Regulation of alternative pre-mRNA splicing, recognized as increasingly important in causing human disease, was studied using the CD44 gene, whose splice variants have been implicated in tumor progression. We identified heterogeneous ribonucleoprotein (hnRNP) A1 as a protein interacting in vitro and in vivo with regulatory splice elements in CD44 variant exon v5. Transient overexpression of hnRNP A1 prevented v5 exon inclusion, dependent on the exonic elements. HnRNP A1-dependent repression was exon-specific and could be relieved by coexpression of oncogenic forms of Ras and Cdc42. The results define hnRNP A1 as a decisive part of an oncogene-regulated splice-silencing complex, which can select between multiple alternatively spliced exons.

Preadipocyte factor 1 (Pref-1/Dlk1) inhibits in vitro adipocyte differentiation and has been recently reported to be a paternally expressed imprinted gene at human chromosome 14q32. Studies on human chromosome 14 deletions and maternal uniparental disomy (mUPD) 14 suggest that misexpression of a yet-to-be-identified imprinted gene or genes present on chromosome 14 causes congenital disorders. We generated Pref-1 knockout mice to assess the role of Pref-1 in growth and in vivo adipogenesis and to determine the contribution of Pref-1 in mUPD. Pref-1-null mice display growth retardation, obesity, blepharophimosis, skeletal malformation, and increased serum lipid metabolites. Furthermore, the phenotypes observed in Pref-1-null mice are present in heterozygotes that harbor a paternally inherited, but not in those with a maternally inherited pref-1-null allele. Our results demonstrate that Pref-1 is indeed paternally expressed and is important for normal development and for homeostasis of adipose tissue mass. We also suggest that Pref-1 is responsible for most of the symptoms observed in mouse mUPD12 and human mUPD14. Pref-1-null mice may be a model for obesity and other pathologies of human mUPD14.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5′-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs. Local native livestock breeds, such as pigs and cattle, are important reservoirs of genetic variation, because they have adapted to local conditions and have not been as strongly selected as commercial livestock breeds. Therefore, the study of local native breeds is of utmost interest to identify causative mutations for traits in which large differences exist between local native breeds and breeds used internationally for meat production. Herein, we apply these principles to meat quality related traits in pigs. A combined linkage-linkage disequilibrium analysis identified the MYH3 gene on pig chromosome 12, which encodes myosin heavy chain 3 protein. Subsequently, we discovered a regulatory mutation in MYH3, which occurs at high frequency in Korean Native Pigs compared to the frequency at which it occurs in Western pig breeds. We confirmed the effect of MYH3 on muscle fiber-type composition and intramuscular fat content using transgenic mice and transfection assays in porcine fibroblast cells. This is the first causal mutation identified in livestock for these traits and the result is immediately applicable for the genetic improvement of pork quality. These results also enhance our understanding of the genetic basis regarding the regulation of myofiber type ratios and associated changes in intramuscular fat contents in pigs and mammals in general.

Nutrient sensing in the hypothalamus is tightly related to food intake regulation. However, the mechanisms by which the nutrient-sensing cells of the brain translate this signal of energy need into feeding behavior via regulation of neuropeptide expression are not known. To address this issue, we investigated two neuronal cell lines expressing agouti-related protein (AgRP), ex vivo hypothalamic tissues, and in vivo whole animals. Maintaining cells in a low cellular ATP concentration generated by low glucose, 2-deoxyglucose (2-DG), ATP synthesis inhibitor, and 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside increased phosphorylation of AMP-activated protein kinase (AMPK) and increased AgRP expression, whereas maintaining cells in high ATP status by high glucose and pyruvate supplementation in 2-DG-treated cells decreased phosphorylation of AMPK and decreased AgRP expression. Overexpression of a dominant-inhibitory mutant of AMPK significantly decreased low-glucose- or 2-DG-induced AgRP expression. Furthermore, ex vivo hypothalamus culture in high glucose concentrations decreased both expression and phosphorylation of AMPK and expression of both AgRP and neuropeptide Y, whereas pyruvate supplementation suppressed a 2-DG-induced AgRP expression. Finally, our in vivo studies clearly show that central administration of pyruvate dramatically delayed 2-DG-induced food intake. These data indicate that modulation of ATP levels in neuronal cells triggers a cascade of events via AMPK that modulate feeding behavior to restore energy status of cells.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry. Significance Current methods for CRISPR/Cas9-mediated gene knockout in avian species require isolation, culture, and genome modification of primordial germ cells in vitro for the subsequent injection into the extraembryonic blood vessel of the egg. These processes require highly skilled techniques and the establishment of optimized conditions. In the present study, the adenoviral CRISPR/Cas9 vector, optimized for expression of gRNA and the Cas9 gene in avian cells, was injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin gene, resulting in gray plumage of homozygous mutant quail. Implementation of this simplified avian knockout process can accelerate avian knockout studies involved in diverse areas of research and lead to the advancement of future agricultural applications.

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1^−/− mice, but not males, were resistant to high-fat (HF) diet–induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1^−/− mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet–induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.