Disruption of the imprinted Grb10 gene leads to

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... Florentia M. Smith, William R. Bennett*, Tracey E. Crew, Francesca Mackenzie†, ..... Wild-type and mutant portal triads were of equivalent size, and hepatocyte ...
Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism Marika Charalambous, Florentia M. Smith, William R. Bennett*, Tracey E. Crew, Francesca Mackenzie†, and Andrew Ward‡ Developmental Biology Program and Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom Communicated by Mary F. Lyon, Medical Research Council, Oxon, United Kingdom, April 14, 2003 (received for review January 22, 2003)

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 ⬇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 ⬇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 insulinlike 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. adapter protein 兩 cell signaling 兩 genomic imprinting 兩 growth factor receptor-bound protein 兩 insulin-like growth factor

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he growth factor receptor-bound protein 10 (Grb10) belongs to a small family of proteins including Grb7 and Grb14 (1). These molecules are commonly referred to as adapter proteins, as they have no catalytic function but contain numerous protein binding motifs; thus, they are predicted to mediate interactions between disparate proteins. Studies carried out on cells in culture have described the binding of Grb10 to activated tyrosine kinase receptors, such as the insulin receptor (2–4), the insulinlike growth factor I receptor (2, 4), and the receptors for epidermal growth factor (1) and hepatocyte growth factor (c-Met) (5). These interactions are mediated by the Srchomology 2 and the ‘‘between pleckstrin-homology and Srchomology 2’’ domains of Grb10. Grb10 is thought to bring together activated receptors with downstream signaling pathway components [Mek1 and Raf1 (6) and Akt (7)] and possibly interact with the protein ubiquitination pathway (8). Whether Grb10 acts positively (3, 5) or negatively (2, 9) on cellular signaling has not been resolved. Uniparental disomies of mouse chromosome 11, in which both homologous chromosomes are inherited either maternally (mUPD11) or paternally (pUPD11), result in discrete and opposite growth phenotypes. mUPD11 results in growth retardation of the embryo and the placenta, whereas pUPD11 concepti are overgrown (10). These data are consistent with the existence of an imprinted gene in this region, either a paternally expressed growth enhancer or a maternally expressed growth suppressor. Mouse Grb10 resides on proximal chromosome 11 8292– 8297 兩 PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14

(1) and is maternally expressed (11); thus, it is a candidate imprinted growth suppressor. In humans, GRB10 has been mapped to chromosome 7p11.2– p12 (12). mUPD7 is observed in ⬇10% of Silver–Russell syndrome (SRS) cases. This heterogeneous pediatric condition is characterized by severe growth retardation with relative sparing of the cranium (reviewed in ref. 13). While the imprinting status of human GRB10 seems complex and isoform-specific (14, 15), overexpression of GRB10 could result in the severe growth retardation seen in SRS. A correspondence between the opposite growth effects and direction of imprinting of the insulin-like growth factor 2 (IGF2) and its binding protein the IGF2 receptor was consistent with the proposal that antagonistic coevolution between parental genomes could drive the evolution of imprinted genes (16). This parent–offspring conflict hypothesis predicts that paternally expressed genes should act to increase embryonic兾placental growth, whereas maternally expressed genes should limit growth. Maternally expressed Grb10 is thus expected to limit growth, possibly by means of a direct interaction with IGF2 signaling. Here, we show that, when maternally inherited, loss of Grb10 function in the mouse results in fetal and placental overgrowth, demonstrating its role as a growth suppressor. This effect occurs during embryogenesis and results in disproportionate overgrowth of the liver with relative sparing of the brain. Expression of Grb10 is not exclusively from the maternally inherited allele, and in fact the imprinting status of the mouse gene closely resembles that of human GRB10. Moreover, growth inhibition mediated by Grb10 does not act on the major embryonic and placental growth pathway involving IGF2. Instead, Grb10 acts within a separate growth axis in a manner consistent with the parent–offspring conflict hypothesis Methods Embryonic Stem (ES) Cells and Mice. Grb10⌬2–4 gene-trap ES cells were grown in the absence of feeder cells, essentially as described (17). Chimeras were made by using an ES cell兾embryo coculture method based on that of Wood et al. (18). The founder male chimera was mated with (C57BL兾6 ⫻ CBA) F1 females, and the Grb10⌬2–4 line was subsequently maintained on a mixed C57BL兾 6:CBA genetic background. Animals were genotyped by PCR using primers that identified the ␤-geo insertion (␤GEOF 5⬘-TTCAACATCAGCCGCTACAG-3⬘ and ␤GEOR 5⬘-CTCGTCCTGCAGTTCATTCA-3⬘) and using primers that amplified wild-type

Abbreviations: Grb10, growth factor receptor-bound protein 10; IGF, insulin-like growth factor; SRS, Silver–Russell syndrome; en, embryonic day n; D1, (neonatal) day 1. *Present address: U.K. Centre for Tissue Engineering, Room 3.446, Stopford Building, Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom. †Present

address: Mammalian Genetics Unit, Medical Research Council, Harwell, Didcot, Oxon OX11 0RD, United Kingdom.

‡To

whom correspondence should be addressed. E-mail: [email protected].

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Mapping and Characterization of the Grb10⌬2– 4 Deletion. Predic-

tions of intron兾exon boundaries of mouse Grb10 and protein domain predictions were taken from the Ensembl genome server (www.ensembl.org). The Grb10 ␣ , Grb10 ␦ , and exon 1atranscript sequences were found in the National Center for Biotechnology Information database with accession numbers U18996, AF022072, and BC016111, respectively. CpG island predictions were performed by the GRAIL algorithm. Genomic DNA from wild-type and lacZ-positive sibs was screened by Southern blot analysis (21) using probes that were generated by PCR to correspond to 2- to 10-kb intervals along the length of the Grb10 gene. Once the 5⬘ extent of the deletion was mapped, DNA from homozygous Grb10⌬2–4 mice was used in a PCR assay to delineate the 3⬘ limit of the deletion and to screen the remainder of the gene for rearrangements (none were found). Northern hybridization (22) was carried out on total RNA prepared from freshly dissected embryonic day 12.5 (e12.5) embryos and individual organs at day 1 (D1), using TRI reagent (Sigma). Probe A was amplified from cDNA by using primers spanning exon 11 to exon 16 (pAF 5⬘-CTGACCTGGAAGAAAGCAGC-3⬘ and pAR 5⬘-GCGAGGAGTCTCACAGGATC-3⬘). Probe B corresponds to part of the 3⬘ UTR of the Grb10 mRNA contained within exon 18 (amplified by using pBF 5⬘-T TA A A AT TGGGGGAGGGA AG-3⬘ and pBR 5⬘GGGCAAGAGTTCATTTCCAA-3⬘). Western Blot Analysis of Embryo Lysates. Whole embryo lysates

were prepared by homogenizing e12.5 embryos in buffer containing 150 mM NaCl, 1% IGEPAL CA-630 (Sigma), and 50 mM Tris at pH 8.0, and Complete Proteinase Inhibitor Mixture tablets (Roche Biochemicals). Lysates were cleared by centrifugation (15,000 ⫻ g for 10 min at 4°C), and equal amounts of protein were subjected to SDS兾PAGE analysis before transfer onto poly(vinylidene difluoride) Western Blotting membranes (Roche). Membranes were blocked at room temperature for 1 h (block solution: 4% skimmed milk兾0.1% Tween 20), followed by incubation with Grb10A18 rabbit polyclonal antibody (Santa Cruz Biotechnology) overnight at 4°C. Bound antibodies were detected with goat anti-rabbit alkaline phosphatase conjugate (Novagen) for 1 h at room temperature, and reactive bands were visualized by using FAST BCIP兾NBT buffered substrate tablets (Sigma). Grb10 Expression Analysis. Embryos and tissues were analyzed for lacZ expression according to Ainscough et al. (23). Paraformaldehyde-fixed material from both lacZ analysis and obtained for in situ hybridization analysis and immunohistochemistry, was stored in 70% ethanol, embedded in paraffin, and sectioned (7 ␮m). Sections obtained from lacZ analysis were rehydrated through an ascending ethanol series, then counterstained in nuclear fast red (Vector Laboratories), according to the manufacturer’s instructions, and mounted in DPX (BDH). In situ hybridization for Grb10 mRNA was carried out by using sense and antisense probes corresponding to Grb10 exons 14–16, labeled with digoxigenin by using the Roche digoxigeninnucleotide labeling kit according to the manufacturer’s instructions. Hybridization was carried out for 18–48 h at 65°C in a humidified chamber. The signals were detected by using alkaline phosphatase immunohistochemistry, and the sections were counterstained briefly with nuclear fast red. No hybridization Charalambous et al.

signal was detected after treatment with the sense probe. Immunohistochemistry was performed on rehydrated sections that had been permeabilized by microwave treatment (15 min in 10 mM sodium citrate, pH 6.0). After quenching of endogenous peroxidase and blocking, sections were incubated with Grb10A18 rabbit polyclonal antibody (Santa Cruz Biotechnology) overnight at 4°C in a humidified chamber. Primary antibody detection was performed by using a biotinylated goat anti-rabbit IgG (Vector Laboratories) and the Vector Elite ABC Reagent (Vector Laboratories) according to the manufacturer’s instructions. Sections were then counterstained with Mayer’s hematoxylin (Sigma). Results Genomic Structure of Mouse Grb10. Mouse Grb10 spans ⬇110 kb on

chromosome 11 (Fig. 1a). Two major transcripts, mGrb10␣ (1) and mGrb10␦ (4), initiate at exon 1. These transcripts differ in their splicing of exon 5; mGrb10␣ contains this exon, whereas mGrb10␦ does not. A second promoter gives rise to transcripts with the alternative leader exon 1a. The translational start in exon 3 and translation termination in exon 18 are common to the above transcripts. Maternal Inheritance of Grb10⌬2– 4 Results in Ablation of Full-Length Grb10 Transcripts. To investigate the function of Grb10, we used

a gene-trap embryonic stem cell line (24) to establish mice with a mutant Grb10 allele. The gene-trap construct is composed of a splice acceptor, a ␤-geo cassette, and a polyadenylation sequence. 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 (designated Grb10⌬2–4), removing ⬇36 kb that includes the translation initiation codon in exon 3 (Fig. 1a). The deletion was mapped by PCR and by restriction length polymorphism analysis on Southern-blotted genomic DNA from ␤-geo-positive and -negative animals, using probes designed at 2to 10-kb intervals along the length of the genomic region. No further genomic rearrangements were discovered from 10 kb upstream of exon 1 to 10 kb downstream of exon 18 (not shown). We examined the effect of maternal and paternal transmission of Grb10⌬2–4 on Grb10 transcript levels in embryos at e12.5, by Northern blot analysis using a probe corresponding to exon 18 that is present in all Grb10 variants described to date (probe B, Fig. 1a). After maternal transmission of Grb10⌬2–4, we could not detect the 5.5-kb product that corresponds to Grb10 transcripts ␣ and ␦ (Fig. 1b). We therefore confirm maternal-specific expression of this gene and demonstrate that the insertion of the gene-trap cassette results in ablation of Grb10 mRNA. Unexpectedly, probe B also detected a novel transcript at 1.5 kb (designated mGrb10␫), whose transcription was not affected by the gene-trap insertion (Fig. 1b). The identity of mGrb10␫ was confirmed by ribonuclease protection analysis (not shown). This transcript could not be detected with probe A and therefore must lack sequences between exons 11 and 16, which encode the between pleckstrin-homology and Src-homology 2 domains of the protein. To confirm that Grb10⌬2–4 prevents the formation of functional protein, we carried out Western blot analysis on lysates from e12.5 embryos (Fig. 1c). The anti-Grb10 antibody detects three bands (75 kDa to 55 kDa) in wild-type samples that are absent from embryos lacking a wild-type Grb10 gene. Prolonged exposure of the Western blot revealed small immunoreactive fragments that may correspond to translation of mGrb10␫ (not shown). We conclude that the deletion in Grb10⌬2–4 results in loss of all of the full-length Grb10 isoforms that are predicted to mediate its adapter function. The Grb10⌬2–4 allele does not, however, allow us to address the function of the mGrb10␫ transcript or the short Grb10-related peptides. PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14 兩 8293

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genomic sequence deleted from the Grb10⌬2–4 allele (MP11F 5⬘-AGCCCATGTGCTGTCTTTCT-3⬘ and MP11R 5⬘-AGGGACAGACGGTTCAGAGA-3⬘). Igf2⌬ mice (19) were maintained for several generations on the same mixed C57BL兾6:CBA genetic background and were genotyped by using a PCR assay described previously (20). All mice were housed under the standard conditions given previously (20).

Fig. 1. (a) Schematic representation of the genomic structure of mouse Grb10. Two major transcripts, mGrb10␣ and mGrb10␦, initiate at exon 1 (arrow). These transcripts differ in their splicing of exon 5; mGrb10␣ contains this exon, whereas mGrb10␦ 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 ␤-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 (⫹兾⌬2– 4), maternally (⌬2– 4兾⫹), or from both parents (⌬2– 4兾⌬2– 4), and from a wild-type littermate (⫹兾⫹). Probe B detects a 5.5-kb band (the predicted size of the previously described fulllength 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, mGrb10␫, 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 (⫹兾⫹) and homozygous mutants (⌬2– 4兾⌬2– 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 Grb10⌬2– 4. An anti-actin antibody was used as a loading control. Protein molecular mass markers (M) are shown in the right lane.

Maternal Transmission of Grb10⌬2– 4 Results in Placental and Embryonic Overgrowth. Growth regulation by Grb10 was assessed by

measuring the weights of placentas and embryos during the second half of gestation (Fig. 2). Maternal transmission of Grb10⌬2–4 resulted in placental growth enhancement that was statistically significant from e14.5 (118 ⫾ 5% of wild-type littermates at e14.5, 120 ⫾ 3% at e16.5, and 130 ⫾ 5% at e17.5, in each case P ⬍ 0.05 by Wilcoxon’s tests on five to six litters, comparing paired means of wild-type and mutant embryos within the same litter). The growth enhancement after maternal transmission of Grb10⌬2–4 was also apparent in the embryo, first detectable at e12.5, and increasing until just before birth (127 ⫾ 10% of wild-type weight at e12.5, 140 ⫾ 8% at e14.5, 8294 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1532175100

Fig. 2. Growth curves comparing placental (a) and embryonic (b) weight between wild-type, maternally transmitted Grb10⌬2– 4, and paternally transmitted Grb10⌬2– 4 concepti. For each embryonic stage (e10.5, e12.5, e14.5, e16.5, and e17.5), wild-type and Grb10⌬2– 4 embryos and placentas were collected and weighed from crosses of ⫹兾⌬2– 4 females with wild-type males and from ⫹兾⌬2– 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.

144 ⫾ 6% at e16.5, and 146 ⫾ 3% at e17.5, in each case P ⬍ 0.05 by Wilcoxon’s test on five to six litters, as above). Paternal transmission of Grb10⌬2–4 did not result in a significant deviation from wild-type weights in either the placenta or the embryo. Grb10⌬2– 4兾ⴙ Neonates Are Overgrown with Relative Sparing of the Brain and Disproportionate Overgrowth of the Liver. At birth, the

growth enhancement after maternal transmission of Grb10⌬2–4 is maintained. D1 neonates are 136 ⫾ 8% the weight of their wild-type sibs (Fig. 3a) and are increased in crown-rump length (Fig. 3b). However, the increase in total body weight at D1 is not due to proportionate overgrowth of all tissues. Grb10⌬2–4兾⫹ brains are only 112 ⫾ 8% of wild-type weight, whereas Grb10⌬2– 4兾⫹ livers are 247 ⫾ 14% of wild-type weight. Heart, lung, and kidney show no deviation from allometry (i.e., overgrowth of these organs is proportional with body weight, Fig. 3a). Thus, there is relative overgrowth of the liver and sparing of the brain. Deviations from Allometry Correlate with Levels of Grb10 Expression in Neonatal Organs. Total RNA was prepared from brains and

livers of wild-type and maternal Grb10⌬2–4 neonates, as well as from wild-type and Grb10⌬2–4兾Grb10⌬2–4 embryos at e12.5. Charalambous et al.

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 Grb10⌬2– 4 heterozygous female with a wild-type male were collected at e14.5, fixed, bisected sagittally, and stained with a colorigenic substrate for ␤-galactosidase (LacZ). Blue staining indicates the sites of expression of the ␤-geo gene-trap. (j) LacZ expression levels compared between e12.5 embryos inheriting the Grb10⌬2– 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 Grb10⌬2– 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.

Wild-type brain at D1 expressed relatively little Grb10, whereas wild-type livers expressed a high level of the 5.5-kb transcript (Fig. 3c). After maternal transmission of the deletion, the Grb10 5.5-kb transcript could not be detected in either brain or liver. Therefore, deviations from allometry correlate with levels of full-length Grb10 mRNA expression. Histological Examination of Organs at D1. We investigated the disproportionately overgrown livers histologically (Fig. 3d). Wild-type and mutant portal triads were of equivalent size, and hepatocyte size was not affected by the deletion. Relatively pale eosin staining in the mutant liver was indicative of increased deposition of glycogen (confirmed by periodic acid–Schiff’s Charalambous et al.

staining, not shown). Approximately 12% of animals with a maternally inherited Grb10⌬2–4 allele died soon after birth. The perinatal mortality was associated with blood-filled alveoli and trachea (Fig. 3d) and might therefore be a consequence of suffocation because of abnormal lung development. Tissue-Specific Expression and Imprinting of Grb10. The insertion of

a ␤-geo cassette into the Grb10⌬2–4 allele provided us with a method of evaluating parental allele-specific expression. To validate the reporter gene expression profile, we first investigated the tissue-specific expression of endogenous Grb10 by using both mRNA in situ hybridization (Fig. 4 a–d) and immunohistochemistry (Fig. 4 e–h). In situ hybridization was performed on paraffin sections by using a probe complementary to exons 14–16 that detects only the long isoforms of Grb10 (probe A, see Fig. 1). Immunohistochemistry was performed on adjacent sections (Fig. 4 e–h), by using an antibody raised against the C terminus of Grb10 that can detect any Grb10 product that contains the terminal exon 18, presumed to include any products of mGrb10␫. Both methods revealed that Grb10 is expressed at high levels in a variety of muscle tissue (Fig. 4 a and e), including that of the face and trunk, the intercostal muscles, the diaphragm and cardiac muscle, and the tongue and the limb (not shown). In the brain, Grb10 expression is most abundant in the subependymal layers, in the meninges, and in the choroid plexus (both the epithelium and the mesenchyme, Fig. 4 b and f ). The liver, bronchioles, and cartilage of the atlas, ribs, and long bones all express high levels of Grb10 (Fig. 4 c and g). In the kidney (Fig. 4 d and h), Grb10 expression is limited to the developing tubules and the mesenchyme. Grb10 expression can also be detected in the adrenal gland (Fig. 4g) and the pancreatic bud (not shown). PNAS 兩 July 8, 2003 兩 vol. 100 兩 no. 14 兩 8295

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Fig. 3. (a) Neonatal weights of six litters of crosses from ⫹兾⌬2– 4 females vs. wild-type males and six litters from ⫹兾⌬2– 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. Grb10⌬2– 4兾⫹ (left) and wild-type (⫹兾⫹, right) littermates on the day of birth. (c) Northern hybridization on total RNA was performed from brains and livers of wild-type (ⴙ) and maternal Grb10⌬2– 4 (m) neonates, as well as from wild-type (ⴙ) and Grb10⌬2– 4兾Grb10⌬2– 4 (m兾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 Grb10⌬2– 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.

The anti-Grb10 antibody detects additional expression in the embryo, notably in the brain (Fig. 4e), probably because of wider distribution of the shorter protein products encoded by mGrb10␫. Embryos from a cross of a Grb10⌬2–4 heterozygous female vs. a wild-type male were collected at e14.5, fixed, bisected sagittally, and stained with a colorigenic substrate for ␤-galactosidase. The sites of expression of lacZ exactly mirrored the result of the in situ hybridization (Fig. 4i, compare with Fig. 4a). We were thus confident that lacZ expression was a reliable reporter of the long, adapter-encoding transcripts of Grb10. We then went on to investigate expression from the paternal Grb10 allele. At e12.5, paternal transmission of the reporter gene results in staining in the cartilage of the limbs, ribs, and face and in the meninges. The level of gene expression in these tissues, as ascertained by stain intensity, is lower than that of a maternally transmitted lacZ reporter (Fig. 4j). At e14.5, the paternally transmitted reporter gene is expressed in the cartilage of the axis, ribs, head, and long bones; in the heart, lungs, gut, umbilicus, and tongue; and in the meninges of the fourth ventricle (Fig. 4k). We could not detect gene expression in the skeletal muscle. Paternal gene expression was thus present in a subset of tissues where maternal Grb10 is found, and the level of gene expression in most of these tissues is lower than that of the maternally transmitted lacZ reporter. We therefore show that the long isoforms of Grb10 are expressed from both parental alleles in a number of tissues at midgestation in the mouse. Grb10 Is Not Epistatic to IGF2. Finally, both the expression pattern of Grb10 and its time of action appeared similar to that of IGF2. IGF2 is a major embryonic and placental growth factor whose loss of function results in severe growth retardation (19). Igf2 is an imprinted gene, with almost exclusive expression from the paternally derived allele (25). IGF2 mediates its embryonic growth effects predominantly through the IGF1 receptor and its placental growth effects through an uncharacterized receptor termed receptor X (RX) (26, 27). We investigated whether Grb10 was interacting with either of these receptors by performing a cross between ⫹兾Grb10⌬2–4 females and Igf2⌬兾⫹ males, with progeny collected and weighed at D1 (Fig. 5 Left), and at e16.5 (Fig. 5 Right). This cross gave rise to four genotypes: wild types, large Grb10⌬2–4兾⫹ animals, small ⫹兾Igf2⌬ animals, and double mutants (Grb10⌬2–4兾⫹; ⫹兾Igf2⌬). If Grb10 were interacting by means of an IGF2 receptor, we would expect the double mutants to be indistinguishable from ⫹兾Igf2⌬ animals. At D1, Grb10⌬2–4兾⫹ and ⫹兾Igf2⌬ offspring were larger and smaller, respectively, than their wild-type littermates, as expected. Double mutants displayed an additive effect of both parental genotypes and significantly differed from neonates with the ⫹兾Igf2⌬ genotype (Mann–Whitney U test, P ⬍ 0.005, n ⫽ 18, individuals from six litters). Organs (brain, heart, lung, liver, and kidney) were dissected at D1, and their weights are expressed as a percentage of total body weight in Fig. 5 Lower Left. Deviations from allometry were observed after loss of expression of both Igf2 and Grb10 in brain and liver, but allometry was restored in the double mutants. At e16.5, double mutants displayed an additive effect of the two gene deletions in both the embryo and the placenta, and in both cases the weights of the double mutants significantly differed from those of the ⫹兾Igf2⌬ genotype (Mann– Whitney U test, P ⬍ 0.01 for embryos and P ⬍ 0.05 for placentas, n ⫽ 18 individuals from six litters). We therefore concluded that a major part of Grb10’s growth effect is mediated through a mechanism that is likely to be independent of IGF1 receptor and RX.

Discussion Grb10 is an adapter protein capable of binding various activated tyrosine kinase receptors and linking them with downstream signaling proteins. We have investigated the role of this gene in 8296 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1532175100

Fig. 5. Crosses testing genetically for an interaction between Grb10 and the IGF signaling system were performed between ⫹兾Grb10⌬2– 4 females and Igf2⌬兾⫹ 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, ⫹兾⫹; maternally transmitted, Grb10⌬2– 4兾⫹; paternally transmitted, ⫹兾Igf2⌬; 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 ⫹兾Igf2⌬ and double mutants by performing a Mann–Whitney U test.

growth and development by the creation of a deletion兾insertion allele of Grb10, designated Grb10⌬2–4. The deletion removes exons 2–4, including the only known translational start in exon 3, thereby ablating the major adapter-encoding transcripts but not a short transcript that includes exon 18. Maternal transmission of Grb10⌬2–4 resulted in placental growth enhancement that was statistically significant from e14.5 of gestation onward, whereas embryonic growth enhancement was also apparent, first detectable at e12.5, then increasing until e18.5. Paternal transmission of Grb10⌬2–4 did not result in a significant deviation from wild-type weights in either the placenta or the embryo. The timing and magnitude of growth enhancement after maternal transmission of Grb10⌬2–4 was very similar to that observed in mice with paternal uniparental disomy of proximal chromosome 11 (10), suggesting that loss of Grb10 function could entirely account for the growth effect resulting from pUPD11. Furthermore, both fetal and placental overgrowth has been shown to commonly occur in mammals cloned by nuclear transfer (28) and has been correlated with epigenetic defects in both sheep (29) and mice (30–33). In cloned mice, the extent of the resulting deregulation of imprinted genes has been shown to differ according to the cell type used as the nuclear donor. However, it has not escaped our notice that silencing of Grb10 can be consistently correlated with overgrowth even when other imprinted genes, including Igf2 and Igf2r, retain faithful patterns of expression (31, 32). In light of the growth-inhibitory action that we demonstrate, loss of Grb10 expression might therefore contribute to the overgrowth associated with various species of cloned mammals. The overgrowth observed during embryonic stages is also apparent at birth. Grb10⌬2–4兾⫹ neonates are 30–40% larger than their wild-type sibs. However, all tissues of the neonate are not equally overgrown; the liver is relatively more overgrown and Charalambous et al.

the brain is spared. Notably, the growth retardation in SRS leaves the cranium relatively unaffected (13). The overgrown livers in Grb10⌬2–4兾⫹ neonates have a normal portal triad structure, but the hepatocytes had a very high glycogen content. This is in agreement with experiments in which a human Grb10 isoform was overexpressed in rat hepatocytes, resulting in the specific inhibition of insulin-stimulated glycogen synthase (34). In addition, it lends support for previous in vitro data demonstrating an interaction between Grb10 and the insulin receptor (2–4). Grb10 is expressed at high levels in several embryonic tissues, including in a variety of muscles, the exchange tissues of the brain, and the liver, kidney, and cartilage. Biallelic expression of human GRB10 in growth plate cartilage has been suggested to disqualify this gene as a candidate for the small stature phenotype of SRS (15). We show that mouse Grb10 is also expressed from both parental alleles in the cartilage, yet there was an effect on overall body size in maternal Grb10 mutants (Fig. 3b), indicating that perturbation of Grb10 dosage can affect stature. It will be of interest to discover whether overexpressing Grb10 in mice can cause growth retardation. Human Grb10 is maternally expressed in embryonic skeletal muscle and biallelically expressed in many other embryonic tissues, including the cartilage (14, 15). We observed widespread expression of our reporter gene from the paternal allele during embryonic stages, but expression of the paternal allele was notably absent from skeletal muscle. Previous reports of exclusively maternally derived expression of Grb10 (11, 14) used an RT-PCR strategy with primers that amplified exon 18 sequence. High levels of maternal-specific mGrb10␫ may have masked the amplification of paternally expressed long transcripts. Both the expression pattern of Grb10 and its time of action seemed similar to that of IGF2. IGF2 is a major embryonic and placental growth factor whose loss of function results in severe

We thank Bill Skarnes for the Grb10⌬2–4 embryonic stem cells; Argiris Efstratiadis for providing the Igf2⌬ mice; James Dutton, Haymo Kurz, Kim Moorwood, Steve Sheardown, Catherine Skuse, and the staff of 5WL1 for advice and technical assistance; and Laurence Hurst, Robert Kelsh, and Jonathan Slack for critical reading of the manuscript. The work was supported by grants from the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Association for International Cancer Research.

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growth retardation. IGF2 exerts its effects predominantly through the IGF1 receptor in the embryo and through an uncharacterized receptor (RX) in the placenta (26). We found that Grb10 acts to control growth in a manner that is largely independent of IGF2 signaling in both the embryo and the placenta. Grb10 is therefore unlikely to regulate growth by means of the IGF1 receptor or the RX. Based on the expression of Grb10 in many insulin-responsive tissues, its strong in vitro interaction, and the effects of Grb10⌬2–4 on glycogen metabolism in the liver, we suggest that one physiological role of Grb10 may be to inhibit signaling by the insulin receptor. It is, however, notable that insulin receptor knockout mice show only a small effect on growth (27). Another strong candidate for a Grb10associated receptor is c-Met, which has been shown to bind Grb10 in vitro (5). Loss of c-Met signaling in the mouse leads to reduction in liver size and placental defects, as well as failure of muscle migration (35, 36). Hyperactive signaling through c-Met caused by loss of Grb10 may lead to the liver and placental hyperplasia that we describe above. In summary, our findings that mouse Grb10 is a potent embryonic and placental growth suppresser with similar imprinting profiles in mouse and human support a causal role for GRB10 in SRS. In addition, the independence of Grb10 and IGF2 signaling lends further evidence (37) for a major fetal growth pathway, influenced by genomic imprinting, that is independent of the IGF pathway.