Embryonic Skeletal Development Research Articles

Abstract LB403: Suppression of antigen presentation in pediatric rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that is diagnosed based on the tumor cell's resemblance to embryonic or poorly differentiated skeletal muscle progenitors. Multiple immunotherapy clinical trials have found no evidence of immune reactivity against RMS, which has been attributed to the relatively low tumor mutation burden in these cancers. However, we made an unexpected observation that many patient RMS tumors had a near absence of MHC-I expression, a key requisite for anti-tumor CD8+ T lymphocytes and many immunotherapies. In this study, we explore the etiology of low antigen presentation in RMS and systematically evaluate therapeutic interventions that sensitize RMS to immunotherapy. Methods: Surface MHC-I expression was evaluated on pediatric tumor cell lines (n=65) from five major pediatric tumor types. RNA-seq, whole exome and genome sequencing data were analyzed from RMS tumors (total of n=247) and iPSC models of skeletal muscle development (PMID: 32011235). Epigentic drugs targeting DNA methylation (decitabine), EZH2 (tazemetostat), Class I/IV HDACs (mocetinostat), and LSD1 (GSK-LSD1) were used at sub-cytotoxic doses on RMS cell lines (n=10) and analyzed for surface MHC-I expression with flow cytometry and gene expression. T cell cytotoxicity assays were performed using engineered human T cells expressing an optimized TCR which recognizes an HLA*A2:01 restricted peptide from PRAME. Results: RMS cell line models exhibited a range of MHC-I expression with most having low or undetectable MHC-I expression, similar to that of a known MHC-I negative tumor type MYCN-amplified neuroblastoma. Exome sequencing of RMS tumors showed no evidence of loss-of-function alterations in essential antigen processing and presentation (APP) pathway genes. Gene expression profiling by RNAseq and western blotting, however, revealed an absence of core APP genes in many RMS tumors and cell lines. We uncovered that during a specific window of embryonic myogenesis, APP is suppressed and that RMS tumors mimicking this state of development show the deepest loss of APP. Multiple classes of epigenetic drugs were evaluated to rescue APP in RMS. Treatment of RMS using a DNA demethylating agent or HDAC inhibitor exhibited the strongest MHC-I restoration across RMS models with no changes observed using LSD1 or EZH2 inhibitors. As a proof-of-concept, we demonstrate that targeting the RMS antigen PRAME using TCR therapy is enhanced by the inclusion of epigenetic therapy. Conclusions and Significance: We revealed that epigenetic suppression of APP is a new hallmark of RMS and normal embryonic skeletal muscle development. Pharmacological reversal of APP pathway downregulation could be achieved using drugs targeting DNA methylation or class I/IV HDACs, and enhanced T cell cytotoxicity against RMS when used in combination. Overall, these findings may explain the historical lack of RMS responsiveness to immunotherapy and unlocks new immunotherapy strategies against RMS that rely on targeting MHC-I antigens. Citation Format: David Milewski, Hsien-Chao Chou, Vineela Gangalapudi, Meijie Tian, Yong Kim, Young Song, Jun Wei, Javed Khan. Suppression of antigen presentation in pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB403.

Ontogeny of the autopodial skeleton of the gecko Tarentola (Squamata: Phyllodactylidae)

Squamates exhibit evident diversity in their limb morphology. Gekkotans are a particularly diverse group in this respect. The appearance of toepads in gekkotans usually cooccurs with the reduction or loss of claws. The gecko Tarentola (Phyllodactylidae) shows a unique combination of features among geckos, with toepads, hyperphalangy, and dimorphism of claw expression (claws are retained on digits III and IV, but lost (manus) or strongly reduced (pes) on the remaining digits). Despite being a candidate model for studying embryonic skeletal development of the autopodium, no studies have investigated the autopodial development of the gecko Tarentola in detail. Here, we aim to follow up the development of the autopodial skeleton in T. annularis and T. mauritanica using acid-free double staining. The results indicate that the terminal phalanges of claw-bearing digits III and IV ossify earlier than in the remaining digits. This confirms the differential ossification as a result of claw regression in Tarentola. The strongly reduced second phalanges of digits IV in both the fore- and hindlimbs are the last ossifying phalanges. Such late ossification may precede the evolutionary loss of this phalanx. If this is correct, the autopodia of Tarentola would be an interesting example of both the hyperphalangy in digit I and the process of phalanx loss in digit IV. Delay in ossification of the miniaturised phalanx probably represents an example of paedomorphosis.

Systematic Identification of Long Noncoding RNAs during Three Key Organogenesis Stages in Zebrafish.

Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related functions have not yet been elucidated. In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos that are important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2), and 36 hpf (T3). LncRNA gas5, associated with muscle development, was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied through qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: (1) A total of 1486 differentially expressed lncRNAs were identified between T2 and T1, among which 843 lncRNAs were upregulated and 643 were downregulated. The comparison with T3 and T2 resulted in 844 differentially expressed lncRNAs, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. A total of 2137 differentially expressed lncRNAs were found between T3 and T1, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. (2) The results of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebrafish. (3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were more highly affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. (4) The results of the expression of the skeletal myogenesis marker myod showed that the expression of myod in myotomes was abnormal, suggesting that skeletal myogenesis was affected after lncRNA gas5 knockout. The results of this study provide an experimental basis for further studies on the role of lncRNA gas5 in the embryonic skeletal muscle development of zebrafish.

Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

The Molecular Mechanism of the TEAD1 Gene and miR-410-5p Affect Embryonic Skeletal Muscle Development: A miRNA-Mediated ceRNA Network Analysis.

Muscle development is a complex biological process involving an intricate network of multiple factor interactions. Through the analysis of transcriptome data and molecular biology confirmation, this study aims to reveal the molecular mechanism underlying sheep embryonic skeletal muscle development. The RNA sequencing of embryos was conducted, and microRNA (miRNA)-mediated competitive endogenous RNA (ceRNA) networks were constructed. qRT-PCR, siRNA knockdown, CCK-8 assay, scratch assay, and dual luciferase assay were used to carry out gene function identification. Through the analysis of the ceRNA networks, three miRNAs (miR-493-3p, miR-3959-3p, and miR-410-5p) and three genes (TEAD1, ZBTB34, and POGLUT1) were identified. The qRT-PCR of the DE-miRNAs and genes in the muscle tissues of sheep showed that the expression levels of the TEAD1 gene and miR-410-5p were correlated with the growth rate. The knockdown of the TEAD1 gene by siRNA could significantly inhibit the proliferation of sheep primary embryonic myoblasts, and the expression levels of SLC1A5, FoxO3, MyoD, and Pax7 were significantly downregulated. The targeting relationship between miR-410-5p and the TEAD1 gene was validated by a dual luciferase assay, and miR-410-5p can significantly downregulate the expression of TEAD1 in sheep primary embryonic myoblasts. We proved the regulatory relationship between miR-410-5p and the TEAD1 gene, which was related to the proliferation of sheep embryonic myoblasts. The results provide a reference and molecular basis for understanding the molecular mechanism of embryonic muscle development.

Histopathological and immunohistochemical study to determine the effects of monosodium glutamate on skeletal muscle development in chickens

The aim of this report was to investigate the effects of MSG administration on embryonic skeletal muscle development in chickens, using histological, histopathological, and immunohistochemical methods. A total of 410 fertilized chicken eggs were used and were divided into five groups: negative control, vehicle control, low-dose group (0.12 mg/g egg MSG), medium-dose group (0.6 mg/g egg MSG), and high-dose group (1.2 mg/g egg MSG). At incubation days 15, 18, and 21, eggs from each group were opened and six live embryos were obtained. The embryos were sacrificed by decapitation, and skeletal muscle tissue samples (musculus fibularis longus and musculus sternocoracoideus pectoralis) were obtained. Sections were stained with hematoxylin-eosin. Moreover, caspase-3 reactivity was determined using the immunohistochemical method. As a result, muscle development was delayed in the MSG groups and the number of caspase-3-positive cells was higher (p < 0.05) than in the controls. Histopathological examinations revealed degenerative and necrotic changes in the skeletal muscle. Muscle degeneration (edema, muscle fiber degeneration, Zenker’s necrosis, and mononuclear cell infiltrations) were observed in all groups, except for the control groups. It was concluded that MSG may have adversely affected the development of the skeletal muscle.

Ectomesenchymal Six1 controls mandibular skeleton formation.

Craniofacial development requires intricate cooperation between multiple transcription factors and signaling pathways. Six1 is a critical transcription factor regulating craniofacial development. However, the exact function of Six1 during craniofacial development remains elusive. In this study, we investigated the role of Six1 in mandible development using a Six1 knockout mouse model (Six1 -/- ) and a cranial neural crest-specific, Six1 conditional knockout mouse model (Six1 f/f ; Wnt1-Cre). The Six1 -/- mice exhibited multiple craniofacial deformities, including severe microsomia, high-arched palate, and uvula deformity. Notably, the Six1 f/f ; Wnt1-Cre mice recapitulate the microsomia phenotype of Six1 -/- mice, thus demonstrating that the expression of Six1 in ectomesenchyme is critical for mandible development. We further showed that the knockout of Six1 led to abnormal expression of osteogenic genes within the mandible. Moreover, the knockdown of Six1 in C3H10 T1/2 cells reduced their osteogenic capacity in vitro. Using RNA-seq, we showed that both the loss of Six1 in the E18.5 mandible and Six1 knockdown in C3H10 T1/2 led to the dysregulation of genes involved in embryonic skeletal development. In particular, we showed that Six1 binds to the promoter of Bmp4, Fat4, Fgf18, and Fgfr2, and promotes their transcription. Collectively, our results suggest that Six1 plays a critical role in regulating mandibular skeleton formation during mouse embryogenesis.

Recent Developments in Polymer Nanocomposites for Bone Regeneration.

Most people who suffer acute injuries in accidents have fractured bones. Many of the basic processes that take place during embryonic skeletal development are replicated throughout the regeneration process that occurs during this time. Bruises and bone fractures, for example, serve as excellent examples. It almost always results in a successful recovery and restoration of the structural integrity and strength of the broken bone. After a fracture, the body begins to regenerate bone. Bone formation is a complex physiological process that requires meticulous planning and execution. A normal healing procedure for a fracture might reveal how the bone is constantly rebuilding as an adult. Bone regeneration is becoming more dependent on polymer nanocomposites, which are composites made up of a polymer matrix and a nanomaterial. This study will review polymer nanocomposites that are employed in bone regeneration to stimulate bone regeneration. As a result, we will introduce the role of bone regeneration nanocomposite scaffolds, and the nanocomposite ceramics and biomaterials that play a role in bone regeneration. Aside from that, recent advances in polymer nanocomposites might be used in a variety of industrial processes to help people with bone defects overcome their challenges will be discussed.

Capra hircus outliers markers in Brazil: Searching for genomic regions under the action of natural selection.

Identifying genome regions subject to selection in local breeds of Brazil is an opportunity to elucidate the C. hircus genome plasticity, flowing the colonization history of the country. Using SNP Bead Chip Illumina 50K genotyping of Brazilian Anglo-Nubian (standardized breed) and Marota (local endangered population from the semiarid area of Brazil) to show outliers loci in genome regions subject to selection. After applying data quality control, 45,600 SNPs were included in this investigation. Principal component analysis (PCAdapt) and FDIST2 analysis signalized 14 genomic regions more affected by selection in the Brazilian dry zone environment. The genome study signalized regions that are close to the sequences of genes related to growth and embryonic skeletal development (FGF12, AMPD2, OSTN). In addition, flagged regions close to the genes UTSB2 and SLC5A2 contribute to research on functional adaptation with low water needs and poor nutritive diet to survive. Both genes encode proteins that act on osmotic pathways and avoid cell flooding on stress cell responses. Further studies on the genetic role and involvement of these outliers' genomic regions, building a robust conceptual high-resolution map of natural selection drives, help to achieve hardiness candidate genes linked to the evolutionary history of Capra hircus in the semiarid area of Brazil.

Polymorphism, Expression, and Structure Analysis of a Key Gene ARNT in Sheep (Ovis aries).

Growth traits are influential factors that significantly affects the development of the sheep industry. A previous TMT proteomic analysis found that a key protein in the HIF signaling pathway, ARNT, may influence embryonic skeletal muscle growth and development in sheep. The purpose of this study was to better understand the association between the polymorphisms of ARNT and growth traits of sheep, and the potential function of ARNT. Real-time qPCR (qRT-PCR) of ARNT was carried out to compare its expression in different developmental stages of the muscle tissues and primary myoblasts in the Hu, Chinese merino, and Gangba sheep. The genetic variance of ARNT was detected using the Illumina Ovine SNP 50 K and 600 K BeadChip in the Hu and Ujimqin sheep populations, respectively. The CDS sequence of the ARNT gene was cloned in the Hu sheep using PCR technology. Finally, bioinformatic analytical methods were applied to characterize the genes and their hypothetical protein products. The qRT-PCR results showed that the ARNT gene was expressed significantly in the Chinese merino embryo after 85 gestation days (D85) (p < 0.05). Additionally, after the sheep were born, the expression of ARNT was significant at the weaning stage of the Hu sheep (p < 0.01). However, there was no difference in the Gangba sheep.In addition, six SNP loci were screened using 50 K and 600 K BeadChip. We found a significant association between rs413597480 A > G and the Hu sheep weight at weaning and backfat thickness in the 5-month-old sheep (p < 0.05), and four SNP loci (rs162298018 G > C, rs159644025 G > A, rs421351865 G > A, and rs401758103 A > G) were also associated with growth traits in the Ujimqin sheep (p < 0.05). Interestingly, we found that a G > C mutation at 1948 bp in the cloned ARNT CDS sequence of the Hu sheep was the same locus mutation as rs162298018 G > C identified using the 600 K BeadChip, which resulted in a nonconservative missense point mutation, leading to a change from proline to alanine and altering the number of DNA, protein-binding sites, and the α-helix of the ARNT protein. There was a strong linkage disequilibrium between rs162298018 G > C and rs159644025 G > A, and the ARNT protein was conserved among the goat, Hu sheep, and Texel sheep. And, we propose that a putative molecular marker for growth and development in sheep may be the G > C mutation at 1948 bp in the CDS region of the ARNT gene. Our study systematically analyzed the expression, structure, and function of the ARNT gene and its encoded proteins in sheep. This provides a basis for future studies of the regulatory mechanisms of the ARNT gene.

Profiling Analysis of N6-Methyladenosine mRNA Methylation Reveals Differential m6A Patterns during the Embryonic Skeletal Muscle Development of Ducks.

Simple SummaryRecent studies show that N6-methyladenosine (m6A) modification, the most common RNA chemical modification, influences the modification, processing, transport, and translation of RNA. N6-methyladenosine is an epigenetic modification that influences skeletal myogenesis and skeletal muscle development. However, the N6-methyladenosine modification profile and its function during poultry muscle development is unclear, and there is only one report about m6A modification in ducks, which focuses on duck hepatitis A virus infection. Here, we compared the m6A modification profiles between E13 (embryonic day 13) and E19 (embryonic day 19) in duck breast muscle differentiation using MeRIP-seq, and evaluated the expression profile of the methyl transferase METTL14 and its cofactors during breast muscle development. This is the first study of N6-methyladenosine modification patterns in duck muscle tissue. The current study not only elucidates the regulation mechanisms of duck skeletal muscle development, but also lays the groundwork for studying the role of RNA modification in poultry muscle development.N6-Methyladenosine is a reversible epigenetic modification that influences muscle development. However, the m6A modification profile during poultry skeletal muscle development is poorly understood. Here, we utilized m6A-specific methylated RNA immunoprecipitation sequencing to identify m6A sites during two stages of breast muscle development in ducks: embryonic days 13 (E13) and E19. MeRIP-seq detected 19,024 and 18,081 m6A peaks in the E13 and E19 groups, respectively. Similarly to m6A distribution in mammalian transcripts, our results revealed GGACU as the main m6A motif in duck breast muscle; they also revealed that m6A peaks are mainly enriched near the stop codons. In addition, motif sequence analysis and gene expression analysis demonstrated that m6A modification in duck embryo skeletal muscles may be mediated by the methyltransferase-like 14. GO and KEGG analysis showed that m6A peaks containing genes at E19 were mainly enriched in muscle-differentiation- and muscle-growth-related pathways, whereas m6A peaks containing genes in E13 were mainly enriched in embryonic development and cell proliferation pathways. Combined analysis of MeRIP-seq and RNA-seq showed that the mRNA expression may be affected by m6A modification. Moreover, qRT-PCR analysis of the expression of METTL14 and its cofactors (WTAP, ZC3H13, RBM15 and VIRMA) during duck embryonic skeletal muscle development in breast and leg muscle samples revealed a significant downward trend as the developmental age progressed. Our results demonstrated that m6A mRNA methylation modifications control muscle development in ducks. This is the first study of m6A modification patterns in duck muscle tissue development, and it lays the foundation for the study of the effects of RNA modification on poultry skeletal muscle development.

Ontogeny of the paraphalanges and derived phalanges of Hemidactylus turcicus (Squamata: Gekkonidae).

Gekkotan lizards of the genus Hemidactylus exhibit derived digital morphologies. These include heavily reduced antepenultimate phalanges of digits III and IV of the manus and digits III-V of the pes, as well as enigmatic cartilaginous structures called paraphalanges. Despite this well-known morphological derivation, no studies have investigated the development of these structures. We aimed to determine if heterochrony underlies the derived antepenultimate phalanges of Hemidactylus. Furthermore, we aimed to determine if convergently evolved paraphalanges exhibit similar or divergent developmental patterns. Herein we describe embryonic skeletal development in the hands and feet of four gekkonid species, exhibiting a range of digital morphologies. We determined that the derived antepenultimate phalanges of Hemidactylus are the products of paedomorphosis. Furthermore, we found divergent developmental patterns between convergently evolved paraphalanges.

3-PEP promotes bone regeneration by up regulating BCL-2 expression via ERK phosphorylation.

Bone healing and regeneration is a complex process that recapitulates embryonic skeletal development and is delayed in diseases like osteoporosis. Bone healing therapies like recombinant bone morphogenetic-2 protein (rhBMP-2) and parathyroid hormone (PTH), an approved bone anabolic therapy reduces fracture risks but are fraught with high cost and several side effects. Thus, there is an unmet need for cost-effective bone healing agents. In this study, we have synthesized 3-piperidinylethoxypterocarpan (3-PEP) which is a hybrid of bone supplement ipriflavone and anti-resorptive drug raloxifene and evaluated its bone regeneration and healing potential. Prior to studies in animal models, the potency of 3-PEP was confirmed in calvarial osteoblast cells. Bromodeoxy uridine cell proliferation and cell viability assay revealed that 3-PEP at 100 pM concentration increased the proliferation and survival of osteoblasts simultaneously inhibiting the apoptosis by involving activation of BCL-2 by phosphorylation at Ser70 site through MEK-ERK pathway. In vivo studies were conducted in estrogen-deficient ovariectomized Balb/c mice and drill hole injury was generated in the mid diaphysis of the femur in all the animals. Treatment with 3-PEP commenced the next day onward and terminated at 7 and 15 days. Micro-CT analysis and calcein labeling of newly generated bone at the drill hole injury site showed that 3-PEP promotes bone healing and new bone formation at a dose of 5 mg/kg at the injury site. These data were also corroborated in non-ovariectomized Balb/c mice cortical defect model. Owing to the side effects associated with rhBMP-2 and PTH, along with the expenses involved, our study proposes an alternative therapeutic option for bone healing.

Investigating the Effects of Vaping and Nicotine’s Block of Kir2.1 on Humerus and Digital Development in Embryonic Mice

Kir2.1 is an ion channel that is important for skeletal development. Nicotine, a common ingredient in electronic cigarettes, blocks Kir2.1. However, little research has been done to investigate how vaping nicotine during pregnancy affects embryonic development. We examined how vaping nicotine during pregnancy would affect embryonic skeletal development in mice. Mice pregnant with embryos of three genotypes—wild‐type, Kir2.1+/KO, or Kir2.1KO/KO—received either no vaping treatment, a 2.4% freebase nicotine vaping treatment, or a 3.6% nicotine salt vaping treatment throughout pregnancy. The embryos were dissected at E18.5, and their skeletons were extracted and stained for imaging. The lengths of the humerus bones and number of digital bones were calculated to quantify differences in bone development between groups. We found significant differences in humerus length between the wild‐type no vape and wild‐type 2.4% freebase nicotine groups, as well as the Kir2.1+/KO no vape and Kir2.1+/KO 2.4% freebase nicotine groups. However, no significant differences were found between wild‐type no vape and wild‐type 3.6% nicotine salt groups, as well as Kir2.1+/KO no vape and Kir2.1+/KO 3.6% nicotine salt groups. We suspect that nicotine salts are metabolized faster than freebase nicotine and thus inhibit Kir2.1’s function for a shorter amount of time, but further research needs to be conducted to test this hypothesis. We conclude that fetal exposure to vaped free base nicotine impacts the development of long bones and reduces long bone length.

Identification of crucial circRNAs in skeletal muscle during chicken embryonic development

BackgroundChicken provides humans with a large amount of animal protein every year, in which skeletal muscle plays a leading role. The embryonic skeletal muscle development determines the number of muscle fibers and will affect the muscle production of chickens. CircRNAs are involved in a variety of important biological processes, including muscle development. However, studies on circRNAs in the chicken embryo muscle development are still lacking.ResultsIn the study, we collected chicken leg muscles at 14 and 20-day embryo ages both in the fast- and slow-growing groups for RNA-seq. We identified 245 and 440 differentially expressed (DE) circRNAs in the comparison group F14vsF20 and S14vsS20 respectively. GO enrichment analysis for the host genes of DE circRNAs showed that biological process (BP) terms in the top 20 related to growth in F14vsF20 were found such as positive regulation of transcription involved in G1/S phase of mitotic cell cycle, multicellular organismal macromolecule metabolic process, and multicellular organismal metabolic process. In group S14vsS20, we also found some BP terms associated with growth in the top 20 including actomyosin structure organization, actin cytoskeleton organization and myofibril assembly. A total of 7 significantly enriched pathways were obtained, containing Adherens junction and Tight junction. Further analysis of those pathways found three crucial host genes MYH9, YBX3, IGF1R in both fast- and slow-growing groups, three important host genes CTNNA3, AFDN and CREBBP only in the fast-growing group, and six host genes FGFR2, ACTN2, COL1A2, CDC42, DOCK1 and MYL3 only in the slow-growing group. In addition, circRNA-miRNA network also revealed some key regulation pairs such as novel_circ_0007646-miR-1625-5p, novel_circ_0007646-miR-1680-5p, novel_circ_0008913-miR-148b-5p, novel_circ_0008906-miR-148b-5p and novel_circ_0001640-miR-1759-3p.ConclusionsComprehensive analysis of circRNAs and their targets would contribute to a better understanding of the molecular mechanisms in poultry skeletal muscle and it also plays an important guiding role in the next research.

An association study on imputed whole-genome resequencing from high-throughput sequencing data for body traits in crossbred pigs.

Body traits are important economic factors in the pig industry. Genome-wide association studies (GWASs) have been widely applied using high-density genotype data to detect QTL in pigs. The aim of the present study was to detect the genetic variants significantly associated with body traits in crossbred pigs using the Illumina Porcine SNP50 BeadChip and imputed whole-genome sequence data. A set of seven body traits - body length, body height, chest circumference, cannon bone circumference, leg buttock circumference, back fat thickness and loin muscle depth - were measured. Moderate to high heritabilities were obtained for most traits (from 0.14 to 0.46), and significant genetic and phenotypic correlations among them were observed. GWAS identified 714significantly associated SNPs located at 39 regions on all autosomes for body traits, and a total of seven functionally related candidate genes: PIK3CD, HOXA, PCGF2, CHST11, COL2A1, BMI1 and OSR2. Functional enrichment analysis revealed that candidate genes were enriched in the estrogen signaling pathway, embryonic skeletal system morphogenesis and embryonic skeletal system development. These results aim to uncover the genetic mechanisms underlying body development and marker-assisted selection programs focusing on body traits in pigs.

31 Gametic Incompatibility: Improving the Success of Mate Allocation in Dairy Cattle

Abstract In the dairy industry, mate allocation is mainly based on the parents’ breeding values and inbreeding coefficients aiming to achieve the producer’s breeding goal. With artificial insemination, the portfolio of sires to choose from is large and the quality of the semen doses is standardized. However, not all sire-dam matings are equally likely to produce a successful pregnancy. Among other reproduction issues, the success of a mating could vary due to the incompatibility of gametes coming from the sire and the dam and could influence the fertilization’s success, additionally to the reproductive capacity of the parents. Considering the gametic incompatibility of the potential parents could be a novel option to improve mating plans. Under the hypothesis that gametic incompatibility has a significant effect on reproduction and reduces the odds of fertilization and pregnancy, this study aimed to determine the genetic background of gametic incompatibility. Transmission ratio distortion (TRD), which detects deviations from Mendelian inheritance expectations, is commonly used to identify deleterious mutations. We adapted the TRD model by including an interaction effect between the gametes leading to the offspring genotype to detect regions with TRD effects and gametic incompatibility. Our dataset contained 436,651 genotyped (50K SNP) Canadian Holstein cattle from 283,817 parents-offspring trios. A total of 482 regions with TRD containing 671 positional genes were found. The functional analysis detected biological pathways associated with uterus development, embryonic skeletal system development, and nervous system development. Additionally, gene ontology terms from the topology-based pathway enrichment analysis were mostly related to the steroid hormones signalling pathway. Although difficult, genes specific to gametic incompatibility could be differentiated from genes underlying other reproduction processes by refining the genetic regions with TRD. With further investigation, we will provide new information to improve mate allocation for the dairy cattle industry.

A Novel Circular RNA circITSN2 Targets the miR-218-5p/LMO7 Axis to Promote Chicken Embryonic Myoblast Proliferation and Differentiation.

Circular RNA (circRNA) is a class of endogenous non-coding RNAs without 5′ and 3′ ends; an increasing number of studies show that circRNA is involved in skeletal muscle development. From our previous sequencing data, the circRNAome in breast muscle of two chicken lines with a distinct rate of muscle development, which included a fast muscle growing broiler (FMGB) and a slow muscle growing layer (SMGL), we found a novel differentially expressed circRNA generated by intersectin 2 (ITSN2) gene (named circITSN2). We verified that circITSN2 is a skeletal muscle-enriched circRNA that promotes chicken primary myoblast (CPM) proliferation and differentiation. Further molecular mechanism analysis of circITSN2 in chicken myogenesis was performed, and we found circITSN2 directly targeting miR-218-5p. Besides, miR-218-5p inhibits CPM proliferation and differentiation, which is contrary to circITSN2. Commonly, circRNAs act as a miRNA sponge to alleviate the inhibition of miRNAs on mRNAs. Thus, we also identified that a downstream gene LIM domain 7 (LMO7) was inhibited by miR-218-5p, while circITSN2 could block the inhibitory effect of miR-218-5p by targeting it. Functional analysis revealed that LMO7 also accelerates CPM proliferation and differentiation, which was similar to circITSN2 but contrary to miR-218-5p. Taken together, these results suggested that circITSN2 promotes chicken embryonic skeletal muscle development via relieving the inhibition of miR-218-5p on LMO7. Our findings revealed a novel circITSN2/miR-218-5p/LMO7 axis in chicken embryonic skeletal muscle development, which expands our understanding of the complex muscle development regulatory network.

Vasoactive Intestinal Peptide Promotes Fracture Healing in Sympathectomized Mice.

Vasoactive intestinal peptide (VIP) as a neuromodulator and neurotransmitter played a significant role in modulating bone homeostasis. Our previous study reported an essential role of VIP in in vitro BMSCs osteogenesis and in vivo bone defect repair. VIP was also revealed to have a promoting effect on embryonic skeletal element development. However, the role of VIP in fracture healing is not known yet. We hypothesized that the disorder of sympathetic nervous system impairs bone structure and fracture healing, whereas VIP may rescue the sympathetic inhibition effects and promote fracture healing. We employed a 6-hydroxydopamine(6-OHDA) induced sympathectomy mice model (sympathectomized mice), in which successful sympathetic inhibition was confirmed by a decreased level of norephedrine (NE) in the spleen. In the sympathectomized mice, the femoral micro-architecture, bone density and mechanical properties were all impaired compared to the vehicle control mice. The femoral fracture was created in the vehicle or sympathectomized mice. Vehicle mice were locally injected with PBS as a negative control, and the sympathectomized mice were treated with injection of PBS or VIP. VIP expression at the fracture site was significantly decreased in sympathectomized mice. The fracture healing was repressed upon 6-OHDA treatment and rescued by VIP treatment. Micro-CT examination showed that the femoral bone micro-architecture at the fracture sites and mechanical properties were all impaired. Simultaneously, the expression level of osteogenic markers OCN and OPN were reduced in sympathectomized mice compared with vehicle group. While the VIP treatment rescued the repression effects of 6-OHDA on bone remodeling and significantly promoted bone quality and mechanical properties as well as increased osteogenesis marker expression in the sympathectomized mice. VIP administration promoted bone fracture healing by inhibiting bone resorption, making it a putative new alternative treatment strategy for fracture healing.

The landscape of chromatin accessibility in skeletal muscle during embryonic development in pigs

BackgroundThe development of skeletal muscle in pigs during the embryonic stage is precisely regulated by transcriptional mechanisms, which depend on chromatin accessibility. However, how chromatin accessibility plays a regulatory role during embryonic skeletal muscle development in pigs has not been reported. To gain insight into the landscape of chromatin accessibility and the associated genome-wide transcriptome during embryonic muscle development, we performed ATAC-seq and RNA-seq analyses of skeletal muscle from pig embryos at 45, 70 and 100 days post coitus (dpc).ResultsIn total, 21,638, 35,447 and 60,181 unique regions (or peaks) were found across the embryos at 45 dpc (LW45), 70 dpc (LW70) and 100 dpc (LW100), respectively. More than 91% of the peaks were annotated within − 1 kb to 100 bp of transcription start sites (TSSs). First, widespread increases in specific accessible chromatin regions (ACRs) from embryos at 45 to 100 dpc suggested that the regulatory mechanisms became increasingly complicated during embryonic development. Second, the findings from integrated ATAC-seq and RNA-seq analyses showed that not only the numbers but also the intensities of ACRs could control the expression of associated genes. Moreover, the motif screening of stage-specific ACRs revealed some transcription factors that regulate muscle development-related genes, such as MyoG, Mef2c, and Mef2d. Several potential transcriptional repressors, including E2F6, OTX2 and CTCF, were identified among the genes that exhibited different regulation trends between the ATAC-seq and RNA-seq data.ConclusionsThis work indicates that chromatin accessibility plays an important regulatory role in the embryonic muscle development of pigs and regulates the temporal and spatial expression patterns of key genes in muscle development by influencing the binding of transcription factors. Our results contribute to a better understanding of the regulatory dynamics of genes involved in pig embryonic skeletal muscle development.