Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies

Abstract

Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28-Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28-Let-7 pathway.

Figure 1. Lin28a inducible mice display increased growth and a proportional increase in organ sizes

(a) Schema for the design of the Lin28a transgenic mice. Animals under study were not induced with doxycycline. (b) Adult Lin28a Tg mice exhibited greater size, wider facies, larger ears, and coarser hair than WT mice. (c-d) Male (4 WT and 5 Tg) and female (6 WT and 4 Tg) weights and crown-rump lengths from weaning until three months of age. (e) Dual Energy X-ray Absorptiometry results showing percent body fat mass, percent lean mass, bone mineral content, and bone mineral density (g/bone area (m²)) for 3 WT and 5 Tg males. (f) Relative organ weights of Tg animals normalized to WT animals (n = 7 and 7). All values represent means +/− SEM (*, p < 0.05; **, p <0.01) and the numbers of mice (n) are shown in the chart or noted in the legend.

Figure 2. Lin28a Tg mice show a delay in the onset of puberty

(a-b) Comparison of the timing of vaginal opening (VO) in WT (blue) and transgenic mice (red) on the CD-1 (a) and C57/B6 backgrounds (b). The cumulative percent of animals with VO is displayed. (c) Weights of WT and Lin28a Tg mice at date of weaning and time of VO. (d) Uterus/ovary weights measured as a percentage of total body weight at day 26 of age (n = 10 and 8). (e) The time to first estrus. (f) The time to first litter. (g) The first litter size from these matings. All values represent means +/− SEM (*, p < 0.05; **, p <0.01) and the numbers of mice (n) are shown in the chart or noted in the legend.

Figure 3. Ectopic expression of Lin28a in transgenic mice

Lin28a mRNA levels in adult (a) and neonatal organs (b) as measured by quantitative PCR (n = 6 and 6 for all analyses). (c-d) Mature let-7a and let-7g levels in the adult liver, skin, and muscle (c) and in the liver and limb of neonates (d). (e) Lin28a immunohistochemistry in neonatal skin and muscle. (f) let-7a and let-7g in the hypothalamic-pituitary-gonadal axis of adult mice. (g) Histology of skin and skull bone in 4 month-old WT and Tg adults. All scale bars are 100μm. All values represent means +/− SEM (*, p < 0.05; **, p <0.01) and the numbers of mice (n) are noted here.

Figure 4. Lin28a Tg mice display increased glucose uptake and insulin sensitivity

(a) Igf2 mRNA levels in adult mouse tissues. Note the break in the y-axis designating a change in scale. (b-c) Glucose concentrations in fasting and fed state mice. Blood glucose and insulin concentrations were determined by tail bleeding in 1 or 3 month-old mice. Experimental groups consisted of 5-10 animals each. (d-e) Results of glucose tolerance tests (GTT) and (f-g) insulin tolerance tests (ITT) for WT and Lin28a Tg mice. GTT and ITT were performed on 1-3 month-old mice with 0.75 units of insulin/kg of body weight and 2 g of glucose/kg of body weight, respectively. (h) Insulin measurements performed during a GTT. (i) Average islet area measurements in 7 week old (n = 2 and 2) or 15 week old pancreata (n = 3 and 3) (8 to 40 islet analyzed per animal). (j-k) 2-deoxy-D-[³H] glucose uptake assay with C2C12 + Lin28a overexpression (j) and C2C12 + Lin28a shRNA knockdown (k). (l) Gene set enrichment analysis of muscle microarray data showing statistically significantly up and downregulated pathways in Tg vs. WT skeletal muscle. (m) Lactate levels were measured using Lactate Scout strips during a GTT experiment. All values represent means +/− SEM (*, p < 0.05; **, p <0.01) and the numbers of experimental animals are listed within the charts.