Fibroblasts are the major mesenchymal cell type in connective tissue and deposit the collagen and elastic fibers of the extracellular matrix (ECM)1. Even within a single tissue fibroblasts exhibit remarkable functional diversity, but it is not known whether this reflects the existence of a differentiation hierarchy or is a response to different environmental factors. Here we show, using transplantation assays and lineage tracing, that the fibroblasts of skin connective tissue arise from two distinct lineages. One forms the upper dermis, including the dermal papilla that regulates hair growth and the arrector pili muscle (APM), which controls piloerection. The other forms the lower dermis, including the reticular fibroblasts that synthesise the bulk of the fibrillar ECM, and the pre-adipocytes and adipocytes of the hypodermis. The upper lineage is required for hair follicle formation. In wounded adult skin, the initial wave of dermal repair is mediated by the lower lineage and upper dermal fibroblasts are recruited only during re-epithelialisation. Epidermal beta-catenin activation stimulates expansion of the upper dermal lineage, rendering wounds permissive for hair follicle formation. Our findings explain why wounding is linked to formation of ECM-rich scar tissue that lacks hair follicles2-4. They also form a platform for discovering fibroblast lineages in other tissues and for examining fibroblast changes in ageing and disease.

Complex multicellular organisms, such as mammals, express two complete sets of chromosomes per nucleus, combining the genetic material of both parents. However, epigenetic studies have demonstrated violations to this rule that are necessary for mammalian physiology; the most notable parental allele expression phenomenon is genomic imprinting. With the identification of endogenous imprinted genes, genomic imprinting became well-established as an epigenetic mechanism in which the expression pattern of a parental allele influences phenotypic expression. The expanding study of genomic imprinting is revealing a significant impact on brain functions and associated diseases. Here, we review key milestones in the field of imprinting and discuss mechanisms and systems in which imprinted genes exert a significant role.

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 Ϸ30% 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 Ϸ10% 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 ͉ cell signaling ͉ genomic imprinting ͉ growth factor receptor-bound protein ͉ insulin-like growth factor

The gene for the atypical Notch ligand Delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms functioning in multiple developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally-inherited chromosome1,2. Here we show that mice deficient in Dlk1 exhibit defects in postnatal neurogenesis within the subventricular zone (SVZ), a developmental continuum resulting in depletion of mature neurons in the olfactory bulb. We show that DLK1 is a factor secreted by niche-astrocytes, while its membrane-bound isoform is present in neural stem cells (NSCs) being required for the inductive effect of secreted DLK1 on self-renewal. Surprisingly, we find a requirement for Dlk1 expressed from both maternal and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ line-derived imprinting control region (IG-DMR). The results emphasize molecular relationships between NSCs and niche-astrocytes identifying a signalling system coded by a single gene functioning co-ordinately in both cell types. The modulation of genomic imprinting in a stem cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche raising wider questions about the adaptability, function, and evolution of imprinting within specific developmental contexts.

OBJECTIVE— Low birth weight (LBW) is associated with increased risk of obesity, diabetes, and cardiovascular disease during adult life. Moreover, this programmed disease risk can progress to subsequent generations. We previously described a mouse model of LBW, produced by maternal caloric undernutrition (UN) during late gestation. LBW offspring (F 1 -UN generation) develop progressive obesity and impaired glucose tolerance (IGT) with aging. We aimed to determine whether such metabolic phenotypes can be transmitted to subsequent generations in an experimental model, even in the absence of altered nutrition during the second pregnancy. RESEARCH DESIGN AND METHODS— We intercrossed female and male F 1 adult control (C) and UN mice and characterized metabolic phenotypes in F 2 offspring. RESULTS— We demonstrate that 1 ) reduced birth weight progresses to F 2 offspring through the paternal line (C♀-C♂ = 1.64 g; C♀-UN♂ = 1.57 g, P < 0.05; UN♀-C♂ = 1.64 g; UN♀-UN♂ = 1.60 g, P < 0.05), 2 ) obesity progresses through the maternal line (percent body fat: C♀-C♂ = 22.4%; C♀-UN♂ = 22.9%; UN♀-C♂ = 25.9%, P < 0.05; UN♀-UN♂ = 27.5%, P < 0.05), and 3 ) IGT progresses through both parental lineages (glucose tolerance test area under curve C♀-C♂ = 100; C♀-UN♂ = 122, P < 0.05; UN♀-C♂ = 131, P < 0.05; UN♀-UN♂ = 151, P < 0.05). Mechanistically, IGT in both F 1 and F 2 generations is linked to impaired β-cell function, explained, in part, by dysregulation of Sur1 expression. CONCLUSIONS— Maternal undernutrition during pregnancy (F 0 ) programs reduced birth weight, IGT, and obesity in both first- and second-generation offspring. Sex-specific transmission of phenotypes implicates complex mechanisms including alterations in the maternal metabolic environment (transmaternal inheritance of obesity), gene expression mediated by developmental and epigenetic pathways (transpaternal inheritance of LBW), or both (IGT).

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.Wilms' tumor, a pediatric malignancy of the kidneys, is the most common solid childhood tumor (reviewed in references 4, 7, 20, 30, and 33). The isolation of genes associated with Wilms' tumor led to the identification of a zinc finger protein, WT1. Subsequently, WT1 was shown to be a transcriptional regulator with putative target genes including those for growth factors and regulators of cell division (5,6,18,21). Approximately 15% of sporadic Wilms' tumors have been found to contain mutations in WT1, while others show aberrant WT1 expression (33).WT1 knockout mice (homozygous null) do not survive gestation, displaying absence or incorrect development of the kidney, gonads, spleen, heart, diaphragm, and retinal ganglia (12,14,35). These findings confirm a major role for WT1 in the formation of the genitourinary system and also a wider role in the development of other tissues.Alternative splicing, RNA editing, and an alternative translation start codon combine to produce a plethora of WT1 isoforms (reviewed in reference 33). One alternative splice inserts three amino acids (KTS) between zinc fingers three and four, resulting in a form of WT1 that associates with RNA processing factors and localizes to regions of RNA processing in the nucleus (17). Thus, the ϩKTS and ϪKTS isoforms of WT1 have been proposed to function in RNA processing and transcription, respectively. These isoforms have both overlapping and distinct roles during development (9, 10). Interestingly, the ϩKTS isoform of WT1 plays the dominant role in the development of the gonad, while the ϪKTS isoform has a more extensive function in kidney formation.The other alternative splice inserts 17 amino acids N terminal to the WT1 zinc fingers and has been shown to have effects on both cell division and cell survival (15,31,32). Specific elimination of this isoform of WT1 in mice does not result in any obvious defects in genitourinary development, suggesting that it may be required specifically for a tumor suppressor role or that it performs a subtle function (28).Several studies have shown ...

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10⌬2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10⌬2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10⌬2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.Insulin controls glucose homeostasis by regulating protein, lipid, and carbohydrate metabolism. Cellular responses to insulin in target tissues, such as skeletal muscle, adipose tissue, and liver, are mediated via the insulin receptor (Insr) (reviewed in reference 51). Activation of the Insr results in tyrosine phosphorylation of intracellular docking proteins such as Shc and IRS-1 through IRS-4, which then bind specific Src homology 2 (SH2) domain-containing enzymes and adapters, leading to the activation of downstream signaling cascades. A critical event mediating insulin regulation of metabolic endpoints is the activation of phosphatidylinositol 3-kinase (PI3K). This stimulates the synthesis of phosphatidylinositol 3,4,5-triphosphate, which induces plasma membrane recruitment and subsequent phosphorylation of protein kinase B (also known as Akt), a key player in the regulation of glucose uptake and glycogen synthesis. Activation of the Insr and downstream signaling results in increased glucose uptake, utilization, and storage in adipose tissue and skeletal muscle, while decreased gluconeogenesis and glycogenolysis and increased glycogen synthesis occur in the liver (reviewed in reference 51). Resistance to these effects of insulin is a defining feature of type 2 diabetes, a polygenic disease afflicting over 110 million people worldwide. Impaired insulin action is also a feature of obesity and predisposes people to arteriosclerosis and cardiovascular diseases, facts which highlight its importance in human health. The fundamental role of the Insr in insulin action was demonstrated following targeted disruption of the receptor (1, 29). To dissect the contribution of individual tissues to gl...

In developed societies, high-sugar and high-fat (HSHF) diets are now the norm and are increasing the rates of maternal obesity during pregnancy. In pregnant rodents, these diets lead to cardiovascular and metabolic dysfunction in their adult offspring, but the intrauterine mechanisms involved remain unknown. This study shows that, relative to standard chow, HSHF feeding throughout mouse pregnancy increases maternal adiposity (+30%, P<0.05) and reduces fetoplacental growth at d 16 (-10%, P<0.001). At d 19, however, HSHF diet group pup weight had normalized, despite the HSHF diet group placenta remaining small and morphologically compromised. This altered fetal growth trajectory was associated with enhanced placental glucose and amino acid transfer (+35%, P<0.001) and expression of their transporters (+40%, P<0.024). HSHF feeding also up-regulated placental expression of fatty acid transporter protein, metabolic signaling pathways (phosphoinositol 3-kinase and mitogen-activated protein kinase), and several growth regulatory imprinted genes (Igf2, Dlk1, Snrpn, Grb10, and H19) independently of changes in DNA methylation. Obesogenic diets during pregnancy, therefore, alter maternal nutrient partitioning, partly through changes in the placental phenotype, which helps to meet fetal nutrient demands for growth near term. However, by altering provision of specific nutrients, dietary-induced placental adaptations have important roles in programming development with health implications for the offspring in later life.