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

Abstract

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.

Figure 1. Comparison of Grb10KO and Grb10Δ2-4 mice.

(A) Structure of Grb10, according to UCSC annotation, showing numbered exons (boxes) and translated regions (filled boxes). The integrated gene-trap cassettes include splice acceptor (SA) and polyadenylation (pA) signals, and a LacZ reporter. (B) Comparative LacZ staining of bisected embryos at e14.5 inheriting the Grb10KO and Grb10Δ2-4 alleles through each of the parental lines. CNS expression observed in Grb10KO ^+/p embryos is not detected in Grb10Δ2-4 ^+/p embryos. (C) Comparative LacZ staining of adult mammary glands at days 7.5 and 12.5 of gestation (G7.5, G12.5) and day 6 of lactation (L6), showing pregnancy-dependent reporter expression in Grb10KO ^m/+ but not Grb10Δ2-4 ^m/+ females. WT (+/+) glands were stained with carmine alum to illustrate morphological changes.

Figure 2. Characterisation of CRM1 and STAT5-mediated expression of Grb10.

(A) In silico identification of conserved elements among selected vertebrate sequences. Conserved intronic sequences between Grb10 homologs are plotted against annotated mouse transcripts. The PReMod track shows the position of the single regulatory module (CRM1). This site aligns with a sequence highly conserved between mouse, human, chimpanzee, cow, and chicken (highlighted). (B) Assay for DNase I hypersensitivity at CRM1, using probe A. A 6 kb BamHI fragment was detected in all samples. A 3.8 kb DNase I digestion fragment was detected in brain, but not liver, chromatin exposed to 200 U DNase I (arrow). The label “B” indicates a BamHI site. (C) In situ hybridisation autoradiographs showing examples of overlapping sites of Grb10 and Stat5b mRNA expression in adult mouse brain, including the arcuate nucleus of the hypothalamus (ARC), dorsomedial nucleus of the hypothalamus (DMH), lateral septal nucleus (LSV), medial amygdaloid nucleus (posteroventral part) (MePV), medial habenular nucleus (MHb), medial preoptic nucleus (MPA), median preoptic nucleus (medial part) (MPOM), periaqueductal grey (PAG), paraventricular thalamic nucleus (PVA), paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus (VMH), and ventraltegmental area (VTA). (D) In vitro transfection assay of the enhancer capability of CRM1. Luciferase activity was measured in cells transfected with a minimal promoter driving luciferase (pGL3-Pro) or with CRM1 cloned upstream of the minimal promoter (pGL3-Pro-CRM1). Only pGL3-Pro-CRM1 responded to increasing doses of constitutively active STAT5b (STAT5b1*6). ***p<0.001 (one-way ANOVA).

Figure 3. Grb10 controls postnatal supply and demand.

(A) WT (+/+) pups born to Grb10KO ^m/+ (m/+) dams gained more weight to postnatal day 15 than WT pups born to WT dams. (B) Grb10KO ^m/+ and WT male siblings on the day of birth. (C) Schematic of dam/pup relationships studied. All crosses used WT sires (white squares). WT dams (white circles) gave birth only to WT pups (i), while Grb10KO ^m/+ dams (half-filled circles) gave birth to mixed litters of WT and Grb10KO ^m/+ pups (ii). Cross-fostering (dashed line) enabled switching of dam/pup genotypes (iii and iv). Pure WT litters were cross-fostered to non-biological WT nurses as a control (v). (D) Effects of nurse or sibling genotype on WT pup growth. (E) Effects of Grb10 genotype interactions between nurse and offspring on pup growth. Values represent means ± standard error.

Figure 4. Functional Grb10 is required in mother and pup for WT offspring body proportions.

(A) Lean/fat mass ratio in a subset of cross-fostered pups indicating that Grb10 ablation in either nurse or pup increases the lean/fat mass ratio relative to WT pups raised by WT nurses. (B) Total lean mass. (C) Total fat mass. (D) Lean/fat mass ratio in a subset of cross-fostered pups, indicating that the body composition of Grb10KO ^m/+ pups raised by Grb10KO ^m/+ nurses is similar to that of WT pups raised by WT or Grb10KO ^m/+ nurses. Data points represent individual animals; mean values are represented by horizontal lines. Datasets in (B) and (C) were analysed using one-way ANOVA with Tukey's post hoc test. **p<0.01. ns, not significant.

Figure 5. Overview of complementary Grb10 functions in mother and pup.

Grb10 expressed in the mother promotes postnatal nutrient supply through the mammary gland, while offspring Grb10 suppresses nutrient demand. Together, this regulation of nutrient acquisition ensures offspring achieve an optimal body size. Body proportions are also influenced by both Grb10 expressed in the mother and in the offspring. Offspring Grb10 suppresses the development of lean mass, while offspring fat mass is promoted by Grb10 expressed in the mother and acting on postnatal nutrient supply, jointly promoting optimal offspring body proportions.