 (e185). Our data show an average intestinal chromosomal ploidy of 33.2 in the wild-type that is close to the predicted value of 32 (Hedgecock & White 1985). This indicates that our results correctly represent intestinal ploidies and suggests that our method in the analysis of chromosomal ploidies is valid. Our data shown in Fig. 3 were obtained with worms stained with 2 µg/mL DAPI for 24 h or more. Flemming et al. (2000) and Morita et al. (2002) stained worms with 7 µg/mL DAPI. We also analysed worms stained with 7 µg/mL DAPI, and the results were similar with our data obtained with 2 µg/mL DAPI but seemed to be more variable (data not shown). They used microdensitometry with a fluorescent microscope for DNA content analysis, while we used a two-photon laser scanning (confocal) fluorescent microscope. Although the latter may have a better resolution to our knowledge, both their method and ours should work. The difference in our results and theirs are likely to be due to differences in the culture conditions and/or strains used. Culture conditions are known to affect hypodermal chromosomal ploidy in C. elegans (A. Leroi, personal communication). Both results agree in that no large increase in the hypodermal ploidy is seen in the same lon-1 mutant as compared to that of the wild-type (15% increase in their result vs. 7% increase in our result).
In the sma-2 mutant, some decrease in the nuclear DNA contents was observed although not statistically significant (decrease by 16% and 30% of the wild-type values in intestine and hypodermis, respectively). In other mutants, no large changes were observed. If only the wild-type, sma-2 and egl-4 mutants are compared, a weak correlation is seen between the total body volume and the hypodermal chromosomal ploidy. However, taking all the small mutants analysed and both hypodermal and intestinal ploidies, our results indicate that nuclear DNA content or chromosomal ploidy does not significantly correlate with body volume. Although Flemming et al. (2000) proposed a role of hypodermal polyploidization in body size regulation, our results do not support their proposal as for C. elegans in general.
In the sma-1 (e30) mutant, total intensities of fluorescence (TIFs) in the nuclei of neurones, intestine and hypodermis were all much lower than those in other strains (the values for neuronal nuclei are described in the legend of Fig. 3). We consider that sma-1 nuclear DNA is generally difficult to stain with DAPI for an unknown reason and that neuronal nuclei are diploid as in other strains. Alternatively, DNA contents in this strain could possibly be decreased compared to those in the wild-type.
In the sma-2, sma-4 and sma-6 mutants of the DBL-1 pathway, body size is much reduced due to corresponding reduction in cell volumes. Wang et al. (2002) discussed four possible explanations for smaller cell size in the small mutants, which were reduced DNA contents, reduced protein synthesis, change in cell cycle and metabolic changes, but without a specific evidence for any of these. On the basis of analyses of nuclear DNA contents and total protein contents in the present paper, we propose that the decrease in cell volume is mainly due to reduction in the level of protein expression. Intimate link between cell size and protein and ribosome synthesis have been suggested in various organisms (Jorgensen et al. 2002; Saucedo & Edgar 2002). Our proposal implies that the DBL-1/TGFß pathway positively controls expression or function of many target genes required for cell growth, or a few critical genes that influence transcription or translation of many mRNAs such as SFP1 in yeast (Jorgensen et al. 2002). In fact, Mochii et al. (1999) identified 22 such transcriptionally controlled genes including sma-6 and lon-1, the latter of which was described in Morita et al. (2002). More genes may be controlled by DBL-1 since a TGFß pathway may control many genes in mammals (Derynck & Zhang 2003).
In contrast, volumes of major organs and their cells are increased in egl-4 mutants (Hirose et al. 2003). Mice deficient in cGMP-dependent protein kinase (cGK) I showed gross distension of intestine (Pfeifer et al. 1998), and mice deficient in GDF-8/TGFß that suppresses growth were significantly larger due to increase in skeletal muscle mass (McPherron et al. 1997). In mice, the cGKI gene is highly expressed in intestine and GDF-8 is specifically expressed in skeletal muscle. These results may be related to those of egl-4 mutants in that a deficiency in a cGMP-dependent protein kinase or up-regulation of a TGFß pathway leads to increase in the mass of the organ in which the gene is expressed in the wild-type.
Change in DNA contents in the two organs and total protein content in the egl-4 mutant is not statistically significant. These results suggest that most of the volume increase in these organs by about 70% in the egl-4 mutant is not due to increase in DNA content or protein content. Based on these results, possible involvement of ion transport in cell size control (Cossins 1991) and on secretary defects of cGKll deficient mice (Pfeifer et al. 1996), we hypothesize that increase of water or fluid content in the cell is a major reason for the volume increase in the egl-4 mutant, which may be supported by the results shown in Fig. 5. This finding is interesting since volume increase in egl-4 mutants is probably related to over-expression of the DBL-1/TGFß pathway. If the fluid content of the cell is increased, it could be due to alteration in ion transport, water uptake or loss, water production or osmotic regulation (Randall et al. 1997). We suggest that the DBL-1 pathway controls such a process. Recently, response of C. elegans to high osmotic media and genes possibly involved in this response were studied (S.T. Lamitina, personal communication). The mechanisms involved in such a response might be related to those involved in volume increase in the egl-4 mutant.
Although DBL-1/TGFß pathway is likely to function downstream of EGL-4, we consider that the explanations presented above for cell size increase in egl-4 mutants and cell size decrease in the small mutants do not necessarily contradict with each other. Egl-4-DBL-1/TGFß pathway could regulate both protein levels and fluid content, but in different modes. For example, most protein levels are positively regulated so that the wild-type levels are high, and inactivation of DBL-1 pathway leads to significant reduction of their levels, while activation by an egl-4 mutation does not much increase their levels. In contrast, a key factor controlling fluid content is regulated so that its wild-type level is low and inactivation of DBL-1 pathway does not lead to much reduction in its level while an egl-4 mutation leads to much increase. If we consider a primary mechanism common to both small and egl-4 mutants, one is alteration in volume regulation, such as regulation of fluid content, which in the small mutants leads to decrease in the protein content to attain normal concentrations of cellular components including proteins. The other is alteration in protein levels, which in egl-4 mutants, although it is slight, leads to increase in cell volume by some unknown mechanism.
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Experimental procedures |
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Bristol strains of C. elegans were used in this work including the wild-type strain N2 (Brenner 1974). The following mutants were used: CB30 sma-1 (e30), CB502 sma-2 (e502), DR1369 sma-4 (e729), CB1482 sma-6 (e1482), and CB185 Ion-1 (e185). These strains were obtained from Caenorhabditis Genetics Centre. Large body size mutant strain FK229 egl-4 (ks61) was isolated in our laboratory (Hirose et al. 2003).
C. elegans worms were handled essentially as described by Brenner (1974) and Sulston & Hodgkin 1988). A few or several adult worms were picked and cultured on 6 cm NGM agar plates seeded with Escherichia coli strain OP50 at 20 °C (Brenner 1974; Sulston & Hodgkin 1988). In this study, worms 4 days after becoming adults were analysed, which are termed 4 day (old) adults in this report. To obtain these 4 day 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 sizes
Total body volume, body length and diameters of the worm were measured as described in Hirose et al. (2003), only body volume being shown here.
Measurement of organ sizes
Volumes of hypodermis, intestine or muscles of worms were measured by analysis of transgenic worms expressing GFP specifically in each organ with a confocal microscope, as described in Hirose et al. (2003). To express GFP in hypodermis, intestine and muscles, col-19p::gfp, dss-1p::gfp and myo-3p::gfp, respectively, were introduced to each of sma-1 (e30), sma-2 (e502), sma-4 (e729) and sma-6 (e1482) mutants in this study, as done for N2 and egl-4 mutants previously (Hirose et al. 2003). The extrachromosomal transgenic strains that were derived from the four small mutants described above and used in this study were FK359, FK365, FK371 and FK377 carrying Ex[col-19p::gfp], FK361, FK367, FK373 and FK379 carrying Ex[dss-1p::gfp], FK363, FK369, FK375 and FK381 carrying Ex[myo-3p::gfp].
Measurement of nuclear or cell numbers
To measure numbers of intestinal nuclei, 4 day adult worms were collected, washed three times in M9 buffer and fixed with Carnoy's solution (60% v/v ethanol, 30% chloroform, 10% acetic acid) overnight at room temperature. The worms were washed by phosphate buffered saline (PBS) containing 0.01% TritonX-100 and stained with 2 mg/mL DAPI at 4 C overnight. Then they were washed and suspended in PBS containing 0.01% TritonX-100, 1.1 mg/mL 2-mercaptoethylamine and 50% glycerol. Numbers of intestinal nuclei were measured in a Zeiss Axiophot II microscope.
To measure hypodermal nuclear numbers, confocal images of worms stained with DAPI as above were collected with an interval of 1 mm with a two-photon laser scanning microscope Zeiss LSM-510 NLO. About 10 optical sections from the upper surface were used to construct a stacked image. Then hypodermal nuclei between pharynx and anus were identified and counted.
Body wall muscle cells were visualized with polarized light under Zeiss Axioplan microscope and their numbers were counted in two quadrants of a 4 day adult, as described by Waterston et al. (1980).
Analysis of DNA contents
To measure nuclear DNA contents in wild-type, small mutants, an egl-4 mutant and a lon-1 mutant, worms stained with DAPI as described above were analysed under two-photon laser scanning microscope Zeiss LSM-510 NLO. An entire series of 1 mm sections were obtained at the resolution of 512 ¥ 512 pixels with a 63¥/1.25 lens. Two photon Mai Tai laser of 785 nm was used and laser power was increased manually from 75% to 100% during the analysis. Gain (contrast) and offset (brightness) parameters were determined so that 30–40% of nuclei in the image have halation and the background is completely clear. For this determination, Range indicator in the Palette tool was used. The halation is shown in red and clear background in blue. Four times of averaging of images each obtained with a scan speed of 1.5–2 s was used to obtain finer pictures.
A series of optical sections were used to reconstruct a 3D image and to calculate the volume of a nucleus and its mean fluorescent density with Zeiss KS 3D-Lite software. Low and high segment limits of 15 and 255 were selected, respectively. Total intensity of fluorescence (TIF) of a nucleus was obtained as the product of the mean fluorescent density and the nuclear volume. The average chromosomal ploidy of hypodermal or intestinal nuclei was determined from the ratio of their average TIF over the average neuronal TIF by assuming diploid (2C) for neuronal nuclei. More than 5 images were taken for each of 10 or more worms for a strain. Calculations were made for all the images of worms.
Standard error of mean (S.E.M.) shown in Fig. 3 was calculated by the formula 
deduced from the general equation 3 for 
Ðx described in p.41 of Bevington & Robinson (2003), where x and Ðx are mean and standard deviation (S.D.) of TIF of hypodermal or intestinal nuclei, and y and Ðy are those of neuronal nuclei. The smaller of the two nuclear numbers was taken as n.
Analysis of protein contents
To measure protein contents in the wild-type, an egl-4 mutant and DBL-1 pathway mutants, twenty 4 day adult animals per each strain were mounted on a cap of a 2 mL microtube containing 10 mL of 1 M NaOH/2% SDS to dissolve the animals. The tubes were incubated at 37 C for 2 h, after which 90 mL of dH2O was added to each sample. Bovine serum albumin (BSA) solutions of 25, 50, 100, 200 and 400 mg/mL in 0.1 M NaOH/0.2% SDS were used as standards. 2 mL of working solution of BCA dye binding assay (Pierce) was added to each and the mixtures were incubated for 30 min. UV/Visible spectro-photometer Ultrospec 3000 (Pharmacia Biotech) was used to measure the absorbance. Standard and sample solutions were analysed with BCA peptide binding program to determine protein concentrations of the samples.
Effect of NaCl concentration on body size
To culture worms with a varied concentration of NaCl, wild-type and egl-4 (ks61) mutant worms were removed from a well grown culture on an NGM plate with M9 buffer when there were a large number of eggs on the plate. Remaining eggs were allowed to hatch for 4 h. New born larvae were collected with M9 buffer, and then placed on to NGM plates containing 0.05 M, 0.15 M, 0.3 M or 0.5 M NaCl. Four-day adult worms were used to measure the total body volumes as described above. On 0.5 M or 0.3 M plates, more than half animals did not grow, and the survivors were analysed.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yohshscb{at}mbox.nc.kyushu-u.ac.jp
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Received: 22 September 2003
Accepted: 28 October 2003
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  N. Watanabe, T. Ishihara, and Y. Ohshima Mutants carrying two sma mutations are super small in the nematode C. elegans Genes Cells, May 1, 2007; 12(5): 603 - 609. [Abstract] [Full Text] [PDF]
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 F. Hofmann, R. Feil, T. Kleppisch, and J. Schlossmann Function of cGMP-Dependent Protein Kinases as Revealed by Gene Deletion Physiol Rev, January 1, 2006; 86(1): 1 - 23. [Abstract] [Full Text] [PDF]
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 N. Watanabe, Y. Nagamatsu, K. Gengyo-Ando, S. Mitani, and Y. Ohshima Control of body size by SMA-5, a homolog of MAP kinase BMK1/ERK5, in C. elegans Development, July 15, 2005; 132(14): 3175 - 3184. [Abstract] [Full Text] [PDF]
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Y. Nakano, Y. Nagamatsu, and Y. Ohshima cGMP and a germ-line signal control body size in C. elegans through cGMP-dependent protein kinase EGL-4 Genes Cells, September 1, 2004; 9(9): 773 - 779. [Abstract] [Full Text] [PDF]
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body wall muscle cells in the two quadrants (n = 10–20); 
intestinal nuclei (n = 12–21); 
open bars for hypodermal nuclei in the upper side of a worm (n = 10). I or T figures represent standard deviations. All the data of the mutants are not significantly different from those of the wild-type N2 in t-tests (P < 0.01) except for the intestinal nuclear number of sma-2.
