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. 2003 Jul 8;100(14):8292-7.
doi: 10.1073/pnas.1532175100. Epub 2003 Jun 26.

Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism

Marika Charalambous  1 , Florentia M Smith, William R Bennett, Tracey E Crew, Francesca Mackenzie, Andrew Ward
Affiliations

Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism

Marika Charalambous et al. Proc Natl Acad Sci U S A. .

Abstract

To investigate the function of the Grb10 adapter protein, we have generated mice in which the Grb10 gene was disrupted by a gene-trap insertion. Our experiments confirm that Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele. Consistent with this, disruption of the maternal allele results in overgrowth of both the embryo and placenta such that mutant mice are at birth approximately 30% larger than normal. This observation establishes that Grb10 is a potent growth inhibitor. In humans, GRB10 is located at chromosome 7p11.2-p12 and has been associated with Silver-Russell syndrome, in which approximately 10% of those affected inherit both copies of chromosome 7 from their mother. Our results indicate that changes in GRB10 dosage could, in at least some cases, account for the severe growth retardation that is characteristic of Silver-Russell syndrome. Because Grb10 is a signaling protein capable of interacting with tyrosine kinase receptors, we tested genetically whether Grb10 might act downstream of insulin-like growth factor 2, a paternally expressed growth-promoting gene. The result indicates that Grb10 action is essentially independent of insulin-like growth factor 2, providing evidence that imprinting acts on at least two major fetal growth axes in a manner consistent with parent-offspring conflict theory.

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Figures

Fig. 1.
Fig. 1.
(a) Schematic representation of the genomic structure of mouse Grb10. Two major transcripts, mGrb10α and mGrb10δ, initiate at exon 1 (arrow). These transcripts differ in their splicing of exon 5; mGrb10α contains this exon, whereas mGrb10δ does not. A second promoter gives rise to transcripts with the alternative leader exon 1a. The translational start (open triangle in exon 3) and translation termination (filled triangle in exon 18) are common to the above transcripts. The correspondence of Grb10 exons to functional domains of the protein is represented by the position of shaded rectangles beneath the line; thus, exon 4 encodes the proline-rich domain (PR), exons 10–13 encode the pleckstrin-homology domain (PH), exons 13–16 encode the between pleckstrin-homology (BPS) and Src-homology 2 (SH2) domain, and exons 16–18 encode the Src-homology domain. Black boxes denote the positions of predicted CpG islands. CpG1 is composed of two blocks: 0.5 kb just upstream of exon 1 and 0.2 kb within exon 1, whereas CpG2 spans 1.2 kb immediately downstream of exon 1a. A (exons 14–16) and B (exon 18) represent the position of cDNA sequences used as probes for Northern and in situ hybridization experiments. The gene-trap insertion construct (open rectangle) contains a splice acceptor (SA), a β-geo cassette, and a polyadenylation sequence (pA). The gene-trap insertion has caused the deletion of genomic sequence from 15 kb upstream of exon 2 to 3 kb downstream of exon 4 (broken lines). (b) Northern hybridization on total RNA from e12.5 embryos inheriting the deletion paternally (+/Δ2–4), maternally (Δ2–4/+), or from both parents (Δ2–4/Δ2–4), and from a wild-type littermate (+/+). Probe B detects a 5.5-kb band (the predicted size of the previously described full-length Grb10 transcripts) in the wild-type and paternal transmission samples, but not after maternal transmission of the deletion. Probe B also detects a further transcript of 1.5 kb, mGrb10ι, that is not affected by the deletion. Hybridization of the same samples with an mGAP probe demonstrates the relative loading. (c) Western analysis of wild-type (+/+) and homozygous mutants (Δ2–4/Δ2–4). Three bands between 75 kDa and 55 kDa are detected with an antibody raised against the C terminus of Grb10 (antibody A18) in protein lysates from e12.5 wild-type embryos. These bands are entirely absent in lysates derived from littermates homozygous for Grb10Δ2–4. An anti-actin antibody was used as a loading control. Protein molecular mass markers (M) are shown in the right lane.
Fig. 2.
Fig. 2.
Growth curves comparing placental (a) and embryonic (b) weight between wild-type, maternally transmitted Grb10Δ2–4, and paternally transmitted Grb10Δ2–4 concepti. For each embryonic stage (e10.5, e12.5, e14.5, e16.5, and e17.5), wild-type and Grb10Δ2–4 embryos and placentas were collected and weighed from crosses of +/Δ2–4 females with wild-type males and from +/Δ2–4 males with wild-type females. Mean weights were calculated for each genotype within a litter, then the weights of mutant embryos as a percentage of wild-type weight were calculated for each litter. Graphs show mean percentage weight of the mutants in five to six litters; error bars show the standard error of the percentage mean. At each embryonic time point, the mutant and wild-type weights of littermates were compared by a paired, nonparametric Wilcoxon's test. Significant differences between the genotypes (P < 0.05) are indicated by asterisks.
Fig. 3.
Fig. 3.
(a) Neonatal weights of six litters of crosses from +/Δ2–4 females vs. wild-type males and six litters from +/Δ2–4 males vs. wild-type females collected on the day of birth, weighed whole (Upper), and dissected for the weights of individual organs (brain, heart, lungs, liver, and kidneys). Organ weights were converted to percentages of total body weight, then mean percentages of total body weight were calculated for each litter by genotype (Lower). Error bars show the standard error of the litter means in each case. (b) Crown–rump length. Grb10Δ2–4/+ (left) and wild-type (+/+, right) littermates on the day of birth. (c) Northern hybridization on total RNA was performed from brains and livers of wild-type (+) and maternal Grb10Δ2–4 (m) neonates, as well as from wild-type (+) and Grb10Δ2–4/Grb10Δ2–4 (m/p) embryos at e12.5, using a probe homologous to exon 18 (probe B). Hybridization of the same samples with an mGAP probe demonstrates the relative loading. (d) Livers (i and ii) and lungs (iii and iv) from wild-type (ii and iv) and maternal Grb10Δ2–4 (i and iii) neonates sectioned and stained with hematoxylin and eosin. Representative sections from both genotypes at ×400 magnification are shown. Arrows in i and ii indicate portal triads. Arrow in iii indicates blood-filled alveoli.
Fig. 4.
Fig. 4.
(a–h) Expression analysis of Grb10 in wild-type embryos at e14.5. Whole embryos (a and e) are shown at a magnification of ×20, fourth ventricle choroid plexus (b and f) and kidney (d and h) at ×200, and lung (c and g) at ×100. (i) Embryos from a cross of a Grb10Δ2–4 heterozygous female with a wild-type male were collected at e14.5, fixed, bisected sagittally, and stained with a colorigenic substrate for β-galactosidase (LacZ). Blue staining indicates the sites of expression of the β-geo gene-trap. (j) LacZ expression levels compared between e12.5 embryos inheriting the Grb10Δ2–4 allele maternally (left) or paternally (right), and a wild-type control (center). Embryos were incubated in colorigenic substrate for an equivalent time. (k) External (Right) and internal (Left) views of e14.5 embryos from a cross of a Grb10Δ2–4 heterozygous male with a wild-type female. ca, Cartilage; cp, choroid plexus; me, meninges; h, heart; g, gut; li, liver; lu, lung; se, subependymal CNS; m, muscle; k, kidney; br, bronchioles; um, umbilicus.
Fig. 5.
Fig. 5.
Crosses testing genetically for an interaction between Grb10 and the IGF signaling system were performed between +/Grb10Δ2–4 females and Igf2Δ/+ males. Progeny were collected and weighed at D1 (Left), and at e16.5 (Right, embryo in Upper and placenta in Lower). Weights of the four resulting genotypes (wild type, +/+; maternally transmitted, Grb10Δ2–4/+; paternally transmitted, +/Igf2Δ; and double mutants) are shown, with percentage of wild-type weight displayed at the top of the bar. At D1, organs (brain, heart, lung, liver, and kidney) were dissected, and their weights are expressed as a percentage of total body weight (Lower Left). In all cases, error bars show the standard error of the mean. For total weights at D1, embryo and placenta weights at e16.5 were compared between +/Igf2Δ and double mutants by performing a Mann–Whitney U test.
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