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Keywords:

  • Lin28a;
  • Lin28b;
  • Dwarfism;
  • Growth;
  • Glucose metabolism;
  • Diabetes;
  • let-7;
  • mTOR

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here, we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO could be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling. STEM Cells 2013;31:1563–1573


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Organismal growth is a highly complex process tied to multiple genetic, nutritional, hormonal, and environmental factors. Recent human genome-wide association studies (GWAS) have reported over a hundred loci relevant to height [1–3]. Among these loci are genes encoding the RNA-binding protein LIN28B, which together with its paralog Lin28A regulate biogenesis of the let-7 family of tumor-suppressor microRNAs, as well as mRNA stability and translation via direct binding of various gene transcripts such as the stem cell factor Oct4, the growth factor Igf2, cell cycle regulators, and ribosomal subunits [4–11]. In addition to Lin28B, genetic variation around loci for at least three validated let-7 targets (Dot1L, HMGA2, and CDK6) has also been associated with human height in GWAS [1], and GWAS has also implicated Lin28B in the timing of onset of human puberty [12–15]. Interestingly, lin-28 and let-7 were originally identified as heterochronic regulators of developmental timing in Caenorhabditis elegans [16–18]. Whereas lin-28 controls let-7 biogenesis, let-7 also regulates lin-28 expression by binding its complementary sequences in the 3′ untranslated region. Thus, lin-28 and let-7 regulate each other and comprise a double negative feedback loop. Their expression is tightly regulated during development, and evolutionarily conserved among worms, flies, frogs, and mammals [19–21].

We have recently shown that transgenic mice that constitutively overexpress Lin28a manifest increased body size and delayed onset of puberty [22], and that overexpression of both murine Lin28a and human LIN28B promotes an insulin-sensitized state that resists diabetes in mice, in direct contrast to overexpression of let-7, which results in insulin resistance and impaired glucose tolerance [23]. Given that Lin28a/b are expressed in distinct spatio-temporal patterns, their precise physiological roles in mammalian development and metabolism remain unclear. Here, we describe Lin28a and Lin28b knockout (KO) mice and characterize the distinct spatio-temporal functions of the Lin28 paralogs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Mice

All animal procedures were approved by the Institutional Animal Care and Use Committee. Design of conditional Lin28a KO mice was reported previously [23]. Design of conditional Lin28b KO mice is shown in Supporting Information Figure S10. For Lin28a and lin28b conditional KO mice, PCR fragments of both gene loci were cloned into a plasmid having two loxP cassetes and a PGK-Neo cassete flanked with frt sequences, and targeting was performed into V6.5 embryonic stem cells (ESCs). Homologous recombination was confirmed by Southern blotting. Chimeric mice were generated by injection of ESCs into Balb/c blastocysts, and then bred to BALB/c females to generate germline-transmitted pups. To delete the floxed allele in the germ line, we used a Ddx4-Cre strain (The Jackson Laboratory, Bar Harbor, ME). LSL-iLet-7 mice were generated by crossing iLet-7 mice [23] and ROSA26-rtTA-IRES-EGFP mice [24]. Ddx4-Cre, Myf5-Cre, Alb-Cre, Ins2-Cre, ubiquitin-Cre/ERT2, and ROSA26-rtTA-IRES-EGFP mice were purchased from The Jackson Laboratory. Tsc1fl/fl mice are kindly provided from Dr. David Kwiatkowski laboratory. For all experiments, littermate controls were used.

Histology

Tissue samples were fixed in 10% buffered formalin or Bouin's solution and embedded in paraffin.

Glucose and Insulin Tolerance Tests

Overnight-fasted mice were given i.p. glucose (2 mg/g b.wt.). For insulin tolerance test (ITT), 5-hour fasted mice were given 0.75 U insulin/kg b.wt. by i.p. injection (Humulin, Eli Lilly, Indianapolis, IN). Blood glucose was determined with a Lifescan One Touch glucometer (Milpitas, CA).

Drug Treatment

Tamoxifen (TAM) (Sigma, St. Louis, MO) was dissolved in corn oil at 20 mg/ml. To delete floxed alleles in E15.5 embryos, a dose of 0.1 mg/g mouse weight was injected intraperitoneally once to the mothers. For 7–9 days old pups, a dose of 0.5 mg/g mouse weight was injected intraperitoneally once. For adult mice, a dose of 0.2 mg/g mouse weight was injected five times in consecutive days.

Quantitative RT-PCR

Total RNA was collected by TRIzol and reverse-transcribed. mRNA and microRNA expression were measured by quantitative PCR using the ΔΔCT method as described previously [23].

Targeted Liquid-Chromatography Mass Spectrometry

Liquid-chromatography mass spectrometry (LC/MS/MS)-based metabolomics analysis was performed as described previously [25]. Whole embryos were harvested in 80% (vol/vol) methanol at −78°C. Insoluble material in lysates was centrifuged at 2,000g for 15 minutes, and the resulting supernatant was evaporated using a refrigerated speed vac. Samples were resuspended using 20 μl LC/MS grade water. Seven microliter was injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/SCIEX, Framingham, MA) coupled to a Prominence HPLC system (Shimadzu, Columbia, MD) using selected reaction monitoring (SRM) of a total of 252 endogenous water-soluble metabolites for analyses of samples. Some metabolites were targeted in both the positive and negative ion mode for a total of 292 SRM transitions. ESI voltage was +4,900 V in the positive ion mode and −4,500 V in the negative ion mode using positive/negative switching. The dwell time was 4 milliseconds per SRM transition, and the total cycle time was 1.82-second producing 9–12 data points per metabolite peak. Samples were delivered to the MS using hydrophilic interaction chromatography using a 4.6 mm internal diameter × 10 cm Amide XBridge column (Waters, Milford, MA) at 300 μl/minute. Mobile phase gradients were run starting from 85% buffer B (LC/MS grade acetonitrile) to 35% buffer B from 0 to 3 minutes; 35% buffer B to 0% buffer B from 3 to 12 minutes; 0% buffer B held from 12 to 17 minutes; 0% buffer B to 85% buffer B from 17 to 18 minutes; and 85% B held for 7 minutes to re-equilibrate the column. Buffer A was comprised of 20 mM ammonium hydroxide and 20 mM ammonium acetate in 95:5 water:acetonitrile (pH = 9.0). Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/SCIEX).

Statistical Analysis

Data are presented as mean ± SEM. Student's t test (two-tailed distribution, two-sample unequal variance), Fisher's exact probability test, or χ2 test was used to calculate p values, with Mendelian ratios as the expected distribution where appropriate. Statistical significance is displayed as *, p < .05 or **, p < .01.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Lin28a KO Mice Manifest Dwarfism Beginning in Embryogenesis

We first generated and analyzed Lin28a KO mice (see Materials and Methods). Lin28a KO mice showed dwarfism as early as E13.5, and by E18.5 showed 20% reduction in weight, relative to heterozygote controls (Fig. 1A, 1B). Although born in the expected Mendelian ratio, over 93% of Lin28a KO mice died within 1 day after birth (Fig. 1C, 1D). By histopathologic analysis, two out of five KO mice which died perinatally harbored a cardiac ventricular septal defect (Supporting Information Fig. S1A), but virtually all other tissues were histologically normal (Supporting Information Fig. S1B). The high perinatal lethality remains unexplained.

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Figure 1. Constitutive deletion of Lin28a leads to persistent growth retardation from embryogenesis through adulthood. (A): Body weights of E13.5 Lin28a +/+, +/−, and -/- embryos. (B): Body weights of E18.5 Lin28a +/+, +/−, and -/- embryos. (C): Representative images of a heterozygous mouse and its Lin28a KO littermate. Bar = 1 cm. (D): Viability of P0.5 newborns and 21-day-old pups born from an inbreeding cross of Lin28a heterozygotes. p-Values were calculated by χ2 test. (E, F): Growth curves for Lin28a heterozygous (solid) versus KO (dashed) mice, both male (blue) and female (red). n = 4–15. (E): Body weight. (F): Body length. (G): Organ weights normalized to total body weight from 4 to 5-month-old males. Epididymal fat pads were used to measure the fat weight. n = 3. (H): DEXA measurements of % lean and % fat weights, relative to total body weight. n = 5–8. (I): DEXA measurement of bone mineral density. n = 5–8. *, p < .05; **, p < .01. Error bars represent SEM. Abbreviation: KO, knockout.

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Because a minority of KO mice survive to adulthood (Fig. 1D), we were able to analyze the postnatal growth of the survivors (Supporting Information Fig. S2A). Both male and female KO mice were 30%–50% smaller in weight and 20%–30% shorter in length, and remained smaller than heterozygote controls throughout the observation period (Fig. 1E, 1F). Organ weights were proportionally lower relative to total body weight, except for fat mass, which was severely decreased in the adult KO mice (Fig. 1G). Dual-energy x-ray absorptiometry imaging confirmed the decreased fat mass and also revealed decreased bone mineral density in the KO animals (Fig. 1H, 1I, Supporting Information Fig. S2B), suggesting that Lin28a KO mice suffer from metabolic dysregulation.

Lin28b Regulates Adult Growth in Males, But Not in Females

We next generated and analyzed Lin28b KO mice, using a similar gene deletion strategy as for Lin28a KO mice, in which exon 2 encoding the functional cold shock domain was engineered to be flanked by LoxP sites and subsequently deleted by Cre recombinase (see Materials and Methods). Although LIN28B is highly expressed in the placenta [26], we did not observe any difference in weights of Lin28b KO embryos or placentae at E18.5 relative to heterozygote controls, indicating that unlike Lin28a, Lin28b is dispensable for embryonic growth (Supporting Information Fig. S3A, S3B). However, Lin28b KO males did show postnatal dwarfism (Fig. 2A, 2B), with organ weights reduced in relative proportion to total body weight (Supporting Information Fig. S3C). In contrast, Lin28b KO females showed no postnatal growth defects, suggesting Lin28b regulates growth in mice in a gender-specific manner.

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Figure 2. Lin28a and Lin28b dosage regulates postnatal growth. (A, B): Postnatal growth curves of Lin28b heterozygous versus KO mice. n = 8–21. (A): Body weight. (B): Body length. (C, D): Postnatal growth curves of Lin28a +/+ versus +/− mice on a Lin28b KO background. n = 11–22. (C): Body weight. (D): Body length. *, p < .05; **, p < .01. Error bars represent SEM. Abbreviation: KO, knockout.

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Combined Dosage of Lin28a/b Is Critical for Postnatal Growth

Lin28a and Lin28b are functionally redundant in that they both block biogenesis of let-7 miRNAs and serve as oncogenes [4, 8, 27-29], although the precise mechanism of let-7 repression has been reported to be distinct for Lin28 paralogs [30]. To discern the importance of gene dosage of Lin28a/b on growth phenotypes, we bred Lin28a+/−b-/- mice and examined their progenies. Weights of embryos and placentae at E13.5 were not significantly different between Lin28a+/−b-/- and Lin28a+/+b-/- (i.e., Lin28b KO) mice (Supporting Information Fig. S3D). However, in both males and females, Lin28a+/−b-/- mice showed postnatal dwarfism compared to Lin28b KO littermates (Fig. 2C, 2D). Haploinsufficiency of Lin28a affected postnatal growth only in a Lin28b null background, since Lin28a+/− mice grew comparably to Lin28a+/+ (i.e., wild-type) controls (Supporting Information Fig. S3E), indicating that the combined dose of Lin28a/b alleles is critical to postnatal growth.

Lin28a/b Double KO Causes Developmental Delay and Embryonic Lethality

To discern the embryonic phenotypes of dual deficiency of Lin28a/b, we analyzed midgestation embryos at different ages and found that double KO (DKO) embryos were smaller in size at E9.5 and E10.5 compared to Lin28a+/+b-/- and Lin28a+/−b-/- embryos (Fig. 3A). Moreover, we observed significantly fewer somites by E10.5 in DKO embryos, suggesting a developmental delay (Fig. 3B). Open neural tubes were found in 2/7 E11.5 DKO embryos, which was also consistent with a developmental delay (Fig. 3C, 3D). Other abnormalities included collapsed ventricles and poor development of the forebrain in some cases. DKO embryos died between E10.5 and E12.5 (Fig. 3E and Supporting Information Table).

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Figure 3. Double knockout of Lin28a/b leads to synthetic lethality in E10.5–E12.5 embryos. (A): Representative images, and (B) Number of somites, in Lin28a +/+ versus Lin28a+/− versus Lin28a-/- embryos on a Lin28b KO background at E9.5–10.5. n = 4–15. (C): Representative images of Lin28a+/− versus Lin28a-/- embryos on a Lin28b KO background at E11.5. Arrow indicates an open neural tube. (D): Hematoxylin-eosin (H&E) staining of Lin28a+/− versus Lin28a-/- embryos on a Lin28b KO background at E11.5. (E): Frequency of viable Lin28a-/-; Lin28b-/- embryos at different ages (Supporting Information Table). Scale bars &Equals; 1 mm (A and C), 500 μm (D). *, p < .05; **, p < .01. Error bars represent SEM. Abbreviation: DKO, double knockout.

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Fetal Expression of Lin28a/b Is Required for Regulating Postnatal Growth

Lin28a and Lin28b are expressed in early embryos but in limited tissues in adults [31]. To test whether embryonic or adult expression regulates postnatal animal growth, we generated TAM-inducible conditional KO mice by crossing a ubiquitin-Cre/ERT2 expressing strain (UBC-CreER) lacking either Lin28a or Lin28b to a strain carrying floxed Lin28afl/fl or Lin28bfl/fl alleles, to generate Lin28afl/−; UBC-CreER (+) or Lin28bfl/−; UBC-CreER (+) mice (Fig. 4A, 4E). We injected TAM to delete the floxed alleles at different ages. PCR analysis confirmed high efficiency allele deletion in multiple tissues (Supporting Information Fig. S4). For Lin28a, Lin28afl/−; UBC-CreER (+) males injected with TAM at E15.5 showed significant postnatal dwarfism, as measured by total body weight and body length (Fig. 4B and Supporting Information Fig. S5A), although the degree of dwarfism at 3 weeks old is more modest than in constitutive Lin28a KO mice. Lin28afl/−; UBC-CreER (+) females injected with TAM at E15.5 showed no dwarfism phenotype. In contrast, when TAM was injected at 7–9 days (neonate) or 6 weeks of age (adult), their subsequent adult growth was indistinguishable from controls (Fig. 4C, 4D and Supporting Information Fig. S5B, S5C). For Lin28b, Lin28bfl/−; UBC-CreER (+) males injected with TAM at E15.5 phenocopied the postnatal dwarfism of constitutive Lin28b KO mice (Fig. 4F and Supporting Information Fig. S5D). In contrast, no growth phenotype was observed when TAM was injected at 7–9 days (neonate) or 6 weeks old (adult) into Lin28bfl/−; UBC-CreER (+) males (Fig. 4G, 4H and Supporting Information Fig. S5E, S5F). These results demonstrate that postnatal growth is dictated by fetal expression of both Lin28a and Lin28b, whereas adult expression is dispensable. Interestingly, deletion of Lin28a at E15.5 does not result in the high frequency of perinatal death observed in constitutive Lin28a KO, nor does it phenocopy the full degree of dwarfism due to constitutive loss of Lin28a, whereas deletion of fetal Lin28b at E15.5 fully phenocopies the dwarfism due to constitutive loss of Lin28b. Taken together, our data suggest that Lin28a acts earlier on organismal growth than Lin28b, such that effects of gene deletion are already apparent in utero.

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Figure 4. Fetal Lin28a/b, not neonatal or adult Lin28a/b, regulates postnatal growth. (A): Breeding strategy to test Lin28a fl/− mice at different ages. n &Equals; 3–12. (B--D): Postnatal growth curves of Lin28a fl/−; UBC-CreER (+) versus Lin28a fl/− mice. Body weights after tamoxifen (TAM) injections at (B) E15.5, (C) P7–9, and (D) 6 weeks. (E): Breeding strategy to test Lin28b fl/− mice at different ages. n &Equals; 5–13. (F--H): Postnatal growth curves of Lin28b fl/−; UBC-CreER (+) versus Lin28b fl/− mice. Body weights after TAM injections at (F) E15.5, (G) P7–9, and (H) 6 weeks. Arrows indicate ages at TAM injection. *, p < .05; **, p < .01. Error bars represent SEM.

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Lin28a KO and Lin28b KO Mice Suffer from Defects in Glucose Metabolism

We have previously shown that Lin28a and LIN28B transgenic mice are more resistant to diabetes, whereas muscle-specific Lin28a KO mice show insulin resistance and impaired glucose uptake [23]. In this study, we found that Lin28a KO mice show a dramatic loss of fat mass by adulthood (Fig. 1G, 1H), although we detect no Lin28a/b expression in fat tissues (Supporting Information Fig. S6). Thus, we further investigated whether Lin28a/b play systemic physiological roles in programming metabolism prior to changes in growth. Glucose tolerance tests (GTT) and ITT demonstrated that muscle-specific loss of Lin28b as well as Lin28a [23] led to insulin resistance and impaired glucose uptake (Fig. 5A, 5B). Since Lin28a KO mice already show a significant growth delay as early as E13.5, we performed metabolomic profiling at E10.5 to determine whether loss of Lin28a affects embryonic metabolism prior to detectable differences in embryonic growth. We observed a relative accumulation of glucose-6-phosphate and fructose-6-phosphate, and significant decreases in glycolytic intermediates after the phosphofructokinase step, suggesting a lower flux in glycolysis (Fig. 5C). This is consistent with our observations of a lower NADH/NAD ratio, and a higher glutathione/glutathione disulfide (GSH/GSSG) ratio, which are indicative of lower rates of glucose catabolism via glycolysis, Krebs cycle, and oxidative phosphorylation, which generate NADH and reactive oxygen species (Fig. 5D). We also found an aberrant drop in dUTP and an accumulation of dTTP, dTDP, dTMP, and thymine, suggesting that loss of Lin28a also leads to dysregulation of pyrimidine metabolism (Fig. 5E).

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Figure 5. Glucose metabolism dysfunction in Lin28a KO and Lin28b KO mice. (A): GTT (n &Equals; 9–10) and (B) ITT (n &Equals; 8–11) of Lin28b fl/fl; Myf5-Cre mice. (C--E): Metabolomic changes in Lin28a+/− versus -/- embryos at E10.5. (C): Glycolysis pathway. (D): Bioenergetics and redox balance. (E): Nucleotide synthesis. +, p < .1; *, p < .05; **, p < .01. Error bars represent SEM. Abbreviations: GTT, glucose tolerance test; ITT, insulin tolerance test; KO, knockout.

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Fetal Muscle-Specific Expression of Lin28b Regulates Adult Growth

Mice engineered to over-express let-7 miRNA are smaller than controls [23, 32]. To understand how Lin28a and Lin28b deficiency affect let-7 levels, we compared let-7 expression levels by quantitative PCR in E8.5 embryos and various tissues of neonatal and adult mice. Lin28a but not Lin28b KO mice expressed 7–18-fold higher levels of let-7a, d, e, f, g, and i but for unclear reasons not b or c, relative to wild-type controls in the E8.5 embryo (Fig. 6A), indicating that Lin28a is the primary regulator of let-7 microRNAs in the early embryo. Let-7 was also higher in the skeletal muscles of neonatal Lin28a KO mice, but no increases in let-7 were observed in adult skeletal muscles nor any other tissue (Fig. 6B and Supporting Information Fig. S7A). This result is consistent with the fact that Lin28a is primarily restricted in its expression to fetal tissues. For Lin28b KO mice, some let-7 family members were expressed at modestly higher levels in adult skeletal muscles in Lin28b KO mice (1.3–2-fold; Fig. 6C and Supporting Information Fig. S7B). These observations indicate that Lin28a potently suppresses let-7 biogenesis during embryogenesis, but its effect decreases postnatally and eventually disappears by adulthood, whereas Lin28b has a smaller but still significant effect on let-7 biogenesis in adulthood, indicating that Lin28a and Lin28b regulate let-7 biogenesis and growth at different developmental stages.

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Figure 6. Muscle-specific Lin28b/let-7 regulates postnatal growth. (A--C): Let-7 levels in (A) Lin28a+/− versus -/- E8.5 embryos, (B) Lin28a+/− versus -/- neonatal muscles, and (C) Lin28b+/− versus -/- adult skeletal muscles. n &Equals; 3–5. (D, E): Postnatal growth curves of LSL-iLet-7s; Myf5-Cre(+) males given DOX from conception. n &Equals; 6–13. Statistical significance was shown for LSL-iLet-7s (controls) versus LSL-iLet-7s; Myf5-Cre(+) mice. (D): Body weights. (E): Body lengths. (F, G): Postnatal growth curves of Lin28b fl/fl; Myf5-Cre males. n &Equals; 13–17. (F): Body weights. (G): Body lengths. *, p < .05; **, p < .01. Error bars represent SEM. Abbreviation: KO, knockout.

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Our previous paper reported that iLet-7 mice, in which a Lin28-resistant form of let-7g is induced with doxycycline, manifest growth retardation if let-7g is induced globally from 3 weeks of age onward [23]. To determine whether skeletal muscle-specific let-7 regulates organismal growth, we generated LSL-iLet-7 mice, in which Cre recombinase removes a Lox-Stop-Lox cassette, thereby enabling tissue-specific let-7g induction with doxycycline (see Materials and Methods). We crossed LSL-iLet-7 mice with skeletal muscle-specific Myf5-Cre mice, and documented sixfold more expression of the transgenic let-7g in neonatal skeletal muscle relative to controls (data not shown). Upon induction with doxycycline from conception onward we observed postnatal dwarfism in both males and females, demonstrating that overexpression of let-7 in skeletal muscle is sufficient to cause growth retardation (Fig. 6D, 6E and Supporting Information Fig. S7C, S7D).

Furthermore, to determine whether the differences in skeletal muscle let-7 between Lin28a and Lin28b KO mice were important for animal growth, we bred skeletal muscle-specific Myf5-Cre mice to Lin28afl/fl or Lin28bfl/fl mice. Whereas muscle-specific Lin28a KO mice (Lin28afl/fl; Myf5-Cre+) were comparable in size to control Lin28afl/fl mice (Supporting Information Fig. S7E), muscle-specific Lin28b KO (Lin28bfl/fl; Myf5-Cre+) males were smaller than control Lin28bfl/fl males, and phenocopied the dwarfism in constitutive Lin28b KO males (Fig. 6F, 6G, compared to Fig. 2A, 2B). Notably, both Lin28a KO and Lin28bfl/fl; Myf5-Cre+ muscles showed normal histology (Supporting Information Fig. S1B, S7F), comparable abundance of fast twitch (type II myosin) muscle fibers, and comparable mitochondrial DNA content and mitochondrial gene expression to controls (Supporting Information Fig. S8). In contrast, liver- or pancreatic β cell-specific Lin28b KO did not affect adult growth (Supporting Information Fig. S9). Our results indicate that absence of Lin28b in the fetal skeletal muscle is sufficient to dysregulate adult growth, whereas Lin28a must be lacking in additional tissues to manifest a change in adult growth.

Lin28b Acts Through Tsc1-mTOR Signaling in Skeletal Muscle

We have previously shown that Lin28a and LIN28B regulate glucose metabolism at least in part via let-7 and the insulin-PI3K-mTOR pathway [23]. Tsc1 is a suppressive regulator of the mTORC1 signaling [33]. Haploinsufficiency of Tsc1 activates mTORC1 signaling. Tsc1 KO mice die during embryonic development, whereas Tsc1+/− mice are viable with increased tumor-susceptibility in various organs [34]. We hypothesized that activation of mTORC1 signaling via Tsc1 haploinsufficiency might rescue the dwarfism of Lin28b KO mice, and thus we crossed Tsc1+/− mice to Lin28b+/− or Lin28b-/- mice and tracked their growth. The growth of Lin28b-/-; Tsc1+/− males was significantly improved compared to Lin28b KO mice, and comparable to that of Lin28b+/− mice (Fig. 7A, 7B). By contrast, Lin28b+/−; Tsc1+/− mice showed no growth advantage relative to Lin28b+/− mice, indicating that the effects of Lin28b deficiency on postnatal growth could be reversed by enhancing mTORC1 signaling. Interestingly, we likewise generated Lin28a-/-; Tsc1+/− mice but found that Tsc1 haploinsufficiency failed to rescue the dwarfism and perinatal lethality phenotypes seen with constitutional Lin28a deficiency.

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Figure 7. Tsc1/mTOR signaling in skeletal muscle can partially rescue aberrant programing of glucose metabolism and organismal growth in Lin28b knockout (KO) mice. (A, B): Postnatal growth curves of Lin28b KO males, with or without Tsc1 haploinsufficiency. n &Equals; 6–14. (A): Body weights. (B): Body lengths. Statistical significance was shown for Lin28b -/-; Tsc1 +/+ versus Lin28b -/-; Tsc1 +/− mice. (C, D): Postnatal growth curves of Lin28b fl/fl; Myf5-Cre males, with or without Tsc1 haploinsufficiency. n &Equals; 21–29. (C): Body weights. (D): Body lengths. Statistical significance was shown for Lin28b fl/fl; Tsc1 fl/+ versus Lin28b fl/fl; Tsc1 fl/−; Myf5-Cre(+) mice on the top, and for Lin28b fl/fl; Tsc1 +/+; Myf5-Cre(+) versus Lin28b fl/fl; Tsc1 fl/−; Myf5-Cre(+) mice on the bottom. (E): GTT of Lin28b fl/fl; Tsc1 fl/+; Myf5-Cre males. n &Equals; 8–11. (F): Summary of the timing and tissue of Lin28a/b expression and the effect on growth and glucose metabolism. Note that pink color denotes the positive effect on normal growth and glucose metabolism. The intensity of color and the number of + signs reflect the magnitude of the effect. “+/−” indicates that Lin28b has the effect on growth only if combined with Lin28a loss. *, p < .05; **, p < .01. Error bars represent SEM. Abbreviation: GTT, glucose tolerance test.

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Next, we tested the hypothesis that Lin28b/Tsc1 effects on growth are skeletal muscle-specific. We generated Lin28b fl/fl; Tsc1 fl/+; Myf5-Cre mice, in which Lin28b KO and Tsc1 haploinsufficiency are achieved only in skeletal muscles. Tsc1 haploinsufficiency in skeletal muscles partially rescued the postnatal dwarfism phenotype seen in skeletal muscle-specific Lin28b KO mice (Fig. 7C, 7D). Moreover, GTT demonstrated modest but significant improvement in Lin28b fl/fl; Tsc1 fl/+; Myf5-Cre mice compared to Lin28b fl/fl; Myf5-Cre mice (Fig. 7E). Taken together, our results suggest that Lin28b expression in fetal muscle programs adult metabolism and growth in part through the Tsc1-mTORC1 signaling pathway.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Heterochronic Metabolism and the Lin28a/b-mTOR Pathway

Both lin-28 and let-7 were originally identified in a C. elegans mutagenesis screen as heterochronic regulators of developmental timing. We and others have reported that overexpression of Lin28a and let-7 affect organismal size [23, 32] and onset of puberty in mice and humans [12–15]. In searching for the mechanism of growth regulation, we found that mammalian Lin28a/b also regulate glucose metabolism, in part through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway [23]. In this study, using genetic KO mouse models we have established that Lin28a and Lin28b are physiologically required for normal glucose homeostasis, albeit with distinct spatio-temporal patterns during mammalian development (Fig. 7F). Furthermore, we have found that fetal Lin28a/b exerts long-lasting effects on adult metabolism and growth, long after the normal expression patterns of these paralogs have extinguished in most tissues. Indeed, Lin28afl/−; UBC-CreER (+) or Lin28bfl/−; UBC-CreER (+) mice revealed that when deleted in adulthood, Lin28a or Lin28b are entirely dispensable with regard to the persistent growth observed with organismal aging in mice. Long-standing adult growth and metabolic effects were observed without concomitant gross defects in mammalian development, nor a disproportionate decrease in tissue mass, save for fat, in which Lin28a/b are not expressed. We found that deficiency of Lin28a/b in fetal muscle dysregulates adult metabolism; although we observe no defects in muscle development. Our finding that haploinsufficiency of Tsc1 can partially rescue the growth and glucose metabolism defects of Lin28b KO mice, further suggests that fetal Lin28b's effects on adult metabolism are mediated, at least in part, by mTORC1 signaling. Loss of embryonic Lin28a led to a general dysregulation of glycolytic flux in early embryos before changes in growth were detected, consistent with previous findings that mTORC1 signaling directly regulates glucose metabolism [33].

Model of Differential Effects of Lin28a and Lin28b on Postnatal Growth

Although our results demonstrate that adult growth is altered by fetal but not adult deficiency of Lin28a or Lin28b, we observed important differences in the phenotypes mediated by temporal and tissue-specific deficiency of these closely related paralogs. Conditional deletion of Lin28a at E15.5 failed to phenocopy constitutive loss of Lin28a, which was associated with perinatal lethality and dwarfism, whereas Lin28b deletion at a similar stage fully phenocopied the dwarfism due to constitutive loss of Lin28b. Furthermore, constitutive loss of Lin28a caused metabolic dysfunction and dwarfism in embryos, whereas constitutive loss of Lin28b did not influence metabolism until later in adults. These data suggest that Lin28a is relevant at an earlier embryonic stage of development than Lin28b, although both are relevant during midgestation as combined loss of both Lin28a and Lin28b led to synthetic lethality first noted around E10.5.

Fetal Lin28a/b and the Barker Hypothesis

The “Barker hypothesis,” originally proposed 2 decades ago, holds that an epigenetic memory of poor fetal and infant nutrition causes permanent changes in metabolism and leads to increased risks in adult life for chronic metabolic diseases such as type 2 diabetes (T2D) [35, 36]. Epidemiological observations support the relationship between fetal metabolism and adult insulin resistance, and it is now generally accepted that environmental factors in early life play a major role in the pathogenesis of T2D [35, 36]. Our conditional KO mice and metabolomics studies here establish that deletion of Lin28a/b during fetal development dictates long-term effects on adult metabolism, resulting in increased insulin resistance and a diabetic phenotype, opposite to the enhanced glucose metabolism which we recently reported as a consequence of hyperfunction of Lin28 in conditional transgenic murine models [23]. The Lin28/let-7 axis thus represents a means by which fetal metabolic regulators can influence life-long growth and metabolism phenotypes. Although we have identified fetal muscle cells as key cellular targets of Lin28 function, and the mTORC1 pathway in the molecular mechanism, how defects in fetal expression of the Lin28/let-7 axis translates into the long-term epigenetic changes that influence adult metabolism remains a mystery. Our several strains of Lin28a/b KO mice should provide powerful tools to study the fetal programming of adult metabolism, with relevance to the ongoing search for new medical interventions in chronic metabolic diseases.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Herein we describe the first comprehensive analysis of Lin28a and Lin28b KO mice. Both strains showed dwarfism starting at different ages. Fetal but not neonatal nor adult deficiency of Lin28 led to aberrant glucose metabolism and growth, indicating that fetal expression of Lin28 exerts life-long effects. Skeletal muscle-specific Lin28b KO mice phenocopied constitutive loss of Lin28b, which we demonstrated acts at least in part through Tsc1-mTOR signaling.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

We thank Mathew William Lensch for invaluable discussions and advice, David J. Kwiatkowski for Tsc1 mice, and Roderick Bronson and the Harvard Medical School Rodent Histopathology Core for mouse tissue pathology. We also thank Min Yuan and Susanne Breitkopf for help with mass spectrometry experiments and Grants NIH 5P01CA120964-04 and NIH DF/HCC Cancer Center Support Grant 5P30CA006516-46 (J.M.A.). The work was supported by grants from the Alex's Lemonade Stand Foundation and the Ellison Medical Foundation to GQD. GQD is an affiliate member of the Broad Institute and an investigator of the Howard Hughes Medical Institute and the Manton Center for Orphan Disease Research. H.Z. is currently affiliated with the Children's Research Institute, Departments of Pediatrics and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_1423_sm_SuppFigure1.pdf2064KSupplementary Fig. 1. Histology of Lin28a KO mice. (A) Some Lin28a KO mice have a cardiac defect. H&E images of Lin28a +/− and −/− P0.5 newborn hearts are shown. IVS interventricular septum, LA left atrium, LV left ventricle, RA right atrium, RV right ventricle, VSD ventricular septal defect. (B) Representative images of different organs/tissues in Lin28a +/− vs. −/− mice. Scale bars 200 μm (A), 100 μm (B). Related to Fig. 1.
STEM_1423_sm_SuppFigure2.pdf898KSupplementary Fig. 2. 7% of Lin28a −/− mice can survive to adulthood on a mixed background. (A) Representative images of Lin28a +/− and −/− mice at different ages. (B) Representative images of Lin28a +/− and −/− mice after DEXA scan. Related to Fig.1.
STEM_1423_sm_SuppFigure3.tiff769KSupplementary Fig. 3. Characterization of Lin28b KO and Lin28a +/−;b −/− mice. (A-B) Lin28b KO embryos and placentae show normal growth. N=9. (A) Weights of E18.5 embryos and placentae in Lin28b +/− vs. −/− mice. (B) Ratio of placenta/embryo weights in Lin28b +/− vs. −/− mice. (C) Organ weights normalized to total body weight in adult Lin28b +/− vs. −/− males. N=3. Epididymal fat pads were used to measure fat weights. (D) Weights of embryos and placentae of Lin28a +/+;b −/− vs. Lin28a +/−;b −/− embryos at E13.5. N=5-9. (E) Postnatal growth curve of Lin28a +/+ vs. +/− males. N=9. * p<0.05. Error bars represent SEM. Related to Fig. 2.
STEM_1423_sm_SuppFigure4.pdf1437KSupplementary Fig. 4. PCR genotyping confirms deletion of Lin28a (A-C) and Lin28b (D-F) alleles in various tissues of Lin28a fl/−; UBC-CreER (+) (A-C) and Lin28b fl/−; UBC-CreER (+) (D-F) mice. Tamoxifen was injected at E15.5 (A and D), P7-9 (B and E), or 6 weeks (C and F). Related to Fig. 4.
STEM_1423_sm_SuppFigure5.tiff770KSupplementary Fig. 5. Fetal Lin28a/b, not neonatal or adult Lin28a/b, regulates postnatal growth. (A-C) Postnatal growth curves of Lin28a fl/−; UBC-CreER (+) vs. Lin28a fl/− mice. Body lengths after tamoxifen (TAM) injections at (A) E15.5, (B) P7-9 and (C) 6 weeks. N=3-12. (D-F) Postnatal growth curves of Lin28b fl/−; UBC-CreER (+) vs. Lin28b fl/− mice. Body lengths after TAM injections at (D) E15.5, (E) P7-9 and (F) 6 weeks. N=5-13. Arrows indicate ages at TAM injection. * p<0.05, ** p<0.01. Error bars represent SEM. Related to Fig. 4.
STEM_1423_sm_SuppFigure6.pdf865KSupplementary Fig. 6. No expression of Lin28a/b in fat. Representative Lin28a (A) and Lin28b (B) immunostaining of wild-type fat tissues. Scale bars 100 μm. Related to Fig. 5.
STEM_1423_sm_SuppFigure7.pdf1304KSupplementary Fig. 7. Overexpression of let-7 in skeletal muscle is sufficient to cause growth retardation. (A) Let-7 levels in adult Lin28a +/− and KO mice. N=4. (B) Let-7 levels in adult Lin28b +/− and KO mice. N=3. (C-D) Postnatal growth curves of LSL-iLet-7s; Myf5-Cre(+) females given DOX from conception. N=4-13. (C) Body weights. (D) Body lengths. (E) Postnatal growth curve of Lin28a fl/fl; Myf5-Cre mice. Body weights. N=4-13. (F) Representative images of skeletal muscles in Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) mice. * p<0.05, ** p<0.01. Error bars represent SEM. Related to Fig. 6.
STEM_1423_sm_SuppFigure8.pdf1221KSupplementary Fig. 8. Characterization of Lin28a KO and Lin28b KO muscles. (A-B) Representative type II myosin immunostaining of gastrocnemius in Lin28a +/− vs. Lin28a KO (A) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (B) mice. (C-D) Mitochondrial DNA content analyzed by quantitative PCR in gastrocnemius (gast) and quadriceps femoris (quad) of Lin28a +/− vs. Lin28a KO (C) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (D) mice. Cox2 DNA was used for mtDNA and normalized to Rsp18 for relative amount calculation. N=3-4. (E-F) Nrf-1, Nrf-2, Pgc-1a, Pgc-1b, Ppara, Tfam, Tfb1m, and Tfb2m mRNA analyzed by quantitative RT-PCR in gastrocnemius of Lin28a +/− vs. Lin28a KO (E) and Lin28b fl/fl vs. Lin28b fl/fl; Myf5-Cre(+) (F) mice. N=3-4. Error bars represent SEM. Scale bars 100 μm. Related to Fig. 6.
STEM_1423_sm_SuppFigure9.tiff625KSupplementary Fig. 9. Hepatocyte- and pancreatic beta cell-specific Lin28b KO mice. (A-B) Postnatal growth curves of Albumin-Cre; Lin28b fl/fl males. N=7-9. (A) Body weights. (B) Body lengths. (C-D) Postnatal growth curves of Ins2-Cre; Lin28b fl/fl males. N=8-10. (C) Body weights. (D) Body lengths. Error bars represent SEM. Related to Fig. 6.
STEM_1423_sm_SuppFigure10.tiff1771KSupplementary Fig. 10. Targeted creation of a conditional Lin28b allele. (A) Upper: Genomic map of the Lin28b locus shows exons, restriction sites. Middle: Lin28b conditional targeting construct. Exon 2 is flanked by loxP sites. PGK-Neo cassette is flanked by a frt site. Lower: Targeted allele following a homologous recombination. Hi=HindIII, Xb=XbaI. (B) Southern blot showing ESC clones with the floxed allele. * denotes a non-specific band. (C) Western blot confirms no Lin28b proteins in Lin28b −/− ESC lysate. (D) PCR genotyping showing wild type (wt), floxed and deleted (null) alleles. Related to Experimental Procedures.
STEM_1423_sm_SuppTable1.tiff567KSupplementary Table. Numbers of Lin28a +/+;b −/− vs. Lin28a +/−;b −/− vs. Lin28a −/−;b −/− embryos collected at different ages. Numbers in parentheses show non-viable embryos, as defined by degenerative tissues or arrested heart. P-values were calculated by Fisher's exact probability test. Related to Fig. 3E.

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