Imprinted Grb10, encoding growth factor receptor bound protein 10, regulates fetal growth independently of the insulin-like growth factor type 1 receptor (Igf1r) and insulin receptor (Insr) genes.

BACKGROUND: Optimal size at birth dictates perinatal survival and long-term risk of developing common disorders such as obesity, type 2 diabetes and cardiovascular disease. The imprinted Grb10 gene encodes a signalling adaptor protein capable of inhibiting receptor tyrosine kinases, including the insulin receptor (Insr) and insulin-like growth factor type 1 receptor (Igf1r). Grb10 restricts fetal growth such that Grb10 knockout (KO) mice are at birth some 25-35% larger than wild type. Using a mouse genetic approach, we test the widely held assumption that Grb10 influences growth through interaction with Igf1r, which has a highly conserved growth promoting role. RESULTS: Should Grb10 interact with Igf1r to regulate growth Grb10:Igf1r double mutant mice should be indistinguishable from Igf1r KO single mutants, which are around half normal size at birth. Instead, Grb10:Igf1r double mutants were intermediate in size between Grb10 KO and Igf1r KO single mutants, indicating additive effects of the two signalling proteins having opposite actions in separate pathways. Some organs examined followed a similar pattern, though Grb10 KO neonates exhibited sparing of the brain and kidneys, whereas the influence of Igf1r extended to all organs. An interaction between Grb10 and Insr was similarly investigated. While there was no general evidence for a major interaction for fetal growth regulation, the liver was an exception. The liver in Grb10 KO mutants was disproportionately overgrown with evidence of excess lipid storage in hepatocytes, whereas Grb10:Insr double mutants were indistinguishable from Insr single mutants or wild types. CONCLUSIONS: Grb10 acts largely independently of Igf1r or Insr to control fetal growth and has a more variable influence on individual organs. Only the disproportionate overgrowth and excess lipid storage seen in the Grb10 KO neonatal liver can be explained through an interaction between Grb10 and the Insr. Our findings are important for understanding how positive and negative influences on fetal growth dictate size and tissue proportions at birth.

Detailed analysis of paternal knockout Grb10 mice suggests effects on stability of social behavior, rather than social dominance.

Imprinted genes are highly expressed in monoaminergic regions of the midbrain and their functions in this area are thought to have an impact on mammalian social behaviors. One such imprinted gene is Grb10, of which the paternal allele is generally recognized as mediating social dominance behavior. However, there has been no detailed study of social dominance in Grb10 +/p mice. Moreover, the original study examined tube-test behavior in isolated mice 10 months of age. Isolation testing favors more territorial and aggressive behaviors, and does not address social dominance strategies employed in group housing contexts. Furthermore, isolation stress impacts midbrain function and dominance related behavior, often through alterations in monoaminergic signaling. Thus, we undertook a systematic study of Grb10 +/p social rank and dominance behavior within the cage group, using a number of convergent behavioral tests. We examined both male and female mice to account for sex differences and tested cohorts aged 2, 6 and 10 months to examine any developments related to age. We found group-housed Grb10 +/p mice do not show evidence of enhanced social dominance, but cages containing Grb10 +/p and wild-type mice lacked the normal correlation between three different measures of social rank. Moreover, a separate study indicated isolation stress induced inconsistent changes in tube test behavior. Taken together, these data suggest future research on Grb10 +/p mice should focus on the stability of social behaviors, rather than dominance per se.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

Developmental programming mediated by complementary roles of imprinted Grb10 in mother and pup.

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk.

Antagonistic roles in fetal development and adult physiology for the oppositely imprinted Grb10 and Dlk1 genes.

BACKGROUND: Despite being a fundamental biological problem the control of body size and proportions during development remains poorly understood, although it is accepted that the insulin-like growth factor (IGF) pathway has a central role in growth regulation, probably in all animals. The involvement of imprinted genes has also attracted much attention, not least because two of the earliest discovered were shown to be oppositely imprinted and antagonistic in their regulation of growth. The Igf2 gene encodes a paternally expressed ligand that promotes growth, while maternally expressed Igf2r encodes a cell surface receptor that restricts growth by sequestering Igf2 and targeting it for lysosomal degradation. There are now over 150 imprinted genes known in mammals, but no other clear examples of antagonistic gene pairs have been identified. The delta-like 1 gene (Dlk1) encodes a putative ligand that promotes fetal growth and in adults restricts adipose deposition. Conversely, Grb10 encodes an intracellular signalling adaptor protein that, when expressed from the maternal allele, acts to restrict fetal growth and is permissive for adipose deposition in adulthood. RESULTS: Here, using knockout mice, we present genetic and physiological evidence that these two factors exert their opposite effects on growth and physiology through a common signalling pathway. The major effects are on body size (particularly growth during early life), lean:adipose proportions, glucose regulated metabolism and lipid storage in the liver. A biochemical pathway linking the two cell signalling factors remains to be defined. CONCLUSIONS: We propose that Dlk1 and Grb10 define a mammalian growth axis that is separate from the IGF pathway, yet also features an antagonistic imprinted gene pair.

High basal γH2AX levels sustain self-renewal of mouse embryonic and induced pluripotent stem cells.

Phosphorylation of histone H2AX (γH2AX) is known to be the earliest indicator of DNA double-strand breaks. Recently, it has been shown that mouse embryonic stem cells (mESCs) have very high basal levels of γH2AX, even when they have not been exposed to genotoxic agents. As the specialized role of high basal γH2AX levels in pluripotent stem cells is still debated, we investigated whether H2AX phosphorylation is important in maintaining self-renewal of these cells. Here, we report that not only mESCs but also mouse-induced pluripotent stem cells (miPSCs), have high basal levels of γH2AX. We show that basal γH2AX levels decrease upon ESC and iPSC differentiation and increase when the cells are treated with self-renewal-enhancing small molecules. We observe that self-renewal activity is highly compromised in H2AX-/- cells and that it can be restored in these cells through reconstitution with a wild-type, but not a phospho-mutated, H2AX construct. Taken together, our findings suggest a novel function of H2AX that expands the knowledge of this histone variant beyond its role in DNA damage and into a new specialized biological function in mouse pluripotent stem cells.

Distinct physiological and behavioural functions for parental alleles of imprinted Grb10.

Imprinted genes, defined by their preferential expression of a single parental allele, represent a subset of the mammalian genome and often have key roles in embryonic development, but also postnatal functions including energy homeostasis and behaviour. When the two parental alleles are unequally represented within a social group (when there is sex bias in dispersal and/or variance in reproductive success), imprinted genes may evolve to modulate social behaviour, although so far no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Here we demonstrate that within the brain Grb10 is expressed from the paternal allele from fetal life into adulthood and that ablation of this expression engenders increased social dominance specifically among other aspects of social behaviour, a finding supported by the observed increase in allogrooming by paternal Grb10-deficient animals. Grb10 is, therefore, the first example of an imprinted gene that regulates social behaviour. It is also currently alone in exhibiting imprinted expression from each of the parental alleles in a tissue-specific manner, as loss of the peripherally expressed maternal allele leads to significant fetal and placental overgrowth. Thus Grb10 is, so far, a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, owing to actions of the two parental alleles in different tissues.

Frequent long-range epigenetic silencing of protocadherin gene clusters on chromosome 5q31 in Wilms' tumor.

Wilms' tumour (WT) is a pediatric tumor of the kidney that arises via failure of the fetal developmental program. The absence of identifiable mutations in the majority of WTs suggests the frequent involvement of epigenetic aberrations in WT. We therefore conducted a genome-wide analysis of promoter hypermethylation in WTs and identified hypermethylation at chromosome 5q31 spanning 800 kilobases (kb) and more than 50 genes. The methylated genes all belong to alpha-, beta-, and gamma-protocadherin (PCDH) gene clusters (Human Genome Organization nomenclature PCDHA@, PCDHB@, and PCDHG@, respectively). This demonstrates that long-range epigenetic silencing (LRES) occurs in developmental tumors as well as in adult tumors. Bisulfite polymerase chain reaction analysis showed that PCDH hypermethylation is a frequent event found in all Wilms' tumor subtypes. Hypermethylation is concordant with reduced PCDH expression in tumors. WT precursor lesions showed no PCDH hypermethylation, suggesting that de novo PCDH hypermethylation occurs during malignant progression. Discrete boundaries of the PCDH domain are delimited by abrupt changes in histone modifications; unmethylated genes flanking the LRES are associated with permissive marks which are absent from methylated genes within the domain. Silenced genes are marked with non-permissive histone 3 lysine 9 dimethylation. Expression analysis of embryonic murine kidney and differentiating rat metanephric mesenchymal cells demonstrates that Pcdh expression is developmentally regulated and that Pcdhg@ genes are expressed in blastemal cells. Importantly, we show that PCDHs negatively regulate canonical Wnt signalling, as short-interfering RNA-induced reduction of PCDHG@ encoded proteins leads to elevated beta-catenin protein, increased beta-catenin/T-cell factor (TCF) reporter activity, and induction of Wnt target genes. Conversely, over-expression of PCDHs suppresses beta-catenin/TCF-reporter activity and also inhibits colony formation and growth of cancer cells in soft agar. Thus PCDHs are candidate tumor suppressors that modulate regulatory pathways critical in development and disease, such as canonical Wnt signaling.

Alternately spliced WT1 antisense transcripts interact with WT1 sense RNA and show epigenetic and splicing defects in cancer.

Many mammalian genes contain overlapping antisense RNAs, but the functions and mechanisms of action of these transcripts are mostly unknown. WT1 is a well-characterized developmental gene that is mutated in Wilms' tumor (WT) and acute myeloid leukaemia (AML) and has an antisense transcript (WT1-AS), which we have previously found to regulate WT1 protein levels. In this study, we show that WT1-AS is present in multiple spliceoforms that are usually expressed in parallel with WT1 RNA in human and mouse tissues. We demonstrate that the expression of WT1-AS correlates with methylation of the antisense regulatory region (ARR) in WT1 intron 1, displaying imprinted monoallelic expression in normal kidney and loss of imprinting in WT. However, we find no evidence for imprinting of mouse Wt1-as. WT1-AS transcripts are exported into the cytoplasm and form heteroduplexes with WT1 mRNA in the overlapping region in WT1 exon 1. In AML, there is often abnormal splicing of WT1-AS, which may play a role in the development of this malignancy. These results show that WT1 encodes conserved antisense RNAs that may have an important regulatory role in WT1 expression via RNA:RNA interactions, and which can become deregulated by a variety of mechanisms in cancer.

A CTCF-binding silencer regulates the imprinted genes AWT1 and WT1-AS and exhibits sequential epigenetic defects during Wilms' tumourigenesis.

We have shown previously that AWT1 and WT1-AS are functionally imprinted in human kidney. In the adult kidney, expression of both transcripts is restricted to the paternal allele, with the silent maternal allele retaining methylation at the WT1 antisense regulatory region (WT1 ARR). Here, we report characterization of the WT1 ARR differentially methylated region and show that it contains a transcriptional silencer element acting on both the AWT1 and WT1-AS promoters. DNA methylation of the silencer results in increased transcriptional repression, and the silencer is also shown to be an in vitro and in vivo target site for the imprinting regulator protein CTCF. Binding of CTCF is methylation-sensitive and limited to the unmethylated silencer. Potentiation of the silencer activity is demonstrated after CTCF protein is knocked down, suggesting a novel silencer-blocking activity for CTCF. We also report assessment of WT1 ARR methylation in developmental and tumour tissues, including the first analysis of Wilms' tumour precursor lesions, nephrogenic rests. Nephrogenic rests show increases in methylation levels relative to foetal kidney and reductions relative to the adult kidney, together with biallelic expression of AWT1 and WT1-AS. Notably, the methylation status of CpG residues within the CTCF target site appears to distinguish monoallelic and biallelic expression states. Our data suggest that failure of methylation spreading at the WT1 ARR early in renal development, followed by imprint erasure, occurs during Wilms' tumourigenesis. We propose a model wherein imprinting defects at chromosome 11p13 may contribute to Wilms' tumourigenesis.

Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele.

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.