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U-515 INSERM, Hôpital Saint-Antoine (M.H., P.L., B.D., L.P., M.B., Y.L.B.), 75571 Paris; and U-380, INSERM, Faculté de Médecine Cochin-Port Royal (G.H.), 75014 Paris, France
Address all correspondence and requests for reprints to: Dr. Martin Holzenberger, U-515, INSERM, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: holzenberger{at}st-antoine.inserm.fr
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functions of the IGF system in vivo have been investigated by transgenic ligand overproduction using ubiquitous and tissue-specific promoters. The ubiquitous overproduction of IGF-I leads to postnatal overgrowth (5). Tissue-specific overproduction of IGF-I leads to overgrowth of the targeted organ only, despite higher levels of circulating IGF-I (6). Similar results have been obtained with IGF-II (7). IGFs play important roles in the central nervous system (8), where IGF overproduction increases myelin content (9) and stimulates brain growth in a region-dependent fashion (10). Similarly, the transgenic overproduction of IGF-I in myocytes causes muscle overgrowth (11). However, as IGF is known to have both endocrine and paracrine functions, the results of transgenic gain of function experiments may not represent the full spectrum of IGF activity in vivo.
Classical inactivation of the receptor results in a potent loss of function model, in which the effects of IGF action can be directly related to specific target cells. The phenotype, however, is strongly cumulative, with significant effects on growth from embryonic day 11 onward, birth weights only 45% those of the wild-type (WT), and perinatal death due to respiratory failure (12, 13). To study the function of IGF signaling in the target tissues in vivo, we initiated a program to inactivate the IGF-IR genetically in a conditional, inducible, tissue-specific, partial, or mosaic manner using the Cre-loxP system (14, 15).
Here, we report the development of the principal component of the model, the IGF-IRlox mouse, and describe a phenotype that appears to result from a genetically fixed reduction in the number of functional IGF-IR due to the insertion of a neomycin resistance cassette into the IGF-IR gene. We describe the phenotype and the effects on the other components of the IGF system. We also explore the molecular mechanism responsible for this partial receptor invalidation.
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Materials and Methods |
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). A neomycin selection cassette,
driven by a PGK promoter and equipped with loxP sites on
both sides, was inserted into the EcoRI site 154 bp upstream
from exon 3. A third loxP site, associated with an
I-SceI site, was inserted into the HindIII site
350 bp downstream from exon 3. The BamHI site 0.5 kb
downstream from HindIII was used to insert a PGK
promoter-driven TK (thymidine kinase) cassette. The TK and
neo genes were inserted in the opposite orientation to the
IGF-IR gene. This construct was amplified in a 2.9-kb plasmid backbone
(details of the construction process can be obtained from the authors).
The entire exon 3, the RNA splice lariat region, and the three
loxP sites that had been designed for this construct and
assembled from synthetic oligonucleotides, were sequenced (Sequenase
2.0, United States Biochemical Corp., Cleveland, OH). The
linearized construct was then used to electroporate the mouse embryonic
stem (ES) cells.
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Animals
We obtained several germline chimeras that transmitted the
mutation in a 129/Sv genetic background. We also crossed these germline
chimeras with C57BL/6 females to generate a population with a mixed
genetic background. Mice heterozygous for the mutated receptor were
mated to create a population consisting of WT, heterozygous, and
homozygous animals. Mice were housed in standard conditions (25 C, 12-h
light, 12-h dark cycle, water/food ad libitum), and pups
were weaned at 25 days. Body weight (±0.1 g) was measured weekly.
Blood samples (100–200 µl) were taken by retroorbital puncture using
Pasteur pipettes prerinsed with 0.5 M EDTA, and
plasma samples were stored at -20 C.
IGF-IRneo mice were crossed with an EIIa-Cre transgenic line to excise the floxed regions in vivo. EIIa-Cre, used here in a 129/Sv genetic background, produces the recombinase ubiquitously, but at moderate levels during early development. In the F1 generation we searched for partial (neo) and total (neo and exon 3) excisions by Southern blotting. In the F2 generation, we identified animals that had inherited an allele with either neo excision (IGF-IRlox) or total excision (IGF-IRex3-). Total excision is the equivalent of the classical IGF-IR knockout (KO) (12, 13). Animals of these two types were then mated to produce homozygous IGF-IRlox and IGF-IRex3-/-.
Detection of the targeted receptor gene in mice by Southern
blot
DNA was prepared from 10-day-old animals using standard
procedures. Tail biopsy samples were digested overnight with proteinase
K (Eurobio, Les Ulis, France) and centrifuged, and the
supernatant was mixed with an equal volume of isopropanol. The
precipitate was washed with 75% ethanol, dried, and resuspended in 10
mmol Tris (pH 8.0). DNA (8 µg) from each animal was digested with
HincII, subjected to electrophoresis in a 1.0% agarose gel,
transferred to Nylon membranes (Hybond+,
Amersham Pharmacia Biotech, Aylesbury, UK) by capillary
action, and hybridized with a genomic probe (radiolabeled using
Rediprime, Amersham Pharmacia Biotech) that recognized a
0.8-kb intronic region directly upstream from the inserted neomycin
resistance cassette. The expected fragments were 2.4 kb for the
wild-type IGF-IR and 4.3 kb for the targeted IGF-IRneo allele.
Receptor binding assays
Recombinant human IGF-I (rhIGF-I; GroPep Pty. Ltd.,
Adelaide, Australia) was labeled with 125I
(Amersham Pharmacia Biotech) using the chloramine-T method
(SA, 
100 µCi/µg). All chemicals were obtained from
Sigma. Crude membranes were prepared from whole brain as
previously described (20). After decapitation, brains were quickly
removed, and homogenized. The homogenate was centrifuged twice at 4 C;
the pellet was resuspended, and the suspension was immediately used for
binding assays. Proteins were measured using the bicinchoninic acid
protein assay from Pierce Chemical Co. (Rockford, IL).
Binding assays were performed in 0.5 ml 50 mM Tris-HCl
buffer containing the membrane preparation (300 µg protein) and the
iodinated ligand. Nonspecific binding was determined in the presence of
200 nM rhIGF-I. Competition experiments were performed
using 15–20 pM [125I]rhIGF-I and
various amounts of unlabeled rhIGF-I. Incubation was stopped after 120
min at 25 C (by which time steady state had been reached), and the
mixture was centrifuged. The pellet was washed twice and counted in a
-counter (1275 MiniGamma, LKB Wallac, Turku,
Finland).
IGF-I assay
Plasma samples (10–25 µl) were incubated in 0.01
M HCl for 30 min at room temperature and ultrafiltered
using Centricon 30 columns (Amicon, Millipore Corp.,
Bedford, MA). The ultrafiltrate was lyophilized, resuspended in
0.1 M phosphate buffer and 1 mg/ml BSA (pH 7.4) and
incubated for 2 days with [125I]rhIGF-I (3000
cpm/tube) and a polyclonal antihuman IGF-I antibody (dilution, 1:120
000) that cross-reacts with murine IGF-I (21, 22) (a gift from J.
Closset, CHU, Liège, Belgium). Samples were tested at five
concentrations, each in duplicate (22). After incubation, free and
bound IGFs were separated using albumin-coated charcoal, as previously
described (23). The detection threshold of the assay was 1–2 ng/ml
plasma. Intraassay variation was 5%, and interassay variation was
10%.
Western ligand blotting of IGFBPs
Plasma samples (3 µl/animal) were subjected to 12.5%
nonreducing PAGE (24). Proteins were electrotransferred onto
nitrocellulose membranes and incubated with
[125I]IGF-I and -II (500,000 cpm each). Blots
were washed and placed against x-ray film (Eastman Kodak Co., Rochester, NY) at -80 C. A pooled normal sample was
included on each gel to make it possible to compare different
experiments. Western ligand blots were quantified using a STORM 850
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA)
and ImageQuant 5.0 software.
RT-PCR, amplicon subcloning, and sequencing
To detect the splicing of sequences from the neomycin resistance
cassette into mature IGF-IR transcripts, we extracted total RNA (25)
from the brain of an adult homozygous IGF-IRneo male and produced first
strand complementary DNA using Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc.) following the
manufacturer’s instructions. In a final reaction volume of 20 µl we
used 500 ng RNA and 20 µg/ml of a reverse complementary
oligonucleotide (5'-GAAGGACAAGGAGACCAAG-3') corresponding to a region
within exon 5 of the receptor gene. All oligonucleotides used were
obtained from Genset (Paris, France). We added 0.5 µl of the
RT products to a tube containing 50 µl of a PCR mixture. The upstream
PCR oligonucleotide corresponded to a sequence in exon 2
(5'-GAAGACCACCATCAACAAT-3'); the downstream oligonucleotide
(5'-ACCACCAAGCGAAACATC-3') was designed to anneal within the neomycin
resistance cassette such that the 5'-half of the cassette (1 kb)
could be tested for splice events. PCR cycling was as follows: 10 min
at 94 C denaturation/enzyme activation step, 40 PCR cycles (94 C for 1
min, 58 C for 1 min, 72 C for 1.5 min), and a final 7-min elongation
step, performed in a thermal cycler (model 480, Perkin-Elmer Corp.) using AmpliTaq Gold (PE). Amplification products were
separated on 1.2% agarose gels, and the regions containing the bands
of interest were cut out for DNA electroelution. The recovered
amplification products were blunted with Pfu DNA polymerase
and inserted into PCR-Script Amp SK+ (Stratagene).
Minipreps (QIAGEN, Hilden, Germany) from
ampicillin-resistant clones were screened based on their restriction
enzyme digestion profiles, and selected clones were sequenced
(GenomeExpress, Paris, France), using T3 and T7 primers.
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Results |
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) and were tested by Southern blotting
with an internal probe for the long arm and an external probe for the
short arm (Fig. 1, C and D). One clone (no. 45) showing the expected
profile was then tested with additional probes covering the entire
targeted region confirming correct integration. The karyotype of this
clone was normal, showing a mean of 40.0 ± 0.12
(±SEM) chromosomes in 31 metaphase spreads analyzed and no
evidence of chromosome translocation or loss (results not shown). Passage of the mutation into the germline. Blastocyst injections of clone 45 yielded 16 male chimeras, 8 of which had 98–100% agouti coat color. Their germline transmission rate was 85–100%. Two of the 100% chimeras were used to establish a heterozygous F1 population of this mouse line that we named IGF-IRneo. The frequency of transmission of the targeted allele was 49% (n = 171), independently of genetic background (129/Sv, or F1 of crosses between 129/Sv and C57BL/6).
Fertility and reproduction of IGF-IRneo mice. Crosses of heterozygous IGF-IRneo generated 43% heterozygous, 29% homozygous, and 28% wild-type animals in a total of 61 animals. These frequencies are within the limits of variability expected for normal Mendelian inheritance. Litter size was normal (a mean of 8.1 pups/litter ± 1.4 SD), and newborns began to suckle within hours of birth. Two pups died within the first 24 h; 1 was wild-type, and the other homozygous IGF-IRneo. We observed no difference between pure (129/Sv) and mixed (F1 between C57BL/6 and 129/Sv) backgrounds. Homozygous IGF-IRneo were fertile when mated together (females from 7 weeks and males from 8 weeks onward) and produced homozygous litters of normal size (7.0 ± 1.0 pups/litter). Fourteen of the 15 homozygous females were fertile. All 11 homozygous males that we tested were fertile.
Postnatal growth of IGF-IRneo mice
At 10 days of age there was no difference in weight between WT
animals and those hetero- and homozygous for IGF-IRneo and no
differences between males and females (as is normal at this age). At 4
weeks of age, we observed a 5% difference in weight between males and
females (for WT, homozygous, and heterozygous IGF-IRneo taken
together); such a difference is normal. However, we also found that the
differences in weight among males of homozygous, heterozygous, and
wild-type genotypes were increasing. Between 4 and 6 weeks after birth
the growth curves of the males continued to separate, such that at 6
weeks, the order of mean body weight was (from lowest to highest):
homozygous females < heterozygous females < wild-type
females < homozygous males < heterozygous males <
wild-type males (Fig. 2A). This order did
not change between 6 and 9 weeks. Differences were greatest at 8 and 9
weeks after birth for the male group; mutant homozygous males were
87%, and heterozygous males were 92% of the weight of the WT males.
Weight differences progressed little thereafter and stabilized at 83%
of WT weight in mutant homozygous adults at 5 months of age (91% for
heterozygotes; Fig. 2B). Homozygous adults gained 4.3 g in weight
from 9 weeks to 5 months of age, which is somewhat less than the weight
gained by 129/Sv wild-type (6.8 g) and IGF-IRneo heterozygotes (6.7 g).
For the females, weight differences were greatest at 6 weeks of age,
with homozygotes being 93% and heterozygotes 97% the weight of WT
females. We ceased to study weight differences between homozygous and
WT females once they had had their first litter. Note that
F2 animals were genotyped on day 10 by Southern
blotting (Fig. 2C). The WT and IGF-IRneo bands were similar in
intensity after passage of the mutation into the germline, indicating
that the two alleles were equally represented in DNA from the targeted
mouse genome.
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). In homozygous IGF-IRneo males these
organs were 10% lighter than those of WT animals. These differences
were significant for the brain and liver, but not so for the other
tissues, possibly due to the small number of observations. Heterozygous
animals showed approximately half the growth retardation of homozygous
animals.
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Receptor binding assay. We assessed receptor binding in whole
brain extracts, as the brain is known to be rich in IGF-IR in young
adult animals. We used the brains of four wild-type, four heterozygous,
and four homozygous males (the same mice used to check that growth was
proportional) and those of two WT and two homozygous females.
[125I]IGF-I binding was specific and saturable,
and receptor affinity did not depend on genotype
(Kd range, 0.47–0.60 nM). The number
of IGF-I-binding sites per cell, however, was lower in hetero- and
homozygous animals than in WT (Fig. 4 and
Table 1). The mean number of specific
binding sites in IGF-IRneo homozygotes was only 59% of that in the WT
(P < 0.01), whereas in heterozygotes it was 88% (not
significant). The number of receptors did not depend on gender, and the
decrease in number was similar for male and female homozygotes. From
our data, we estimate the mean number of receptor binding sites to be
8 x 105/cell in WT and 5 x
105/cell in homozygous IGF-IRneo brains, all cell
types included.
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). However, in adult males homozygous
for IGF-IRneo, the mean plasma IGF-I concentration was 26% higher than
that in WT (P < 0.05). Plasma IGFBP-1, -2, -3, and -4
concentrations, estimated by Western ligand blotting (Fig. 5), did not differ significantly in
hetero- and homozygous IGF-IRneo females from those in the WT animals
(Table 2). Adult homozygous IGF-IRneo males had significantly lower
plasma IGFBP-4 concentrations than WT animals (P <
0.01), but plasma IGFBP-1, -2, and -3 concentrations were similar to
those in the WT.
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). Once
RNA splicing has begun in the reverse neo sequence, there
are two possibilities. 1) RNA splicing of the neo sequence
continues until the 3'-end of exon 3 is reached and then splices into
exon 4, and so forth. The resulting messenger RNA (mRNA) contains
normal 5'- and 3'-untranslated regions (UTR) and additional sequences
from neo. Whatever the splice site (827, 842, or other
positions in the neo open reading frame), the stop codons in
the reverse neo will invariably produce invalidating
truncations of the translation products (see Fig. 6 for details). 2)
RNA splicing may leave the neo sequence before exon 3 is
reached, then continue normally by accepting the 5'-end of exon 3, and
so on. Again, mRNAs will contain normal 5'- and 3'-receptor UTR and
additional neo sequences of various lengths. Most of these
mRNAs will produce receptor proteins truncated in the region
corresponding to neo, exon 3, or exon 4, and the rest will
have random insertions between exons 2 and 3; thus, none of them is
likely to contribute to IGF signaling.
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). Their birth weight was 50.3% that of
heterozygous and WT littermates. All homozygous animals were born alive
but unable to breath. They invariably died within the first few minutes
of extrauterine life. They had generalized hypotrophia, with
particularly underdeveloped intercostal muscles. We performed
receptor binding assays with end-gestational embryos; IGF-IR binding
was undetectable in homozygous IGF-IRex3-/-
embryos (n = 2), whereas in heterozygous
IGF-IRex3- (n = 5) and WT (n = 3)
embryos it was 113 ± 53 (±SEM) and
221 ± 66 fmol/mg protein, respectively.
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Discussion |
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In homozygous IGF-IRneo mice, we observed 41% fewer than normal receptors (in brain tissue) and a growth deficit of 13% in young adult males (6% in females). We investigated whether aberrant RNA splicing due to cryptic RNA splice sites in neo occurred with the IGF-IRneo allele and found evidence that this was indeed the case. Other studies have shown that aberrant neo splicing occurs in the adult and embryo, and that it is not specific for a particular gene, developmental stage, or tissue (33, 34, 35). The insertion of bacterial DNA into mammalian genes interferes with normal expression in a stochastic fashion, and the gene product appears to be invalidated at the translational level due to aberrant RNA splicing. We found that neo insertion reduced receptor density and growth in heterozygous and homozygous animals. The effect on growth retardation and receptor density in heterozygotes was about half that in homozygotes. This is important, as it may shed light on certain aspects of IGF-IR gene regulation.
As the IGF-IR is expressed in a biallelic fashion (unlike IGF-II and the mannose-6-phosphate/IGF type 2 receptor, which are reciprocally imprinted genes in the mouse) (28, 36), we conclude that in IGF-IRneo, there is little or no compensation of receptor expression at the transcriptional and/or posttranscriptional levels. The same seems also to apply to our heterozygous IGF-IRex3- mice. This differs from the observations of Liu and colleagues using classical IGF-IR KO animals (12, 13). In heterozygous classical KO animals, they found normal levels of receptor mRNA and normal growth, suggesting effective up-regulation of the second (intact, WT) allele, probably involving feedback control via the receptor mRNA or protein. This implies that a single functional WT allele of the IGF receptor is sufficient to assure normal expression and growth. Classical IGF-IR KO was achieved by partial ablation of IGF-IR exon 3 and neo insertion into exon 3. As neo and the receptor gene were in the same orientation, all IGF-IR transcripts were truncated by polyadenylation upstream from exon 3. In this study, the neo insertion that created the IGF-IRneo mutation was intronic. Thus, all of the mature receptor mRNAs would have their 3'-UTRs and some simply contain additional neo sequences in the coding region. Thus, decreasing intact receptor mRNA levels seems to increase the production of the second, WT receptor allele. The 5'- and 3'-UTRs of the IGF-IR are unusually large (>1 and >5 kb, respectively) and are well conserved even between distant species (37, 38, 39). It is therefore possible that the UTRs of IGF-IR contain elements that efficiently regulate receptor gene expression and/or mRNA stability.
The relatively modest effects on postnatal growth of this partial receptor invalidation were sufficient to increase the plasma IGF-I concentration (and decrease the plasma IGFBP-4 concentration) in males, but not in females. This suggests that a decrease in the number of receptors has a more pronounced effect on growth regulation in males than in females. A significant growth deficit also seems to be necessary to provoke an IGF system response. It also shows that the relationship between the number of IGF-IR and postnatal growth is not simply proportional.
In IGF-IRneo mice, various tissues are attained to a very similar degree, whether they produce the receptor in large numbers under normal conditions (kidney, brain) or in small numbers (e.g. liver). Thus, it may be misleading to assess the functional importance of this growth factor system for the development of a given tissue from the local physiological expression levels of its receptor. IGF-IRneo mice developed normally until 3 weeks after birth. Similarly, as the number of receptors in most tissues is higher during embryogenesis and early postnatal life than during later postnatal life, one would expect a relative lack of receptors to affect growth primarily early in life. This is clearly not the case, but the number of receptors does become limiting for growth later in postnatal life, when circulating IGF-I levels peak, and physiological receptor expression has already been down-regulated (40). Thus, high levels of IGF-IR do not seem to be a prerequisite for the development of a loss of function phenotype. We conclude that the functional deficit due to the partial invalidation of the receptor is minimal during embryogenesis but becomes relevant during postnatal growth.
High plasma IGF-I concentrations in homozygous IGF-IRneo males indicate that a lack of receptors on the target cells effectively triggers IGF ligand regulation and may reflect the activation of GH-IGF feedback. Both hypothalamic and pituitary sites are targets for IGF-I, which reduces GH synthesis and secretion (41). In IGF-I-/- mice, the pituitary gland exhibits ultrastructural signs of somatotropic stimulation (42). Selective IGF-I gene deletion in the liver, which results in a substantial reduction in the circulating IGF-I concentration, markedly increases the serum GH concentration (32). The high plasma IGF-I concentration in our homozygous IGF-IRneo males can therefore be interpreted as resulting from an increase in GH production due to hypothalamic and/or pituitary IGF-IR deficiency. Higher plasma IGF-I concentrations may also result from peripheral responses in paracrine regulation.
In Western ligand blots, there was no difference in the IGFBP profiles of IGF-IRneo and WT females. In males, it was not possible to detect any increase in IGFBP-3 by Western ligand blotting. The IGFBP-3 concentration normally changes after the IGF-I concentration. Studies in transgenic mice have shown that IGFBP-4 is a functional antagonist of IGF-I in vivo (43). The observed down-regulation of IGFBP-4 may therefore be an additional compensatory mechanism by which receptor-deficient males try to promote growth. The overproduction of IGF-I in transgenic animals increases tissue-specific IGFBP-5 production, but does not affect IGFBP-4 levels (44). This suggests that IGFBP-4 regulation is not secondary to an increase in IGF-I levels, but may be more directly related to the lack of physiological IGF-IR concentrations. In light of the lack of available data (for review, see Ref. 45), more experiments are clearly needed to investigate this further.
These and other findings suggest that the IGF system of the mouse is particularly required during later postnatal growth, but can no longer compensate at this point for the defect caused by the IGF-IRneo allele in males. We conclude that unimpaired, WT expression of the IGF-IR is necessary if male mice are to attain their normal body weight, and that a partial reduction in IGF signaling effectively reduces the probability that males will achieve their full growth potential. Females, in contrast, seem more able to tolerate the relative lack of IGF receptors during postnatal growth.
The somatotropic axis is sexually dimorphic in mammals. It is therefore tempting to interpret the observed growth retardation as a consequence of sex-related differences in GH secretion patterns and changes in other growth mediators (for reviews, see Refs. 46, 47). However, although gender-related differences in growth correlate with circulating IGF-I levels in some species, in many others they do not (46). Similarly, GH-mediated growth effects of androgens, at least on an endocrine level, are also unlikely to account for males being more sensitive to a decrease in IGF-IR levels than females. There is, however, increasing evidence that the growth-promoting effects of androgens result from direct effects on peripheral target cells, involving cooperation with IGF signaling or stimulation of the synthesis of IGF system components. Zung et al. (48) found that testosterone-induced weight gain is not mediated by hepatic IGF-I expression or high plasma IGF-I concentrations. Testosterone did, however, significantly increase GH receptor mRNA levels in the epiphyseal growth plate. This mechanism may, in turn, increase peripheral GH sensitivity and subsequently increase local IGF production and signaling. The importance of the IGF-I produced by peripheral tissues has been demonstrated by the conditional KO of IGF-I in hepatocytes (32). Similarly, androgens increase IGF-I expression and selectively decrease IGFBP-4 in a human osteoblastic cell line (49), a mechanism that may also explain on a paracrine/autocrine level why males are more affected by a decrease in the number of IGF-IR than females. Future experiments using conditional genetic approaches may further clarify some of these aspects.
Both mouse models presented here, IGF-IRneo and IGF-IRlox, are useful for conditional gene invalidation (32, 50). The advantage of IGF-IRlox is that postnatal growth of the mutants is indistinguishable from that of the WT, even in males, so subtle phenotypes may be studied. The advantage of IGF-IRneo is that during Cre recombination of the floxed regions, two intermediate recombination products (selective loss of neo and selective loss of exon 3) may form, thereby creating partial gene excision patterns that provide information about the recombination kinetics of the particular Cre transgene used. The presence of intermediate products indicates low level Cre expression, marks the initial phase of Cre recombination, and is characteristic for progressive cumulative genomic Cre recombination. A lack of intermediate products, in contrast, is associated with Cre transgenes that produce rapid invalidation through high level recombinase expression.
We have shown herein that a functional deficit of the IGF-IR affects the postnatal growth of males more than that of females. Floxed IGF-IR mice offer new perspectives for the in vivo study of IGF signaling. Combinations of floxed IGF-IR mice with other Cre transgenic lines (Holzenberger, M., M. Leneuve, R. Zaoui, and Y. Le Bouc, manuscript in preparation) show that this receptor can be specifically invalidated at various developmental stages, in many tissues, and also in terminally differentiated cells in the adult organism.
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Acknowledgments |
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Footnotes |
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2 Supported by a European Community Research Grant and a Novo Nordisk
Fellowship.
Received November 2, 1999.
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