Comparison of <i>Grb10KO</i> and <i>Grb10Δ2-4</i> mice.

<p>(A) Structure of <i>Grb10</i>, 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 <i>LacZ</i> reporter. (B) Comparative <i>LacZ</i> staining of bisected embryos at e14.5 inheriting the <i>Grb10KO</i> and <i>Grb10Δ2-4</i> alleles through each of the parental lines. CNS expression observed in <i>Grb10KO</i>^+/p embryos is not detected in <i>Grb10Δ2-4</i>^+/p embryos. (C) Comparative <i>LacZ</i> 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 <i>Grb10KO</i>^m/+ but not <i>Grb10Δ2-4</i>^m/+ females. WT (+/+) glands were stained with carmine alum to illustrate morphological changes.</p

Functional <i>Grb10</i> is required in mother and pup for WT offspring body proportions.

<p>(A) Lean/fat mass ratio in a subset of cross-fostered pups indicating that <i>Grb10</i> 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 <i>Grb10KO</i>^m/+ pups raised by <i>Grb10KO</i>^m/+ nurses is similar to that of WT pups raised by WT or <i>Grb10KO</i>^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. **<i>p</i><0.01. ns, not significant.</p

Characterisation of CRM1 and STAT5-mediated expression of <i>Grb10</i>.

<p>(A) <i>In silico</i> identification of conserved elements among selected vertebrate sequences. Conserved intronic sequences between <i>Grb10</i> 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 <i>Bam</i>HI 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 <i>Bam</i>HI site. (C) <i>In situ</i> hybridisation autoradiographs showing examples of overlapping sites of <i>Grb10</i> and <i>Stat5b</i> 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) <i>In vitro</i> 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). ***<i>p</i><0.001 (one-way ANOVA).</p

Overview of complementary <i>Grb10</i> functions in mother and pup.

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