Department of Biology, Faculty of Sciences, Kyushu University Graduate
School, Hakozaki, Fukuoka 812-8581, Japan
* Present address: Research Institute of Microbial Diseases, Osaka University,
Yamadaoka, Suita 565-0871, Japan
Author for correspondence (e-mail:
yohshscb{at}mbox.nc.kyushu-u.ac.jp)
Accepted 6 December 2002
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SUMMARY |
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Key words: cGMP-dependent protein kinase, Body size, Lifespan, C. elegans, EGL-4, Big mutant
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INTRODUCTION |
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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 the cell number are much more
important than those in individual cell size for determining inter-species
differences in the mass (Conlon and Raff,
1999). In lower invertebrates, cell size variation also seems
important (Böhni et al.,
1999
; Flemming et al.,
2000
). Although age and environmental factors such as food intake
affect the body size of an individual, both the number and the size of cells
must be determined primarily by genetic factors.
Although there is little direct evidence for a genetic factor responsible
for body size difference between species, various examples are known for the
change in body or organ size within a species that depends on genetic factors.
Transgenic mice grew twice as big as normal through overexpression of a growth
hormone gene (Palmiter et al.,
1982). Pit1 transcription factor that activates prolactin and
growth hormone genes was shown to be mutated in Snell dwarf mutant
mice (Li et al., 1990
). More
recently, gene disruption was performed in mice for a cyclin-dependent kinase
inhibitor p27Kip1 (Nakayama et al.,
1996
), a cyclic GMP-dependent protein kinase (cGKII)
(Pfeifer et al., 1996
) and a
TGFß superfamily member GDF8
(McPherron et al., 1997
).
cGKII knockout mice were smaller than normal, whereas the other two were
larger because of enlargement of skeletal muscle or various organs. In
Drosophila, mutants in the components of the insulin/insulin-like
growth factor 1 (IGF-1) signaling pathway were reported to be smaller
(Leevers et al., 1996
;
Böhni et al., 1999
). In
C. 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 (receptor) and
dbl-1/cet-1 (ligand) encode components of a TGFß signaling
pathway (Savage et al., 1996
;
Estevez et al., 1993
;
Krishna et al., 1999
;
Suzuki et al., 1999
;
Morita et al., 1999
).
Of the several classes of factors that control cell size or cell growth,
some are included in the results cited above. Chromosomal ploidy is a
universal control factor for cell size
(Brodsky and Uryvaeva, 1985;
Conlon and Raff, 1999
). Somatic
polyploidization has been suggested to be a part of the mechanism for the
evolution of body size in nematodes
(Flemming et al., 2000
). Cell
cycle is generally an important control point determining the cell size.
Protein synthesis and transcription also seem to be important control points,
which may be related to the dependence of cell size on chromosomal ploidy.
Growth hormone, IGF-1, TGFß, other growth factors and their signaling
pathways could also affect cell size, at least in part, by stimulating protein
synthesis or transcription. Decrease in cell size in Drosophila
mutants for phosphoinositide-3 kinase Dp110 or IRS1-4
(Leevers et al., 1996
;
Böhni et al., 1999
) and
increase in GDF-8 mutant mice (McPherron
et al., 1997
) are examples of changes in cell size. Extracellular
and intracellular transport of molecules may be another general determinant of
the cell size (see Cossins,
1991
).
The control mechanisms of cell number or cell proliferation are complex.
Changes in cell numbers were noted in the mutants of insulin and TGF-ß
signaling components (Leevers et al.,
1996; McPherron et al.,
1997
; Böhni et al.,
1999
). The size of an organ, and hence that of the whole body, in
mammals is maintained by keeping the total cell mass, not the cell number,
constant (Conlon and Raff,
1999
). A good example of this is seen in a tetraploid mouse
carrying larger and fewer cells (Henery et
al., 1992
). Various mechanisms keeping the organ size or cell mass
constant have been proposed (Conlon and
Raff, 1999
; Brock and Gomer,
1999
).
When searching the literature for mutations affecting body size, we noted that large mutants were much less frequently reported than small mutants. We find no literature on large body size mutants, either isolated by screening or arising spontaneously. Because cell growth and proliferation are regulated by both positive and negative regulators, one should be able to isolate large mutants carrying a defect in a negative growth regulator. To elucidate mechanisms of body size determination further, we have explored this idea by using C. elegans, and have succeeded in isolating many large mutants. We describe four of them and the common responsible gene, egl-4.
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MATERIALS AND METHODS |
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Isolation of mutants
Approximately 1000 L4 hermaphrodites of wild-type N2 were mutagenized with
50 mM ethylmethane-sulfonate (EMS) for 4 hours. They were cultivated overnight
on three NGM (nematode growth medium) plates
(Brenner, 1974), and each young
adult worm was picked and cultured separately in a plate for 4 to 6 days in a
total of 50 or more plates. To obtain a synchronized population of
F2 progeny, embryos in F1 adults from a parent were
collected using alkaline hypochlorite solution
(Sulston and Hodgkin, 1988
),
and allowed to hatch overnight in M9 buffer. Collected F2 L1 larvae
were grown in ten 9 cm NGM plates for 4 days. Worms that were longer than 1.5
mm were isolated under a dissecting microscope (Olympus SZX-12) equipped with
a micrometer in the eyepiece. Isolated animals were grown and body sizes of 10
or more 4-day old adults in their progeny were measured to confirm their body
size phenotype. These mutants were backcrossed with N2 four or five times to
produce strains FK223 ks60, FK229 ks61 and FK234
ks62 used in this study. ks16 mutant was also isolated with
EMS, but from CE1377 daf-6 (e1377) in a screen for
thermotaxis-defective mutants and backcrossed three times with N2 to give
FK216 ks16. Double mutants carrying ks61 and a mutation in
daf-16, daf-2, daf-3, sma-6 or dbl-1 were made by crossing
FK229 and DR26, GR1307, CB1370, CB1482 or NU3, and all the mutations in the
doubles were confirmed by sequencing the respective genes.
Measurement of body size
To synchronize adult worms, young adults were picked up and grown on NGM
plates. To measure 4-day old adults, worms were transferred once to remove the
progeny. Alternatively, parent adults were allowed to lay eggs for 4-6 hours
and removed, and the remaining eggs were cultured for five days to obtain a
synchronous population (2.5-3 day adults). Animals were put in APF medium
(del Castillo and Morales,
1967) containing 50 mM NaN3 and then put on a sectioned counting
chamber (Erma Burker-Turk 1/10mm deep) after one hour. Their images were taken
with Zeiss Axiophot 2 microscope and analyzed using an image-analyzing device
`Senchu-gazou-kaiseki-souchi SVK-3A' (Showa Denki Co. Ltd, Fukuoka, Japan). In
this system, an outline and a centerline of an indicated animal are drawn, and
the length of the centerline of any curve is measured as the body length,
automatically. Then an indicated number (such as 50 or 100) of lines
perpendicular to, and equally spaced along, the centerline are drawn to the
outline. The lengths of these perpendiculars are measured automatically to
give average and maximum diameters of the animal. The volume of the animal is
estimated as the sum of the volumes of all the truncated cones obtained by
rotating the divided images around the centerline, assuming that orthogonal
sections of a worm are circular.
Measurement of lifespan
A population of worms synchronized by allowing to hatch for four hours was
grown on a 6 cm plate. At the fifth day, well-grown adults were picked with a
platinum picker onto fresh 3.5 cm NGM plates (five worms per plate, 50 or more
per experiment) and grown in a wet chamber at 20°C. Thereafter, animals
were occasionally transferred to fresh plates to remove the progeny, and the
plates were examined at various times for live, dead and missing individuals.
Missing animals were not included for the calculation of lifespan. Worms were
scored as dead when they were unresponsive to prodding with a picker.
Complementation test and mapping of an egl-4 mutation
For complementation tests between ks16 and each of ks60,
ks61 and ks62, males of ks16; him-5
(e1490) carrying a green fluorescent protein (GFP) marker
flr-1::gfp/nls were crossed with hermaphrodites of ks60, ks61 or
ks62. F1 young adult hermaphrodites carrying the GFP marker were
cultured, and four days later, their volumes were measured and compared with
those of the single mutants, N2 and ks16;him-5 Ex[flr-1::gfp/nls]
(n=20 or more). Complementation between ks16 and
n477 or egl-18 (n162) was similarly examined but
without a GFP marker.
For mapping of an egl-4 mutation based on single nucleotide
polymorphisms (SNPs), FK216 ks16 was crossed with CB4856 and large F2
worms were isolated. Their progeny were assayed for the volume, and
approximately 100 candidate ks16 homozygous recombinant lines were
established. SNPs of these lines were analyzed either by digestion with a
restriction enzyme (RFLP) or by sequencing PCR products as described by Wicks
et al. (Wicks et al., 2001).
Information of SNPs on LGIV was also obtained by sequencing and comparing N2
and CB4856 genomes.
Germline transformation
Germline transformation was done as described by Mello et al.
(Mello et al., 1991). Circular
plasmid DNAs were injected at 50 or 100 µg/ml and the total DNA
concentration was adjusted to 100 µg/ml with the addition of pBluescript
plasmid DNA. For rescue experiments, a kin-8::gfp plasmid
(Koga et al., 1999
) was
co-injected as a marker.
Molecular biology
Mutation sites of egl-4 (ks16, ks61, ks62, n477, n478 and
n612) were identified in cDNA synthesized by RT-PCR from each mutant and
that of ks60 was identified in PCR products obtained from genomic
DNA. The mutation sites for ks16, ks61 and n477 were
confirmed in the genomic DNA.
To obtain egl-4 promoter::gfp fusion genes, 5.5kb and 1.4kb sequences upstream of the initiation codons for PKGa and PKGb, respectively, were amplified by PCR and subcloned between PstI and BamHI sites of pPD95.69 vector carrying a gene for GFP (S65C) and SV40 NLS to produce Ppkga and Ppkgb plasmids. For expression of PKGa2 and PKGb1 cDNA, cDNA was amplified from yk341c9 and yk87g12 clones respectively, obtained from Y. Kohara, and then inserted at a KpnI site downstream of a PstI-BamHI fragment carrying promoter a or promoter b in pPD49.26 vector to produce Ppkga-PKGa and Ppkgb-PKGb. pPD95.69 and pPD49.26 vectors were provided by A. Fire.
DNA sequencing was done in an ABI Prism Genetic Analyzer 310 or 3100.
Isolation and analysis of DNA and RNA were done basically as described by
Sambrook et al. (Sambrook et al.,
1989).
Antibody preparation
The cDNA fragment of PKGa corresponding to amino acids 35-138 was subcloned
into pGEX4T3 (Amersham Pharmacia Biotech) and pMALc-2 (New England BioLabs) to
generate glutathione S-transferase (GST) and maltose-binding protein
(MBP)-fusion proteins, respectively. GST and MBP-fusion proteins were
expressed in E. coli DH5 and purified by affinity
chromatography with glutathione sepharose and amylose resin, respectively. To
produce anti-PKG antibody, rabbits were immunized with the GST fusion protein,
and the PKG-specific antibody was affinity purified with an Affigel 10
(Bio-Rad) column coupled with the MBP fusion protein.
Immunoprecipitation and kinase assay
Wild-type N2 or egl-4 (ks16) mutant worms were washed
twice and disrupted by sonication in lysis buffer containing 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1%
Triton X-100 and one tablet of Complete Mini Protease Inhibitor Cocktail
(Roche)/6 ml. After centrifugation at 15,000 g for 30 minutes,
protein concentration of a lysate was determined by BCA assay kit (Pierce).
The lysate containing 1 mg protein was incubated with 2 µl of purified
anti-EGL-4 antibody and the immunocomplexes were collected with 20 µl of a
50% suspension of protein A Sepharose (Amersham). After washing three times
with lysis buffer by centrifugation for 1 minute at 2000 rpm, immunocomplexes
were examined by kinase assay and western blotting probed with anti-PKG
antibody (western blot data not shown). The kinase assay was performed in 50
mM Tris-HCl (pH 7.2), 10 mM MgCl2, 1 mM ditiothreitol, an indicated
concentration of cGMP, 5 µCi [-32P]ATP and 100 mM
KEMPTIDE (LRRASLG, Sigma) as substrate at 30°C for 10 minutes. Reaction
mixtures were absorbed on P81 phosphocellulose papers (Whatman) and they were
washed four times with 75 mM H3PO4. The papers were
dried and the radioactivities were measured by Cherenkov scintillation
counting.
Morphological analysis of organs and cells
For anatomy of intestine, hypodermis or muscle, dss-1p::gfp,
col-19p::gfp or myo-3p::gfp reporter plasmid was injected at 100
or 50 µg/ml into wild type, FK216 egl-4 (ks16) and FK229
egl-4 (ks61) strains to obtain fluorescent transgenic lines.
The C. elegans dss-1 gene in YAC Y119D3 on LGIII was cloned as a
homolog of human SHFM1/DSS-1 gene
(Crackower et al., 1996) by K.
Ishii and T. Tani in our laboratory (unpublished). A 3.0 kb genomic fragment
including 1.4 kb sequence upstream of the initiation codon was cloned into
PstI/BamHI sites of pPD95.75 vector plasmid (A. Fire) to
produce a dss-1p::gfp promoter fusion gene. This reporter is
expressed exclusively in the entire intestine. col-19p::gfp reporter
plasmid carrying 845 bp sequence upstream of the initiation codon of the
col-19 gene was constructed by inserting a PCR product from plasmid
pJA1 into PstI/BamHI sites of pPD95.77. The promoter drives
expression of GFP specifically in adult hypodermis as described for
lacZ expression (Liu et al.,
1995
). A myo-3p::gfp reporter carrying approximately 2 kb
upstream sequence of myo-3 gene was made by T. Ishihara by exchanging
the lacZ gene in pPD18.49 (Fire
et al., 1990
) for a GFP (S65C) gene. 4-day old fluorescent adults
were picked from agar plates and put directly into 50 mM sodium azide for
analysis.
For anatomy of gonad, adult worms were dissected and fixed essentially as
described by Francis et al. (Francis et
al., 1995). The fixed worms were stained with 5 µg/ml FITC and
50 mM NH4Cl in 1 ml PBS at 4°C overnight, transferred into a
hole slide glass (Toshinriko Co. Ltd., Japan) and extruded gonads were cut
from uteri to obtain half gonads for observation.
Anesthetized worms expressing GFP, and gonads stained with FITC, were analyzed under a Zeiss LSM-410 confocal laser-scanning microscope. An entire series of 1 µm optical sections were obtained usually at the resolution of 512x512 (1024x1024 for Fig. 6A-D) with 20x/0.75 objective lens and pinhole of 12 (20 for gonads). For entire intestine, hypodermis or muscle of a worm, two or three separate sectionings were needed. A fluorescent muscle cell was examined within a single section using a 40x/0.75 lens. Contrast and brightness parameters for intestine, hypodermis or muscle were selected as those that gave correct volumes of FITC-stained L3 larvae, the volumes of which were also obtained by the automatic volume measurement system described before. Most typical combinations of contrast and brightness were 250/9000 and 250 or 225/9500. A series of optical sections obtained as above were analyzed in a Zeiss KS 3D-Lite three-dimensional (3D) image processing system equipped with 3D for LSM software, to reconstruct a 3D image and to calculate the volume of an object. Segment (threshold) parameters for 3D image construction and volume measurement were selected to give correct volumes and normal morphology: low thresholds were 36 or 20 for GFP and 55 for gonad, and the high threshold was 255. The precision of the volume calculation by this KS 3D-Lite was confirmed in model experiments using fluorescent Polybead Microparticles YG90 (Polyscience, Inc.; 87.9±9.14 µm in diameter) or an optical segment of an FITC-stained worm of a specified volume.
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Numbers of intestinal nuclei were determined after staining with
diamidino-phenylindole (DAPI) (Sulston and
Hodgkin, 1988). Body wall muscle cells were counted using
polarized light microscopy (Waterston et
al., 1980
).
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RESULTS |
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The average size of the mutants in 4-day old adults (4 days after becoming adults) is approximately 1.7 mm long, 90-100 µm in maximum diameter and 7-8 nl in volume in contrast to 1.4 mm, 80 µm and 4.5 nl, respectively, in the wild type. The mutations are recessive because heterozygotes (/+) have a normal body size. ks61;him-5 males are also bigger than him-5 males (Fig. 1A). lon-1, lon-2 and lon-3 mutants are not larger, or are smaller, than the wild type (Fig. 1A), although longer. Known small body size mutants daf-4, sma-6, sma-2, sma-3 and sma-4 are really smaller (Fig. 1A and data not shown).
Morphology of the mutants is grossly normal as noted by normal ratios of body length to maximum diameter (around 18) and by Nomarsky photomicrographs (Fig. 1B), but the intestine is darker (Din phenotype).
Growth of the mutants and the wild type was monitored by their volumes (Fig. 2A; results on ks16 and ks62 are similar and not shown). The mutant embryos and larvae are not significantly larger than those of the wild type. The mutant body size markedly increases in the adult stage. Although growth of the wild type ceases approximately 2 days after the adult stage is reached, the mutants continue to grow for 4 or 5 days.
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The four mutants have weak egg-laying (Egl) phenotypes
(Trent et al., 1983). Namely,
egg-laying is slightly delayed on the average, resulting in more and
irregularly arranged embryos in the uterus in 2-day old adults (data not
shown), and the brood size is slightly decreased (number of larvae born per
parent = 316±48 for N2, 200±37 for ks60, 229±36
for ks61 and 227±25 for egl-4 (n477), n=10).
We found that all four mutants had an extended lifespan: the mean and maximum
lifespans were increased by approximately 50% over those of the wild type N2
(Fig. 2B and data not
shown).
The four mutants are egl-4 mutants
Because the four mutants showed similar phenotypes, complementation tests
were done. ks16 did not complement with each of the other three in
the body sizes as described under Materials and Methods. We initially mapped
ks16 with Tc1 polymorphisms
(Williams et al., 1992), and
further with SNPs on the left arm of chromosome IV. In this region, there are
two known egg-laying defective genes egl-4 and egl-18. ks16
did not complement with egl-4 (n477), and did complement
with egl-18 (n162) in body size and Egl phenotypes.
egl-4 mutants were reported to have an increased body length
(Daniels et al., 2000
).
egl-4 mutants n477 and n478 had an increased body
volume (Fig. 1A). These
results, together with identification of mutation sites in the mutants,
indicate that all the four big mutants independently isolated are
egl-4 mutants.
Identification of the egl-4 gene
To identify the egl-4 gene, we mapped egl-4
(ks16) using SNPs to a region between cosmids K07A9 and F55A8
(Fig. 3A). DNA from yeast cells
carrying YAC Y39C2 located in this region, but not DNA for another YAC Y53H5,
completely rescued body size, Egl and Din phenotypes of ks16
mutation, thus supporting the notion that egl-4 is located in this
region. This region spans approximately 50 kb and contains only two probable
candidate genes F55A8.2a and F55A8.2b. These genes encode homologues of a
mammalian cyclic GMP-dependent protein kinase (PKG) on the basis of the genome
sequence databases. We sequenced cDNAs obtained by RT-PCR and cDNA clones
obtained from Y. Kohara, to identify four alternative forms of the cDNA.
F55A8.2a and F55A8.2b encoding PKGa and PKGb differ in the promoters and the
first exons. Use of an alternative splice donor site at the edge of exon 8
results in three additional amino acids in PKGa2 and PKGb2
(Fig. 3A). PKGa1 is predicted
to consist of 780 amino acids with a glycine-rich domain, two tandem cyclic
nucleotide-binding domains and a kinase domain, whereas PKGb1 consists of 735
amino acids with the glycine-rich domain deleted
(Fig. 3B,C). These PKGs are
highly conserved in other species such as man and Drosophila. C.
elegans PKGa1 has 48.3% and 41.6% amino acid identity over the entire
protein, and 63.8% and 58.7% identity in the kinase domain, with the human
cGKIa and cGKII (Accession numbers D45864, D70899), respectively.
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We identified mutation sites for the seven egl-4 alleles (Fig. 3C). The alleles ks61, ks16 and ks62 carry a nonsense mutation near the N terminus, and are probably null. ks60 has a 958 bp deletion including the entire last exon. The allele n477 has an eight-base pair insertion between V464 and T465, which causes a frameshift and a stop codon and leads to loss of the kinase domain. n478 and n612 have a missense mutation in the kinase domain. The glycine 481 and cysteine 576 for which n478 and n612, respectively, have a substitution are conserved among species. This cysteine 576 might form a disulfide bond essential for structural integrity of the protein. As to the body size, n478 is the biggest among those examined, n612 is the smallest (a weak allele) and the other five have intermediate and similar body sizes (Fig. 1A and data not shown for n612).
egl-4 cDNA a2 and b1 were expressed under promoters a and b, respectively, in ks16. Expression under promoter a completely rescued body size, Egl and Din phenotypes, but expression under promoter b failed to rescue (volumes were 5.0±0.39 nl for N2, 7.1±0.66 nl for ks16, 4.3±0.73 nl for ks16;Ex[Ppkga/PKGa] and 6.9±0.57 nl for ks16;Ex[Ppkgb/PKGb] 120 hours after hatch, n=16-33).
Expression patterns
To examine expression patterns of the egl-4 gene, we prepared
constructs to express a GFP fusion protein with a nuclear localization signal
(NLS) under the control of promoter a (5.5 kb) or promoter b (1.4 kb). Under
promoter a, GFP was predominantly expressed in head neurons
(Fig. 4A,B), a few cells in the
tail (Fig. 4A) and hypodermis
except for seam cells (Fig.
4A,C). A total of 30-50 fluorescent neurons of varying intensities
were observed in the head. Stronger or more consistent expression was observed
in RMDV, RMD, SMDV, RIB, ASK and probably RMDD and SMDD neurons. Expression in
AWC, ASE, AVJ and ASH was sometimes seen. In the tail, DVB and DVC neurons
were probably expressing. GFP was also expressed in ventral cord neurons
(Fig. 4D) and intestine,
typically near the tail (Fig.
4D,E). L2 and L3 larvae show extensive expression most notably in
head neurons and hypodermis, and the expression is weaker in L4 and adults.
Expression is also seen in later embryos
(Fig. 4F). Under promoter b,
GFP was expressed in body wall muscles
(Fig. 4G).
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EGL-4 proteins have protein kinase activity
We developed polyclonal antibodies to a common N-terminal part (35-138 in
PKGa1) of the EGL-4 proteins. The purified antibodies detected a single
protein band of approximately 80 kDa in a wild-type extract
(Fig. 5A), the size of which is
consistent with the molecular masses of the predicted products of the
egl-4 gene (735 and 780 amino acids). The protein band was not
detected in ks16 nonsense and n477 frameshift (and nonsense)
mutants, and the amount of the protein was significantly decreased in
n478 and n612 missense mutants. Instability of mutant
products might be a major reason for decrease of the 80 kDa protein band in
n478 and n612 mutants and absence of a protein band in
n477 in Fig. 5A. These
results showed specificity of the antibodies used.
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EGL-4 proteins immunoprecipitated with the antibodies were assayed for kinase activity on KEMPTIDE peptide carrying a serine residue, in the presence of various concentrations of cGMP (Fig. 5B). An extract of the wild-type N2 showed little or no kinase activity in the absence of cGMP (lane 2) as compared to the activity of a ks16 mutant extract (lane 1), and the activity was stimulated by cGMP dependent on the concentration (lanes 3, 4 and 5). These results indicate that endogenous EGL-4 proteins have protein kinase activity.
Some organs and cells in the mutants have an increased volume
Because egl-4 mutants have a markedly increased body size in 4-day
old adults, the number or size of some types of cells is probably increased.
We analyzed morphology of four major organs. To do this, a whole animal
expressing GFP specifically in intestine, hypodermis or muscle, or dissected
and stained gonad, was examined using a confocal laser-scanning microscope.
Based on a series of sectional fluorescent images, a 3D image was
reconstructed and its volume was calculated with an image processing system as
described in Materials and Methods. Fig.
6 shows examples of 3D images of these organs in the wild type.
The morphology of intestine, hypodermis and muscle in the egl-4
mutants in the 4-day adults looked normal as compared to those of the wild
type. The morphology of a dissected gonad was somewhat abnormal: a mutant
gonad was more irregular, sometimes partly bloated and more fragile (data not
shown).
Results of volume measurements are presented in
Table 1. The volume of whole
intestine is increased by 60-80% in the 4-day adult mutants. The average
number of intestinal nuclei did not change in the mutants even in the 4-day
adults (33.3 and 33.6 for ks16 and wild type, n=21 and 20),
indicating that the number of intestinal cells (20) did not change in the
mutants. Therefore, intestinal cells in the mutants must be 60-80% larger on
average. The volume of hypodermis is increased by approximately 80% in the
mutants. In the muscles expressing myo-3p::gfp (body wall muscles,
vulval, uterine and intestine-associated muscles)
(Okkema et al., 1993), a great
majority of which consists of body wall muscles, the volume increased by 42%
or 30% in the mutants. For body wall muscles, direct analysis of a single,
fluorescently isolated cell was possible because of occasional loss of the
extrachromosomal GFP fusion gene (Fig.
6D). Body wall muscle cell volumes in the mutants increased by 24%
or 36%. The number of body wall muscle cells was not increased in the mutants
(48 or 47 in adjacent two quadrants in ks16 as was in N2). Volume of
a dissected half gonad excluding the uterus in the mutants of 2-day old adults
was similar to that of the wild type, and it was rather decreased in 4-day
adults. This finding and somewhat abnormal morphology suggest a defect in the
mutant gonad. The uterus containing embryos was not precisely analyzed, but it
may be somewhat larger in the mutants because of the Egl phenotype.
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Genetic interaction of the egl-4 gene
Loss of function or reduced function mutations in several genes such as
daf-2 (insulin receptor homolog), age-1 (PI-3 kinase) and
pdk-1 (Akt/PKB kinase) in the insulin-like branch of the pathway
controlling dauer larva formation in C. elegans result in an
increased lifespan (Kenyon et al.,
1993; Paradis et al.,
1999
). Because extension of the lifespan in these mutants is
suppressed by a mutation in a downstream gene daf-16 encoding a
putative transcription factor (Kenyon et
al., 1993
; Ogg et al.,
1997
; Paradis et al.,
1999
), we asked whether a daf-16 mutation also suppresses
extension of the lifespan in an egl-4 mutant. Lifespan of a
daf-16 (m26); egl-4 (ks61) double mutant
was indistinguishable from that of daf-16 (m26) single
mutant and was similar to that of the wild type, indicating that the lifespan
extension was suppressed by a daf-16 mutation
(Fig. 2B). This result was
confirmed for daf-16 (mgDf50) null (complete deletion)
mutation in a separate experiment: the lifespan for wild type, daf-16,
egl-4(ks61);daf-16, and egl-4 was 14.2±2.6,
11.6±2.0, 13.7±2.4 and 18.8±5.7 days, respectively.
Interaction with respect to body size of an egl-4 mutation with daf-16 (m26) was not clear because three independent double mutants gave different results (data not shown). We prepared two double mutant lines carrying egl-4 (ks61) and a null mutation mgDf50 in daf-16. Interestingly, the daf-16 (mgDf50) mutant showed an increased body volume as did egl-4 mutants, and both the double mutant lines were bigger than either single mutant (Fig. 1A; see also Discussion). We also prepared three double mutant lines carrying a reference allele (e1370) of daf-2 and egl-4 (ks61). The daf-2 mutant was slightly smaller than N2 and body sizes of the double mutants were in-between those of daf-2 and egl-4 (Fig. 1A).
Several phenotypes of egl-4 mutants such as Egl and partially
constitutive dauer formation (Daf-c) were reported to be suppressed by
daf-3 (e1376) mutation and partially by daf-5
(e1385) mutation (Trent et al.,
1983; Daniels et al.,
2000
). We made three double mutant lines carrying egl-4
(ks61) and a null mutation daf-3 (mgDf90). The
daf-3 (mgDf90) single mutant showed normal body size, and
this daf-3 mutation did not suppress the increased body volume
(Fig. 1A), nor the body length
(data not shown) of the egl-4 mutant.
We also tested interaction of egl-4 with Sma TGFß pathway (dbl-1-sma-6/daf-4-sma-2, 3 and 4) by constructing a double mutant with sma-6 (e1482) or dbl-1 (nk3) null mutation. Both mutations clearly suppressed egl-4 (ks61) mutation in body size (Fig. 1A).
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DISCUSSION |
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Structure and function of EGL-4 proteins
Before egl-4 was cloned, cGMP-dependent protein kinases had been
isolated and characterized in Drosophila and mammals. Their
structure, conservation, expression and functions were reviewed
(Lohmann et al., 1997),
although much remains unknown as to the roles and related mechanisms.
We found four types of egl-4 cDNA PKGa1, a2, b1 and b2
(Fig. 3A), and Stansberry et
al. (Stansberry et al., 2001)
found three types, CGK-1A, 1B and 1C, without assignment to EGL-4. In a
comparison of the expected products, our PKGa1 (780 amino acids) and PKGb1
(735 amino acids) probably correspond to CGK-1A and CGK-1B, respectively. We
did not find PKG-1C (744 amino acids) and they did not find our PKGa2 and b2.
However, all these expected products differ only in the N terminus or three
amino acids in exon 8, and therefore we presume that their functions are
similar. More important differences among the genes may be their promoters and
expression patterns (see below).
EGL-4 proteins isolated with the purified antibodies from an extract of the
wild-type C. elegans showed cGMP-dependent protein kinase activity
(Fig. 5). The antibodies we
used were prepared against a common part for all the expected products,
suggesting that the proteins we examined were a mixture of endogenous
products. Stansberry et al. (Stansberry et
al., 2001) expressed CGK-1C in cultured cells and examined the
kinase activity in vitro. These kinase activities are probably essential for
the function of the EGL-4 proteins because three mutations n477, n478
and n612 either lead to deletion of, or are found in, the kinase
domain (Fig. 3C). Mammalian
cGKI has a functional NLS of eight amino acids, which resembles that of
interleukin-1
, in the kinase domain
(Gudi et al., 1997
). A
homologous sequence (KALKKKHI) is found in PKGa1 at a corresponding position
(Fig. 3B).
Knockout (KO) mice for a cGMP-dependent protein kinase were reported
previously. cGKII KO mice were slightly dwarf (14% less heavy and 16% shorter
at 8-10 weeks) because of impaired bone growth, and showed intestinal
secretary defects (Pfeifer et al.,
1996). In contrast, cGKI KO mice showed gross intestinal
distention or hypertrophy and showed defects in NO/cGMP-dependent smooth
muscle relaxation (Pfeifer et al.,
1998
). The body size phenotype of egl-4 mutants seems to
be opposite to that of cGKII-deficient mice, and similar to that of cGKI KOs
with respect to volume increase in intestine, although it is not clear if cGKI
KO mice were larger than the wild type.
C. elegans has a gene (C09G4.2) potentially encoding another PKG. The expected product of this gene has 37.7% and 54.7% amino acid identity with EGL-4a1 overall and in the kinase domain, respectively. It has a higher homology to human cGKIa than to cGKII (41.8% compared with 35.5%) as does EGL-4, but the homologies are lower than those between EGL-4 and mammalian counterparts.
Functional foci of the egl-4 gene
Expression of the GFP reporter was observed predominantly in head neurons
and hypodermis under egl-4 promoter a and in body wall muscles under
promoter b (Fig. 4). Functional
foci of the egl-4 gene were suggested to reside in head neurons or
hypodermis by rescue of body size, Egl and Din phenotypes of the ks16
mutant with expression of a cDNA under promoter a but not with promoter b (see
Results). Fujiwara et al. (Fujiwara et
al., 2002) showed that expression of an egl-4 cDNA under
tax-4 promoter rescued all the phenotypes of an egl-4
mutant, including that of body size. L'Etoile et al.
(L'Etoile et al., 2002
)
rescued the defect in olfactory adaptation of n479 mutant by
expression of a cDNA mainly in AWC sensory neurons in the head. These results
suggest that expression of the egl-4 gene is required only in some
neurons in the head for various functions including control of body size.
Thus, the action of EGL-4 on the size of intestine, hypodermis and muscle may
be mediated by molecules secreted by those egl-4 expressing neurons.
Alternatively, EGL-4 could also function cell-autonomously in these major
organs for their size control as well as non cell-autonomously in neurons.
Genetic pathways related to egl-4
We have shown lifespan extension and increased body volume as the novel
phenotypes of egl-4 mutants. Demonstration of volume increase, not
only of increase in body length (Daniels et
al., 2000), is critical in discussing body size because
lon mutants are longer but their volumes are not increased
(Fig. 1A). Increase in both
body size and lifespan resulting from a single mutation could be interesting
in the sense that body size and lifespan are roughly correlated in
vertebrates, although these are genetically separable in egl-4, as
discussed below.
Lifespan extension in egl-4 (ks61) mutant was suppressed by a daf-16 mutation (Fig. 2B and text), suggesting that egl-4 controls lifespan through the insulin-like signaling pathway. However, daf-16;egl-4 double mutants were larger than either single mutant, and daf-2;egl-4 doubles had a size in-between those of the single mutants (Fig. 1A). Both these results are interpreted to mean that egl-4 functions to control body size independently of the insulin-like signal pathway that includes daf-16 and daf-2.
As to the genetic interaction of egl-4 in the body size control,
large body size of an egl-4 mutant was not suppressed by a
daf-3 mutation either suggesting that the pathway in which
egl-4 functions to control body size is different from the TGFß
branch of the dauer control pathway to which daf-3 belongs. However,
suppression of body size of an egl-4 mutant by a sma-6 or
dbl-1 mutation (Fig.
1A) suggests that EGL-4 functions upstream of DBL-1 and SMA-6 that
act in another TGFß pathway as a ligand and a receptor, respectively
(Krishna et al., 1998; Suzuki et al.,
1999; Morita et al.,
1999
). This result seems reasonable because this pathway is known
to control body size (Patterson and
Padgett, 2000
), and is also important to elucidate mechanisms for
the control of body size by egl-4. Thus, functions of egl-4
are related to at least three signaling pathways. These multiple functions of
egl-4 in multiple pathways can be explained if EGL-4 kinase
phosphorylates multiple substrates. Because EGL-4 requires cGMP for effective
kinase activity [Fig. 5B and
Stansberry et al. (Stansberry et al.,
2001
)], a guanylyl cyclase must be a component upstream of
EGL-4.
Hsin and Kenyon (Hsin and Kenyon,
1999) and Patel et al. (Patel
et al., 2002
) reported that germline ablation induced extension of
lifespan, and both lifespan extension and body size increase by approximately
50%, respectively, suggesting that a germline signal represses lifespan and
growth. Extension of lifespan required DAF-16 but gigantism did not. Although
egl-4 mutants probably do not lack this hypothetical germline signal,
EGL-4 could possibly be a component related to this signal.
Mechanisms for the control of body size by egl-4
We found that the volume of intestine or hypodermis was much increased in
the egl-4 mutants (Table
1). Volumes of muscles expressing myo-3 and body wall
muscle cells also increased. Although volume of uterus may be increased in
adults of egl-4 mutants, volume of gonad excluding uterus was not
increased in the mutants (Table
1E). This is important because egl mutants in general
could be regarded to be bigger because of bloating of the gonad. To our
knowledge, this is the first paper reporting volume of an organ in C.
elegans, and we established the methods used for the volume measurement.
In contrast, cell numbers in intestine and body wall muscle were not
increased. We suggest that volume increase in the cells of those major organs
contribute to most of the body size increase in the mutants. In mammals, organ
size seems to be kept constant even when cell size is increased (see
Introduction). The difference between mammals and egl-4 mutants and
Drosophila mutants such as chico
(Böhni et al., 1999) in
organ size control may suggest that invertebrates generally have a different
mechanism. Alternatively, similar cases of increase in organ size may also be
found in mammals. Mechanisms for the control of cell size by egl-4
should be elucidated in the future.
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ACKNOWLEDGMENTS |
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Footnotes |
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