The Impact of Novel BMPR1B Mutations on Litter Size in Short-Tailed Gobi Sheep and Larger-Tailed Ujimqin Sheep.

The significant deposition of tail fat in sheep has a profound impact on the economic benefits of animal husbandry. Furthermore, increasing the litter size is a crucial means of enhancing economic benefits. The BMPR1B and T/Brachyury genes are considered major functional genes that could affect sheep litter size and tail bone number, respectively. In this study, we employed direct sequencing to identify specific mutations of the BMPR1B gene in Gobi short tail sheep and carried out genotyping using MassARRAY technology for each variant of both the BMPR1B and T genes. Significant associations were demonstrated between the c.687G>A mutation of BMPR1B and the litter size in both the Gobi short tail sheep and Ujimqin sheep breeds. Meanwhile, the g.30058882_30058873GCAGATTAAAIndel mutation was significantly associated with the litter size in Gobi short tail sheep. These findings may provide valuable genetic markers for expanding sheep litter size. In addition, we also confirmed that the frequency of tail-bone-number-related T alleles was significantly higher in Gobi short tail sheep than in longer-tailed Ujimqin sheep.

Effects of New Mutations in BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 Genes on Litter Size in Sheep.

Prolificacy is a crucial characteristic of livestock, particularly for species such as sheep that have many births. The objectives of this study were as follows: (1) to investigate the genetic diversity of the 13 new and 7 known variants in the BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 genes in Ujimqin (UM), the F1 population of Dorper × Ujimqin crossbred (DPU), the F1 population of Suffolk × Ujimqin crossbred (SFKU), Sonid sheep (SN), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) sheep breeds/populations; (2) to perform an association analysis of the above 20 variants with litter size in 325 UM, 304 DPU, and 66 SFKU sheep populations; (3) to compare the frequencies of the litter-size-related alleles of these 20 variants among 8 sheep breeds/populations (the above seven sheep breeds + Mongolia sheep breed). With the use of the Sequenom MassARRAY®SNP assay technology, these 20 mutations were genotyped. The association analysis results showed that the c.746A>G (FecB) mutation in BMPR1B was significantly associated with the litter size of UM and DPU, the c.994A>G (FecGA) in GDF9 was significantly associated with the litter size of SFKU, and the c.31_33CTTinsdel (B1) in BMP15 was significantly associated with the litter size of UM. Our findings might provide valuable genetic markers for expanding sheep litter sizes.

Effects of the Expressions and Variants of the CAST Gene on the Fatty Acid Composition of the Longissimus Thoracis Muscle of Grazing Sonid Sheep.

Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene.

Effects of novel variants in BMP15 gene on litter size in Mongolia and Ujimqin sheep breeds.

Reproductive traits, such as ovulation rate and litter size, are important factors influencing the sheep industry. The bone morphogenetic protein 15 (BMP15) is a major gene affecting the reproductive traits in sheep, and multiple mutations in BMP15 gene could affect the ovulation rate and litter size in many sheep breeds, showing high breed specificity. However, identification of novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are still important. In this study, we sequenced the BMP15 gene of Mongolia sheep, and 12 novel variants were detected by direct sequencing and whole-genome resequencing. Among them, the g.50985975 G > A polymorphism in intron and synonymous c.755 T > C (Leu252Pro) in exon 2 of BMP15 were significantly associated with the litter sizes of Mongolia ewes (P < 0.01 and P < 0.01, respectively), as well as the g.50988478C > A and g.50987863G > A in the promoter region of BMP15 were significantly associated with the litter sizes of Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The c.755 T > C mutation is predicted to change the tertiary structure of BMP15. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

The comparative genomic analysis provides insights into the phylogeny and virulence of tick-borne encephalitis virus vaccine strain Senzhang.

Tick-borne encephalitis virus (TBEV) is one of the most dangerous tick-borne viral pathogens for humans. It can cause severe tick-borne encephalitis (TBE), multiple neurological complications, and death. The European subtype (TBEV-Eu), Siberian subtype (TBEV-Sib), and Far-Eastern subtype (TBEV-FE) are three main TBEV subtypes, causing varying clinical manifestations. Though TBEV-FE is the most virulent TBEV subtype, the degree of variation in the amino acid sequence of TBEV polyprotein is not high, leaving an issue without proper explanation. We performed phylogenic analysis on 243 TBEV strains and then took Senzhang strain as a query strain and representative strains of three major TBEV subtypes as reference strains to perform the comparative genomic analysis, including synteny analysis, SNP analysis, InDel analysis, and multiple sequence alignment of their envelope (E) proteins. The results demonstrated that insertions or deletions of large fragments occurred at the 3' end but not at the 5' end or in the CDS region of TBEV Senzhang strain. In addition, SNP sites are mainly located in the CDS region, with few SNP sites in the non-coding region. Our data highlighted the insertions or deletions of large fragments at the 3' end and SNP sites in the CDS region as genomic properties of the TBEV Senzhang strain compared to representative strains with the main subtypes. These features are probably related to the virulence of the TBEV Senzhang strain and could be considered in future vaccine development and drug target screening for TBEV.

Association analysis between novel variants in LEPR gene and litter size in Mongolia and ujimqin sheep breeds.

Prolificacy is an important trait of livestock, especially for species with multiple births, such as sheep. The leptin receptor (LEPR) is considered as a major candidate gene that affects the reproductive traits of sheep. The known R62H mutation in the LEPR gene is associated with the hyperprolific phenotype of Davisdale sheep. However, studies identifying novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are rare. In this study, we sequenced all coding regions of the LEPR gene of Mongolia sheep, and nine novel variants of the LEPR gene were detected by direct sequencing. Among them, the synonymous c.240C > T and c.279C > T mutations were significantly associated with the litter sizes of Mongolia ewes (P < 0.05 and P < 0.01, respectively) and Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The silent c.240C > T and c.279C > T mutations are predicted to change the stability of the mRNA secondary structure by reducing the minimum free energy and are predicted to change the mRNA secondary structure of LEPR. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

Serum Metabolomics of Tick-Borne Encephalitis Based on Orbitrap-Mass Spectrometry.

BACKGROUND: Tick-borne encephalitis virus (TBEV), the most prevalent arbovirus, causes potentially fatal encephalitis in humans. Prevalent in northeast China, tick-borne encephalitis (TBE) poses a major threat to public health, local economies and tourism. There are no biomarkers for TBE, which is classified serologically and clinically. Due to sample heterogeneity of samples and different detection platforms, obtaining stable markers is a great challenge for metabolomics. Accurate annotation is vital for data mining and interpretation. OBJECTIVE: To identify reliable biomarkers of TBEV infection. METHODS: An untargeted metabolomics analysis of serum from 30 TBE patients and 30 healthy controls was carried out. Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics methods were used to characterize the subjects' serum metabolic profiles and to screen and validate TBE biomarkers. RESULTS: A total of 3370 molecular features were extracted from each sample, and the peak intensity of each feature was obtained. Pattern analysis, principal component analysis, partial least squares discriminant analysis were used to screen for potential metabolites. Bilirubin, LysoPC (18:1[9Z]), palmitic acid, and CL (8:0/8:0/8:0/8:0) were significantly different. Pathway enrichment analysis showed that these metabolites were in the fatty acid biosynthesis and glycerophospholipid metabolism pathways. The phospholipid family had a significant difference in both the difference ratio and the abundance. CONCLUSION: Phospholipids may be used to distinguish TBEV patients from healthy controls. TBEV infection affects the normal metabolic activity of host cells, providing insight into the pathogenesis of TBE.

Association between novel variants in BMPR1B gene and litter size in Mongolia and Ujimqin sheep breeds.

Prolificacy is an important trait of animals, specifically for sheep. The Bone morphogenetic protein receptor 1B (BMPR1B) is a major gene affecting the litter size of many sheep breeds. The well-known FecB mutation (Q249R) was associated fully with the hyper prolific phenotype of Booroola Merino. However, the identification of variation in all exonic regions of BMPR1B was rare. In this study, we sequenced all exonic regions of BMPR1B gene of Mongolia sheep breed, and ten novel variants were detected by direct sequencing. Among them, the litter size of the Mongolia ewes with the CC genotype was significantly higher (0.34 additional lambs, p < .05) than those with the TT genotype of the g.29346567C>T single nucleotide polymorphism (SNP). The litter size of the Mongolia ewes with the TT genotype was significantly higher (0.19 additional lambs, p < .05 and .31 additional lambs, p < .01, respectively) than those with the GT and GG genotypes of the c.1470G>T SNP. The silent c.1470G>T mutation is predicted to increase the stability of the mRNA secondary structure through reducing minimum free energy and is predicted to change the mRNA secondary structure of BMPR1B. Our findings may give potentially useful genetic markers for increasing litter size in sheep.

Generation of VEGF knock-in Cashmere goat via the CRISPR/Cas9 system.

Cashmere is a rare and specialised animal fibre, which grows on the outer skin of goats. Owing its low yield and soft, light, and warm properties, it has a high economic value. Here, we attempted to improve existing cashmere goat breeds by simultaneously increasing their fibre length and cashmere yield. We attempted this by knocking in the vascular endothelial growth factor (VEGF) at the fibroblast growth factor 5(FGF5) site using a gene editing technology and then studying its hair growth-promoting mechanisms. We show that a combination of RS-1 and NU7441 significantly improve the efficiency of CRISPR/Cas9-mediated, homologous-directed repair without affecting the embryo cleavage rate or the percentages of embryos at different stages. In addition, we obtained a cashmere goat, which integrated the VEGF gene at the FGF5 site, and the cashmere yield and fibre length of this gene-edited goat were improved. Through next-generation sequencing, we found that the up-regulation of VEGF and the down-regulation of FGF5 affected the cell cycle, proliferation, and vascular tone through the PI3K-AKT signalling pathway and at extracellular matrix-receptor interactions. Owing to this, the gene-edited cashmere goat showed impressive cashmere performance. Overall, in this study, we generated a gene-edited cashmere goat by integrating VEGF at the FGF5 site and provided an animal model for follow-up research on hair growth mechanisms.

MiR-455-5p monitors myotube morphogenesis by targeting mylip.

As a posttranscriptional regulatory factor, microRNA (miRNA) plays an important role in the formation of myotubes. However, little is known about the mechanism of miRNA regulating myotube morphogenesis. Here, we aimed to characterize the function of miR-455-5p in myotube morphogenesis by inducing differentiation in C2C12 myoblasts containing murine Mylip fragments with the miR-455-5p target sequence. We found that miR-455-5p overexpression promoted the differentiation and hypertrophy of myotubes, while miR-455-5p inhibition led to the failure of myotube differentiation and formation of short myotubes. Furthermore, we demonstrated that miR-455-5p directly targeted the Mylip 3'-untranslated region, which plays a key role in monitoring myotube morphogenesis. Interestingly, the expression and function of Mylip were opposite to those of miR-455-5p during myogenesis. Our data uncovered novel miR-455-5p targets and established a functional link between Mylip and myotube morphogenesis. Understanding the involvement of Mylip in myotube morphogenesis provides insight into the function of the gene regulatory network.

Dlgap1 negatively regulates browning of white fat cells through effects on cell proliferation and apoptosis.

BACKGROUND: Obesity is a metabolic imbalance characterized by excessive deposition of white fat. The browning of white fat can effectively treat obesity and related diseases. Although Dlgap1 (Discs, Large (Drosophila) Homolog-Associated Protein 1) is suspected to have an effect on this process, no empirical evidence is available. METHODS: To understand the role of Dlgap1, we cultured white and brown fat cells, then performed overexpression and knockout experiments. RESULTS: We found that Dlgap1 overexpression in brown adipocytes inhibits brown-fat-related gene expression, promotes white-fat-related genes, while also increasing brown-adipocyte proliferation and apoptosis. However, the gene overexpression has no effect on brown adipocyte maturation. Knocking out Dlgap1 in white fat cells promotes the expression and inhibition of brown-fat-related and white-fat-related genes, respectively. Additionally, the knockout inhibits white fat cell proliferation and apoptosis, while also promoting their maturation. CONCLUSIONS: Dlgap1 negatively regulates the browning of white adipocytes by influencing cell proliferation and apoptosis.

Fibronectin type III domain-containing protein 5 promotes proliferation and differentiation of goat adipose-derived stem cells.

Fibronectin type III domain-containing protein 5 (FNDC5) plays an important role in white-to-brown adipose tissue conversion in mice. However, there is no report on the role of this protein in Arbas Cashmere goat adipose tissue. We investigated the effect of FNDC5 on the proliferation and differentiation of goat adipose-derived stem cells (gADSCs). We found that FNDC5 promotes the proliferation of gADSCs and increases the levels of p-ERK and p-p38, while it has no effect on the levels of ERK, p38, AKT and p-AKT. What's more, FNDC5 promotes the differentiation of gADSCs into lipid droplets. Overexpression of FNDC5 increased the protein levels of ASC1, UCP1, PPARγ and SREBP1. Additionally, mRNA levels of PPARγ, SREBP1, ACC, and FABP4 increased significantly. FNDC5 knockdown was associated with opposite effects. These results suggest that FNDC5 promotes the proliferation of gADSCs via MAPK signaling pathway and also induces the differentiation of these cells.

Generation of Tß4 knock-in Cashmere goat using CRISPR/Cas9.

The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tß4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tß4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tß4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tß4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.

Comparison of gene editing efficiencies of CRISPR/Cas9 and TALEN for generation of MSTN knock-out cashmere goats.

The genome editors CRISPR/Cas9 (clustered regularly interspaced short palindromicrepeats/Cas9 nuclease-null) and TALENs (transcription activator-like effector nuclease) are popularly used for targeted modification of the mammalian genome. To date, few comparative studies have been carried out to investigate the differences between the use of CRISPR/Cas9 and TALENs in genome editing for goat breeding. Here, we compared CRISPR/Cas9 and TALEN technologies at multiple levels for generating a knock out (KO) of the Alpas cashmere goat myostatin (MSTN) gene, which negatively regulates the proliferation and differentiation of skeletal muscle cells. The electrotransfection efficiency observed using CRISPR/Cas9 was 8.1% more than that observed using TALEN for generating MSTN KO cells. In addition, the cutting efficiency of CRISPR/Cas9 for editing exon 1 of the MSTN gene was higher than that of TALENs. However, the off-target effects of the CRISPR/Cas9 system were also higher than those of TALENs. Further, we found that the frequency of obtaining MSTN-/- mutations by CRISPR/Cas9 was 8.5 times higher than that by TALEN. The CRISPR/Cas9-edited colonies involved longer deletions (up to 117 bp) than the TALEN-edited colonies (up to 13 bp). Remarkably, when embryos used to generate cloned goat via somatic cell nuclear transfer were compared, we found that the TALEN MSTN KO embryos easily developed to 8 cells and their cleavage rate was significantly higher than that of CRISPR/Cas9-edited embryos. Finally, we produced a MSTN KO lamb using CRISPR/Cas9, which suggested that a high level of targeted gene modification could be achieved in goat using CRISPR/Cas9. Taken together, our study indicates that although TALEN enables a variety of genome modifications and may have some advantages over CRISPR/Cas9, the latter provides a significant advantage by permitting precise and efficient gene editing. Thus, CRISPR/Cas9 has more potential to become a robust gene-engineering tool for application in the breeding of farm animals.

The Overexpression of Tß4 in the Hair Follicle Tissue of Alpas Cashmere Goats Increases Cashmere Yield and Promotes Hair Follicle Development.

Increased cashmere yield and improved quality are some goals of cashmere goat breeding. Thymosin beta-4 (Tß4) plays a key role in the growth and development of hair follicles. For the past ten years, we have evaluated the role of Tß4 by establishing a flock of 15 cashmere goats that specifically overexpress the Tß4 gene in the hair follicles. These Tß4 overexpression (Tß4-OE) cashmere goats had more secondary hair follicles than the WT goats and produced more cashmere. Meanwhile, combined analysis of the skin transcriptome and proteome in cashmere goats suggested that Tß4 may affect hair growth by interacting with keratin type II cytoskeletal 4 epidermal (KRT4) to mediate the extracellular signal-regulated protein kinase (ERK) signaling pathway, thereby promoting the development of secondary hair follicles, and consequently, increasing cashmere yield. Thus, the specific overexpression of Tß4 in the hair follicles of cashmere goats effectively increased the cashmere yield.

CRISPR/Cas9-mediated specific integration of fat-1 at the goat MSTN locus.

Recent advances in understanding the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, no study has reported simultaneous knockout of endogenous genes and site-specific knockin of exogenous genes in large animal models. Using the CRISPR/Cas9 system, this study specifically inserted the fat-1 gene into the goat MSTN locus, thereby achieving simultaneous fat-1 insertion and MSTN mutation. We introduced the Cas9, MSTN knockout small guide RNA and fat-1 knockin vectors into goat fetal fibroblasts by electroporation, and obtained a total of 156 positive clonal cell lines. PCR and sequencing were performed for identification. Of the 156 clonal strains, 40 (25.6%) had simultaneous MSTN knockout and fat-1 insertion at the MSTN locus without drug selection, and 55 (35.25%) and 101 (67.3%) had MSTN mutations and fat-1 insertions, respectively. We generated a site-specific knockin Arbas cashmere goat model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer for the first time. For biosafety, we mainly focused on unmarked and non-resistant gene screening, and point-specific gene editing. The results showed that simultaneous editing of the two genes (simultaneous knockout and knockin) was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become an important and applicable gene engineering tool in safe animal breeding.

The role of Sox9 in maintaining the characteristics and pluripotency of Arbas Cashmere goat hair follicle stem cells.

In our previous work, we isolated Arbas Cashmere goat hair follicle stem cells (gHFSCs) and explored the pluripotency. In this study, we investigated the expression and putative role of Sox9 in the gHFSCs. Immunofluorescence staining showed that Sox9 is predominantly expressed in the bulge region of the Arbas Cashmere goat hair follicle, and also positively expressed in both nucleus and cytoplasm of the gHFSCs. When the cells were transfected using Sox9-shRNA, cell growth slowed down and the expression of related genes decreased significantly, cell cycle was abnormal, while the expression of terminal differentiation marker loricrin was markedly increased; cells lost the typical morphology of HFSCs; the mRNA and protein expression of gHFSCs markers and stem cell pluripotency associated factors were all significantly decreased; the expression of Wnt signaling pathway genes LEF1, TCF1,c-Myc were significantly changed. These results suggested that Sox9 plays important role in gHFSCs characteristics and pluripotency maintenance.

Construction of FGF21 knockout mouse models by the CRISPR/Cas9 system.

Fibroblast growth factors (FGFs) are multifunctional signal molecules between cells, regulating the various physiological functions of the organism. FGF21 is a regulatory factor of the FGF family and has been postulated to play important roles in hair follicle development and hair follicle growth cycle. To evaluate the roles of FGF21, we had established a FGF21 knockout mouse model, using the CRISPR/Cas9 technology. We had constructed a FGF21 targeting vector and microinjected it with Cas9 mRNA and gRNA into fertilized ova of FVB mice. The gRNA was designed to target the exon 1 of the endogenous mouse FGF21 gene. Three lines of Fgf21 -/- mice were obtained from these experiments, and confirmed to harbor Fgf21 -/- genotypes and null expression phenotype, using DNA sequencing, qRT-PCR and Western blotting. FGF21 mRNA and FGF21 protein were not detected in tissues of these Fgf21 -/- mice. Depilation and histochemistry analyses showed that the Fgf21 -/- mice had lower body weight, slower hair regrowth and poorer hair quantities and smaller hair follicles diameters, as compared to WT mice. The Fgf21 -/- mice reported here could provide a useful genetic model for future studies of FGF21 functions in hair follicle development and hair follicle growth cycle.

Role of thymosin beta 4 in hair growth.

Although thymosin beta 4 (Tß4) is known to play a role in hair growth, its mechanism of action is unclear. We examined the levels of key genes in a Tß4 epidermal-specific over-expressing mouse model and Tß4 global knockout mouse model to explore how Tß4 affects hair growth. By depilation and histological examination of the skin, we confirmed the effect of Tß4 on hair growth, the number of hair shafts and hair follicle (HF) structure. The mRNA and protein expression of several genes involved in hair growth were detected by real-time PCR and western blotting, respectively. Changes in the expression of ß-catenin and Lef-1, the two key molecules in the Wnt signaling pathway, were similar to the changes observed in Tß4 expression. We also found that compared to the control mice, the mRNA and protein expression of MMP-2 and VEGF were increased in the Tß4 over-expressing mice, while the level of E-cadherin (E-cad) remained the same. Further, in the Tß4 global knockout mice, the mRNA and protein levels of MMP-2 and VEGF decreased dramatically and the level of E-cad was stable. Based on the above results, we believe that Tß4 may regulate the levels of VEGF and MMP-2 via the Wnt/ß-catenin/Lef-1 signaling pathway to influence the growth of blood vessels around HFs and to activate cell migration. Tß4 may have potential for the treatment of hair growth problems in adults, and its effects should be further confirmed in future studies.

The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor-2C phosphorylation levels through integrin-linked kinase in goat skeletal muscle satellite cells.

Mammalian target of rapamycin (mTOR) signaling pathway plays a key role in muscle development and is involved in multiple intracellular signaling pathways. Myocyte enhancer factor-2 (MEF2) regulates muscle cell proliferation and differentiation. However, how the mTOR signaling pathway regulates MEF2 activity remains unclear. We isolated goat skeletal muscle satellite cells (gSSCs) as model cells to explore mTOR signaling pathway regulation of MEF2C. We inhibited mTOR activity in gSSCs with PP242 and found that MEF2C phosphorylation was decreased and that muscle creatine kinase (MCK) expression was suppressed. Subsequently, we detected integrin-linked kinase (ILK) using MEF2C coimmunoprecipitation; ILK and MEF2C were colocalized in the gSSCs. We found that inhibiting mTOR activity increased ILK phosphorylation levels and that inhibiting ILK activity with Cpd 22 and knocking down ILK with small interfering RNA increased MEF2C phosphorylation and MCK expression. In the presence of Cpd 22, mTOR activity inhibition did not affect MEF2C phosphorylation. Moreover, ILK dephosphorylated MEF2C in vitro. These results suggest that the mTOR signaling pathway regulates MEF2C positively and regulates ILK negatively and that ILK regulates MEF2C negatively. It appears that the mTOR signaling pathway regulates MEF2C through ILK, further regulating the expression of muscle-related genes in gSSCs.