SUMMARY Regeneration capacity declines with age, but why juvenile organisms show enhanced tissue repair remains unexplained. Lin28a, a highly-conserved RNA binding protein expressed during embryogenesis, plays roles in development, pluripotency and metabolism. To determine if Lin28a might influence tissue repair in adults, we engineered the reactivation of Lin28a expression in several models of tissue injury. Lin28a reactivation improved hair regrowth by promoting anagen in hair follicles, and accelerated regrowth of cartilage, bone and mesenchyme after ear and digit injuries. Lin28a inhibits let-7 microRNA biogenesis; however let-7 repression was necessary but insufficient to enhance repair. Lin28a bound to and enhanced the translation of mRNAs for several metabolic enzymes, thereby increasing glycolysis and oxidative phosphorylation (OxPhos). Lin28a-mediated enhancement of tissue repair was negated by OxPhos inhibition, whereas a pharmacologically-induced increase in OxPhos enhanced repair. Thus, Lin28a enhances tissue repair in some adult tissues by reprogramming cellular bioenergetics.

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Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumor suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. However, here we show that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN mRNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN-amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma pathogenesis with broad implications for cancer pathogenesis.

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 can 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.

Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single nucleotide variants in the MKL2, MYH7 and NKX2–5 genes. The MYH7 and MKL2 variants were inherited from the affected-asymptomatic father and the rare NKX2–5 variant (minor allele frequency=0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell–derived cardiomyocytes provided histologic and molecular evidence for the NKX2–5 variant’s contribution as a genetic modifier.

BackgroundThe Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity.ResultsWAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil.ConclusionsWhether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.

Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.