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Delving into porcine embryonic myogenesis is the key to elucidate the complex regulation of breed-specific differences in growth performance and meat production. Increasing evidence proves that pigs with less meat production show earlier embryonic myogenesis, but little is known about the underlying mechanisms. In this study, we examine the longissimus dorsi muscle (LDM) by immunohistochemistry and confirm that the differentiation of myogenic progenitors is increased (

HMGB2, a DNA-binding protein, highly expresses during embryogenesis and plays an important role in development of some organs and tissues. However, it remains to be further investigated weather HMGB2 influences muscle development. In this work, we identified HMGB2 as an essential factor in myogenesis. Compared to wild type (WT) mice, body weights of systemic hmgb2 homozygous knockout (hmgb2-/- ) mice especially males were reduced. Diameter and cross-section area of tibialis anterior (TA) muscle fibers as well as expression of Myogenin and MyHC were all decreased in hmgb2-/- mice. CTX injury model revealed that HMGB2 was required for satellite cell proliferation and muscle regeneration. Moreover, HMGB2 interacted with S6K1 and regulated the kinase activity of S6K1 during cell proliferation. Knockdown and inactivation of S6K1 in C2C12 cells both resulted in impaired proliferation and differentiation. Furthermore, expression of cyclin D1 and Myf5 were both decreased when HMGB2 or S6K1 were knocked down and kinase activity of S6K1 was inhibited. These results indicate that HMGB2 is required for skeletal muscle development and regeneration, and HMGB2 maintains proliferation of myoblasts through regulating kinase activity of S6K1.

Myostatin (MSTN), a member of the transforming growth factor-β superfamily, is a negative regulator of muscle growth and development. Disruption of the MSTN gene in various mammalian species markedly promotes muscle growth. Previous studies have mainly focused on the disruption of the MSTN peptide coding region in pigs but not on the modification of the signal peptide region. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was used to successfully introduce two mutations (PVD20H and GP19del) in the MSTN signal peptide region of the indigenous Chinese pig breed, Liang Guang Small Spotted pig. Both mutations in signal peptide increased the muscle mass without inhibiting the production of mature MSTN peptide in the cells. Histological analysis revealed that the enhanced muscle mass in MSTN+/PVD20H pig was mainly due to an increase in the number of muscle fibers. The expression of MSTN in the longissimus dorsi muscle of MSTN+/PVD20H and MSTNKO/PVD20H pigs was significantly downregulated, whereas that of myogenic regulatory factors, including MyoD, Myogenin, and Myf-5, was significantly upregulated when compared to those in the longissimus dorsi muscle of wild-type pigs. Meanwhile, the mutations also activated the PI3K/Akt pathway. The results of this study indicated that precise editing of the MSTN signal peptide can enhance porcine muscle development without markedly affecting the expression of mature MSTN peptide, which could exert other beneficial biological functions in the edited pigs.

Liver development is a complex process that is regulated by a series of signaling pathways. Three-dimensional (3D) chromatin architecture plays an important role in transcriptional regulation; nonetheless, its dynamics and role in the rapid transition of core liver functions during development and obesity-induced metabolic stress remain largely unexplored. To investigate the dynamic chromatin architecture during liver development and under metabolic stress, we generated high-resolution maps of chromatin architecture for porcine livers across six major developmental stages (from embryonic day 38 to the adult stage) and under a high-fat diet-induced obesity. The characteristically loose chromatin architecture supports a highly plastic genome organization during early liver development, which fundamentally contributes to the rapid functional transitions in the liver after birth. We reveal the multi-scale reorganization of chromatin architecture and its influence on transcriptional regulation of critical signaling processes during liver development, and show its close association with transition in hepatic functions (i.e., from hematopoiesis in the fetus to metabolism and immunity after birth). The limited changes in chromatin structure help explain the observed metabolic adaptation to excessive energy intake in pigs. These results provide a global overview of chromatin architecture dynamics associated with the transition of physiological liver functions between prenatal development and postnatal maturation, and a foundational resource that allows for future in-depth functional characterization.

Fat-related traits have great influences on pork quality. As different fat tissues have different biochemical profiles depending on their location, intramuscular fat contributes to gustatory qualities, while subcutaneous fat is considered as a negative factor associated with growth performance. In this study, both primary intramuscular and subcutaneous vascular stem cells (IVSCs and SVSCs) could be differentiated into mature adipocytes, though the IVSC differentiation efficiency was lower. By comparative analysis of transcriptomes, 2524 differentially expressed genes (DEGs) were found between two VSCs before differentiation, while only 551 DEGs were found and enriched in two pathways including biosynthesis of unsaturated fatty acids after differentiation. This result indicated that differentiated VSCs were more similar. During differentiation, more DEGs existed in IVSCs than that in SVSCs, suggesting that adipogenesis of IVSCs might be more complex. Additionally, the expression level of DEGs involved in the adipogenic process helps to explain the difference of differentiation efficiency between IVSCs and SVSCs.

Protein arginine methyltransferase 1 (PRMT1) methylates a variety of histone and nonhistone protein substrates to regulate multiple cellular functions such as transcription, DNA damage response, and signal transduction. It has been reported as an emerging regulator of various metabolic pathways including glucose metabolism in the liver, atrophy in the skeletal muscle, and lipid catabolism in the adipose tissue. However, the underlying mechanisms governing how PRMT1 regulates adipogenesis remain elusive. Here, we delineate the roles of PRMT1 in mitotic clonal expansion and adipocyte differentiation. Gain and loss of functions demonstrate that PRMT1 is essential for adipogenesis of 3T3-L1 and C3H10T1/2 cells. Mechanistically, we show PRMT1 promotes the expression of transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) by catalyzing histone modification H4R3me2a and impedes the activation of Wnt/β-catenin signaling by increasing the level of Axin to accelerate adipogenic differentiation. In addition, we demonstrate mitotic clonal expansion is suppressed by PRMT1 deficiency. PRMT1 interacts with transcription factor CCATT enhancer-binding protein β (C/EBPβ), and the absence of PRMT1 leads to the depressed phosphorylation of C/EBPβ. Interestingly, we discover PRMT1 acts as a positive regulator of C/EBPβ protein stability through decreasing the level of E3 ubiquitin ligase Smurf2, which promotes the ubiquitination and degradation of C/EBPβ, thus facilitating adipogenesis. Collectively, these discoveries highlight a critical role of PRMT1 in adipogenesis and provide potential therapeutic targets for the treatment of obesity.

Myofibres (primary and secondary myofibre) are the basic structure of muscle and the determinant of muscle mass. To explore the skeletal muscle developmental processes from primary myofibres to secondary myofibres in pigs, we conducted an integrative three-dimensional structure of genome and transcriptomic characterization of longissimus dorsi muscle of pig from primary myofibre formation stage [embryonic Day 35 (E35)] to secondary myofibre formation stage (E80). In the hierarchical genomic structure, we found that 11.43% of genome switched compartment A/B status, 14.53% of topologically associating domains are changed intradomain interactions (D-scores) and 2,730 genes with differential promoter–enhancer interactions and (or) enhancer activity from E35 to E80. The alterations of genome architecture were found to correlate with expression of genes that play significant roles in neuromuscular junction, embryonic morphogenesis, skeletal muscle development or metabolism, typically, NEFL, MuSK, SLN, Mef2D and GCK. Significantly, Sox6 and MATN2 play important roles in the process of primary to secondary myofibres formation and increase the regulatory potential score and genes expression in it. In brief, we reveal the genomic reorganization from E35 to E80 and construct genome-wide high-resolution interaction maps that provide a resource for studying long-range control of gene expression from E35 to E80.

Additional file of Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling

Additional file 1: Seven supplementary figures and one supplementary table. Additional file 1 contains seven supplementary figures and supplementary table 1 in PDF format. Figure S1. Saturation of DGE libraries. Figure S2. The positions of tags in the gene. Figure S3. Effect of library size on the number of genes identified. Figure S4. STC (Series Test of Cluster) analysis of DE genes. Figure S5. Biological process GO terms of profiles 6 and 1. Figure S6. Biological process GO terms of profiles 7 and 0. Figure S7. Differential expression of heat shock genes. Table S1. Tissue distribution of H-PRRSV in infected pigs using QPCR assays. (PDF 987 KB)

Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

BackgroundDelving into porcine embryonic myogenesis is the key to elucidating the complex regulation of breed-specific differences in growth performance and meat production. Increasing evidence proved that pigs with less meat production showed more intense embryonic myogenesis, but little is known about the underlying mechanisms. ResultsIn this study, we confirmed that the differentiation process of myogenic progenitors in Lantang pig (LT, fat) was faster than that in Landrace pig (LR, lean), which resulted in more differentiated myoblasts (Pax7-/MyoD+) but less myogenic progenitors (Pax7+/MyoD-) in LT at 35 days post-conception (35dpc). Additionally, in vitro, embryonic myogenic progenitors isolated from LT showed stronger differentiation capacity with earlier expression of MyoD. Furthermore, the expression levels of genes related to Notch signaling in LR progenitor cells were significantly higher than that of LT, while there was no significant difference between the two breeds in gene expression levels of Wnt and Akt/mTOR pathway. Inhibition of Notch signaling or knockdown of Pax7 promotes myogenic differentiation of primary progenitor cells or myoblasts, while activation of Notch signaling or overexpression of Pax7 has the opposite effects.ConclusionsMyogenic differentiation is more rapid in LT than that in LR at 35dpc. Mechanically, Notch signaling facilitates maintenance of myogenic progenitor cells and antagonizes myogenic differentiation by promoting Pax7 expression but preventing MyoD expression in LR.

Enhancer of zeste homolog 2 (EZH2) has been extensively investigated to participate in diverse biological processes, including carcinogenesis, the cell cycle, X-chromosome inactivation, and early embryonic development. However, the functions of this protein during mammalian oocyte meiotic maturation remain largely unexplored. Here, combined with RNA-Seq, we provided evidence that EZH2 is essential for oocyte meiotic maturation in pigs. First, EZH2 protein expression increased with oocyte progression from GV to MII stage. Second, the siRNA-mediated depletion of EZH2 led to accelerated GVBD and early occurrence of the first polar body extrusion. Third, EZH2 knockdown resulted in defective spindle assembly, abnormal SAC activity, and unstable K-MT attachment, which was concomitant with the increased rate of aneuploidy. Finally, EZH2 silencing exacerbated oxidative stress by increasing ROS levels and disrupting the distribution of active mitochondria in porcine oocytes. Furthermore, parthenogenetic embryonic development was impaired following the depletion of EZH2 at GV stage. Taken together, we concluded that EZH2 is necessary for porcine oocyte meiotic progression through regulating spindle organization, maintaining chromosomal integrity, and mitochondrial function.

The dominant white phenotype in pigs is thought to be mainly due to a structural mutation in the KIT gene, a splice mutation (G > A) at the first base in intron 17 which leads to the deletion of exon 17 in the mature KIT mRNA. However, this hypothesis has not yet been validated by functional studies. Here, we created two mouse models, KIT D17/+ to mimic the splice mutation, and KIT Dup/+ to partially mimic the duplication mutation of KIT gene in dominant white pigs using CRISPR/Cas9 technology. We found that the splice mutation homozygote is lethal and the heterozygous mice have a piebald coat. Slightly increased expression of KIT in KIT Dup/+mice did not confer the patched phenotype and had no obvious impact on coat color. Interestingly, the combination of these two mutations reduced the phosphorylation of PI3K and MAPK pathway associated proteins, which may be related to the impaired migration of melanoblasts observed during embryonic development that eventually leads to the dominant white phenotype.

The FecBB mutation, which increases the ovulation quota, was identified in a hyperprolific sheep breed, and is associated with a single point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor-type 1B (BMPR1B). However, the mechanism of action of the FecBB mutation remains unclear. Here, we describe the application of clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) to introduce the FecBB mutation into the human granulosa cell line COV434 via homology-directed repair (HDR), using single-stranded oligodeoxynucleotides (ssODNs) as template. Upon screening single cell-derived clones, we found one clone containing the FecBB mutation on one allele and knockout of BMPR1B on the other allele, and another clone harbouring an in-frame deletion on one allele and knockout of BMPR1B on the other. These two clones were subjected to functional analysis. We found that the FecBB mutation and knockout of BMPR1B could upregulate the expression of cumulus expansion-related genes, potentially mediated by enhanced activation of the Sma- and Mad-related 1/5 (SMAD1/5) pathway. The FecBB mutation, an in-frame mutation, and knockout of BMPR1B could impair the synthesis of oestradiol in COV434 cells, possibly through inhibition of the SMAD2/3 pathway. Our study provides a useful cell model for further investigation into the mechanism of action of the FecBB mutation.

MicroRNAs are a class of highly conserved ∼20 nucleotides non-coding RNAs that post-transcriptionally regulate gene expression. Many miRNAs were studied in the development of skeletal muscle, such as miR-1, miR-206, and miR-133. In our previous study, miR-127-3p was found highly expressed in porcine fetal skeletal muscle, whereas the detailed functions of miR-127-3p in muscle development is still unclear. In this study, we detected that miR-127-3p also highly expressed in skeletal muscle, cardiac muscle of adult mice and proliferative C2C12 cell lines. Overexpression of miR-127-3p almost has no effects on differentiation of C2C12 cell lines. However, miR-127-3p significantly inhibited the cell proliferation of C2C12 cells. Moreover, we identified KMT5a as a target gene that was down-regulated in both mRNA and protein level when miR-127-3p mimics were introduced. Furthermore, KMT5a overexpression in miR-127-3p treated cells rescued the influence of miR-127-3p on C2C12 proliferation. In brief, our data reveals that miR-127-3p regulates the proliferation of myocytes through KMT5a.

The adipocyte differentiation process, controlled by a tightly regulated transcriptional cascade, contributes partly to determine intramuscular adipose tissue (IMAT) mass, which is associated with meat quality in food animals, as well as obesity and related metabolic complications in human. Thus, this study aimed to characterize genes critical for intramuscular preadipocyte differentiation. Primary intramuscular preadipocytes were isolated from pigs, and mRNA profiles were performed at several key points (0 h, 4 h, 8 h, 1 day, 2 days, and 6 days) during adipogenesis using microarrays. By gene functional analysis, we identified numerous differentially expressed genes among distinct stages of intramuscular preadipocyte differentiation, which included numbers of transcription factors in the early stages. We obtained 4 clusters of differential gene expression pattern, including crucial candidate genes associated with adipogenesis of intramuscular adipocytes. Further, we demonstrated that POSTN and FGFR4 suppressed, whereas AKR1CL1 promoted, the expression of adipogenic marker PPARγ and C/EBPα. Taken together, our data delineated the transcriptome landscape during porcine intramuscular preadipocyte differentiation, which provided a valuable resource for finding the genes responsible for IMAT formation.

Bone morphogenetic protein 15 (BMP15) is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of BMP15 significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor (Fshr) expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide in vivo evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.

Objectives Mixed lineage leukaemia protein‐1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis. Materials and methods The expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT‐PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 (Myf5) promoter. A cardiotoxin (CTX)‐mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo. Results MLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7‐positive satellite cell proliferation and muscle repair. Conclusion MLL1 facilitates proliferation of myoblasts and Pax7‐positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.

The dominant white phenotype in pigs is thought to be mainly due to a structural mutation in the KIT gene, a splice mutation (G > A) at the first base in intron 17 which leads to the deletion of exon 17 in the mature KIT mRNA. However, this hypothesis has not yet been validated by functional studies. Here, we created two mouse models, KIT D17/+ to mimic the splice mutation, and KIT Dup/+ to partially mimic the duplication mutation of KIT gene in dominant white pigs using CRISPR/Cas9 technology. We found that the splice mutation homozygote is lethal and the heterozygous mice have a piebald coat. Slightly increased expression of KIT in KIT Dup/+mice did not confer the patched phenotype and had no obvious impact on coat color. Interestingly, the combination of these two mutations reduced the phosphorylation of PI3K and MAPK pathway associated proteins, which may be related to the impaired migration of melanoblasts observed during embryonic development that eventually leads to the dominant white phenotype.

Background Myogenic Differentiation 1 (MyoD) is a crucial master switch in regulating muscle-specific gene transcription. Forced expression of myoD is equipped to induce several cell lineages into myoblast, which then differentiate and fuse into myotube. Pig is one of the most significant livestock supplying meat, and has been classified into lean, fat and miniature pig breeds. However, the mechanisms underlying muscle mass variation among different pig breeds have remained unclear. Considering the important effect of MyoD on muscle development, it remains to be investigated whether the difference in muscle mass is caused by its single nucleotide polymorphisms (SNPs) which are the major differences among pig breeds at DNA level. Results In this study, we identified the locations of porcine myoD regulatory regions including proximal regulatory region (PRR), distal regulatory region (DRR), and core enhancer (CE) region. There are 8 SNPs in the regulatory regions and 6 SNPs in gene body region, which were identified from lean, fat and miniature pig populations. However, these SNPs have no effects on its temporal expression and transcriptional activity which might lead to the distinction in postnatal muscle mass. In addition, overexpression of myoD clones across from amphibious to mammals including xenopus tropicalis, chicken, mouse and pig whose gene identities vary from 68 to 84%, could promote myogenesis in NIH3T3 fibroblasts cells. Conclusions These results proved that myoD nucleotide variations from different pig populations have no effect on muscle mass, suggesting that the function of myoD is highly conserved not only among different pig breeds, but also across different species. Thus, it would be futile to discover SNPs affecting muscle mass in pig populations with normal muscle development. Electronic supplementary material The online version of this article (10.1186/s12863-019-0772-6) contains supplementary material, which is available to authorized users.

Bone morphogenetic protein 15 (BMP15) is a multifunctional oocyte-specific secreted factor. It controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role on follicular development and ovulation rate of mono-ovulatory mammalian species, but has minimal impact on poly-ovulatory mice. However, whether this is true in non-rodent poly-ovulatory species need to be validated. To investigate this question, we generated a BMP15 knockdown pig model. We found that BMP15 knockdown gilts showed markedly reduced fertility accompanied with phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation. Molecular and transcriptome analysis revealed that knockdown of BMP15 significantly suppressed cell proliferation, differentiation, Fshr expression, leading to premature luteinization and reduced estradiol production in GCs, and simultaneously decreased the quality and meiotic maturation of oocyte. Our results provide in vivo evidences for the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.

Dominant white phenotype in pigs is considered to be caused by two structural mutations inKITgene, including a 450-kb duplication encompassing the entireKITgene, and a splice mutation (G > A) at the first base in intron 17, which leads to the deletion of exon 17 in matureKITmRNA, and the production of KIT protein lacking a critical catalytic domain of kinase. However, this speculation has not yet been validated by functional studies. Here, by using CRISPR/Cas9 technology, we created two mouse models mimicing the structural mutations ofKITgene in dominant white pigs, including the splice mutation mouse modelKITD17/+with exon 17 of one allele ofKITgene deleted, and duplication mutation mouse modelKITDup/+with one allele ofKITgene coding sequence (CDS) duplicated. We found that each mutation individually can not cause dominant white phenotype. Splice mutation homozygote is lethal and heterozygous mice present piebald coat. Inconsistent with previous speculation, we foundKITgene duplication mutation did not confer the patched phenotype, and had no obvious impact on coat color. Interestingly, combination of these two mutations lead to dominant white phenotype. Further molecular analysis revealed that combination of these two structural mutations could inhibit the kinase activity of the KIT protein, thus reduce the phosphorylation level of PI3K and MAPK pathway associated proteins, which may be related to the observed impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.Author summaryKIT plays a critical role in control of coat colour in mammals. Two mutation coexistence inKITare considered to be the cause of theDominant whitephenotype in pigs. One mutation is a 450-kb large duplication encompassing the entireKITgene, another mutation is a splice mutation causing the skipping of KIT exon 17. The mechanism of these two mutations of KIT on coat color formation has not yet been validated. In this study, by using genome edited mouse models, we found each structural mutation individual does not lead dominant white phenotype, but combination of these two mutations could lead to a nearly complete white coat colour similar to pig dominant white phenotype, possibly due to the inhibition of the kinase activity of the KIT protein, thus its signalling function on PI3K and MAPK pathways, leading to impaired migration of melanoblasts during embryonic development, and eventually lead to dominant white phenotype. Our study provides a further insight into the underlying genetic mechanisms of porcine dominant white coat colour.

Here, we performed whole-genome bisulfite sequencing of longissimus dorsi muscle from Landrace and Wuzhishan (WZS) miniature pigs during 18, 21, and 28 d postcoitum. It was uncovered that in regulatory regions only around transcription start sites (TSSs), gene expression and methylation showed negative correlation, whereas in gene bodies, positive correlation occurred. Furthermore, earlier myogenic gene demethylation around TSSs and earlier hypermethylation of myogenic genes in gene bodies were considered to trigger their earlier expression in miniature pigs. Furthermore, by analyzing the methylation pattern of the myogenic differentiation 1(MyoD) promoter and distal enhancer, we found that earlier demethylation of the MyoD distal enhancer in WZSs contributes to its earlier expression. Moreover, DNA demethylase Tet1 was found to be involved in the demethylation of the myogenin promoter and promoted immortalized mouse myoblast cell line (C2C12) and porcine embryonic myogenic cell differentiation. This study reveals that earlier demethylation of myogenic genes contributes to precocious terminal differentiation of myoblasts in miniature pigs.-Zhang, X., Nie, Y., Cai, S., Ding, S., Fu, B., Wei, H., Chen, L., Liu, X., Liu, M., Yuan, R., Qiu, B., He, Z., Cong, P., Chen, Y., Mo, D. Earlier demethylation of myogenic genes contributes to embryonic precocious terminal differentiation of myoblasts in miniature pigs.

Bone morphogenetic protein 15 (BMP15), a member of the transforming growth factor beta superfamily, plays an essential role in ovarian follicular development in mono-ovulatory mammalian species. Studies using a biallelic knockout mouse model revealed that BMP15 potentially has just a minimal impact on female fertility and ovarian follicular development in polyovulatory species. In contrast, our previous study demonstrated that in vivo knockdown of BMP15 significantly affected porcine female fertility, as evidenced by the dysplastic ovaries containing significantly decreased numbers of follicles and an increased number of abnormal follicles. This finding implied that BMP15 plays an important role in the regulation of female fertility and ovarian follicular development in polyovulatory species. To further investigate the regulatory role of BMP15 in porcine ovarian and follicular development, here, we describe the efficient generation of BMP15-edited Yorkshire pigs using CRISPR/Cas9. Using artificial insemination experiments, we found that the biallelically edited gilts were all infertile, regardless of different genotypes. One monoallelically edited gilt #4 (Δ66 bp/WT) was fertile and could deliver offspring with a litter size comparable to that of wild-type gilts. Further analysis established that the infertility of biallelically edited gilts was caused by the arrest of follicular development at preantral stages, with formation of numerous structurally abnormal follicles, resulting in streaky ovaries and the absence of obvious estrous cycles. Our results strongly suggest that the role of BMP15 in nonrodent polyovulatory species may be as important as that in mono-ovulatory species.

Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.

Table S4. Sequence size and location of myoD regulatory regions. (DOCX 13 kb)