Single-nucleus transcriptional and chromatin accessible profiles reveal critical cell types and molecular architecture underlying chicken sex determination.

INTRODUCTION: Understanding the sex determination mechanisms in birds has great significance for the biological sciences and production in the poultry industry. Sex determination in chickens is a complex process that involves fate decisions of supporting cells such as granulosa or Sertoli cells. However, a systematic understanding of the genetic regulation and cell commitment process underlying sex determination in chickens is still lacking. OBJECTIVES: We aimed to dissect the molecular characteristics associated with sex determination in the gonads of chicken embryos. METHODS: Single-nucleus RNA-seq (snRNA-seq) and ATAC-seq (snATAC-seq) analysis were conducted on the gonads of female and male chickens at embryonic day 3.5 (E3.5), E4.5, and E5.5. RESULTS: Here, we provided a time-course transcriptional and chromatin accessible profiling of gonads during chicken sex determination at single-cell resolution. We uncovered differences in cell composition and developmental trajectories between female and male gonads and found that the divergence of transcription and accessibility in gonadal cells first emerged at E5.5. Furthermore, we revealed key cell-type-specific transcription factors (TFs) and regulatory networks that drive lineage commitment. Sex determination signaling pathways, dominated by BMP signaling, are preferentially activated in males during gonadal development. Further pseudotime analysis of the supporting cells indicated that granulosa cells were regulated mainly by the TEAD gene family and that Sertoli cells were driven by the DMRT1 regulons. Cross-species analysis suggested high conservation of both cell types and cell-lineage-specific TFs across the six vertebrates. CONCLUSIONS: Overall, our study will contribute to accelerating the development of sex manipulation technology in the poultry industry and the application of chickens as a unique model for studying cell fate decisions.

Comprehensive Atlas of Alternative Splicing Reveals NSRP1 Promoting Adipogenesis through CCDC18.

Alternative splicing (AS) plays a crucial role in regulating gene expression, function, and diversity. However, limited reports exist on the identification and comparison of AS in Eastern and Western pigs. Here, we analyzed 243 transcriptome data from eight tissues, integrating information on transcription factors (TFs), selection signals, splicing factors (SFs), and quantitative trait loci (QTL) to comprehensively study alternative splicing events (ASEs) in pigs. Five ASE types were identified, with Mutually Exclusive Exon (MXE) and Skipped Exon (SE) ASEs being the most prevalent. A significant portion of genes with ASEs (ASGs) showed conservation across all eight tissues (63.21-76.13% per tissue). Differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) exhibited tissue specificity, with blood and adipose tissues having more DASGs. Functional enrichment analysis revealed coDASG_DEGs in adipose were enriched in pathways associated with adipose deposition and immune inflammation, while coDASG_DEGs in blood were enriched in pathways related to immune inflammation and metabolism. Adipose deposition in Eastern pigs might be linked to the down-regulation of immune-inflammation-related pathways and reduced insulin resistance. The TFs, selection signals, and SFs appeared to regulate ASEs. Notably, ARID4A (TF), NSRP1 (SF), ANKRD12, IFT74, KIAA2026, CCDC18, NEXN, PPIG, and ROCK1 genes in adipose tissue showed potential regulatory effects on adipose-deposition traits. NSRP1 could promote adipogenesis by regulating alternative splicing and expression of CCDC18. Conducting an in-depth investigation into AS, this study has successfully identified key marker genes essential for pig genetic breeding and the enhancement of meat quality, which will play important roles in promoting the diversity of pork quality and meeting market demand.

Uncovering Evolutionary Adaptations in Common Warthogs through Genomic Analyses.

In the Suidae family, warthogs show significant survival adaptability and trait specificity. This study offers a comparative genomic analysis between the warthog and other Suidae species, including the Luchuan pig, Duroc pig, and Red River hog. By integrating the four genomes with sequences from the other four species, we identified 8868 single-copy orthologous genes. Based on 8868 orthologous protein sequences, phylogenetic assessments highlighted divergence timelines and unique evolutionary branches within suid species. Warthogs exist on different evolutionary branches compared to DRCs and LCs, with a divergence time preceding that of DRC and LC. Contraction and expansion analyses of warthog gene families have been conducted to elucidate the mechanisms of their evolutionary adaptations. Using GO, KEGG, and MGI databases, warthogs showed a preference for expansion in sensory genes and contraction in metabolic genes, underscoring phenotypic diversity and adaptive evolution direction. Associating genes with the QTLdb-pigSS11 database revealed links between gene families and immunity traits. The overlap of olfactory genes in immune-related QTL regions highlighted their importance in evolutionary adaptations. This work highlights the unique evolutionary strategies and adaptive mechanisms of warthogs, guiding future research into the distinct adaptability and disease resistance in pigs, particularly focusing on traits such as resistance to African Swine Fever Virus.

S-dinotefuran affects the social behavior of honeybees (Apis mellifera)and increases their risk in the colony.

The toxic effects of neonicotinoid pesticides on honeybees is a global concern, whereas little is known about the effect of stereoisomeric pesticides among honeybee social behavior. In this study, we investigated the effects of stereoisomeric dinotefuran on honeybee social behavior. We found that honeybees exhibit a preference for consuming food containing S-dinotefuran, actively engage in trophallaxis with S-dinotefuran-consuming peers, and consequently acquire higher levels of S-dinotefuran compared with R-dinotefuran. In comparison to R-dinotefuran, S-dinotefuran stimulates honeybees to elevate their body temperature, thereby attracting more peers for trophallaxis. Transcriptome analysis revealed a significant enrichment of thermogenesis pathways due to S-dinotefuran exposure. Additionally, metabolome data indicated that S-dinotefuran may enhance body temperature by promoting lipid synthesis in the lysine degradation pathway. Consequently, body temperature emerges as a key factor influencing honeybee social behavior. Our study is the first to highlight the propensity of S-dinotefuran to raise honeybee body temperature, which prompts honeybee to preferentially engage in trophallaxis with peers exhibiting higher body temperatures. This preference may lead honeybees to collect more dinotefuran-contaminated food in the wild, significantly accelerating dinotefuran transmission within a population. Proactive trophallaxis further amplifies the risk of neonicotinoid pesticide transmission within a population, making honeybees that have consumed S-dinotefuran particularly favored within their colonies. These findings may contribute to our understanding of the higher risk associated with neonicotinoid use compared with other pesticides.

Genome-Wide Detection of Copy Number Variations Associated with Miniature Features in Horses.

Copy number variations (CNVs) are crucial structural genomic variants affecting complex traits in humans and livestock animals. The current study was designed to conduct a comprehensive comparative copy number variation analysis among three breeds, Debao (DB), Baise (BS), and Warmblood (WB), with a specific focus on identifying genomic regions associated with miniature features in horses. Using whole-genome next-generation resequencing data, we identified 18,974 CNVs across 31 autosomes. Among the breeds, we found 4279 breed-specific CNV regions (CNVRs). Baise, Debao, and Warmblood displayed 2978, 986, and 895 distinct CNVRs, respectively, with 202 CNVRs shared across all three breeds. After removing duplicates, we obtained 1545 CNVRs from 26 horse genomes. Functional annotation reveals enrichment in biological functions, including antigen processing, cell metabolism, olfactory conduction, and nervous system development. Debao horses have 970 genes overlapping with CNVRs, possibly causing their small size and mountainous adaptations. We also found that the genes GHR, SOX9, and SOX11 may be responsible for the miniature features of the Debao horse by analyzing their overlapping CNVRs. Overall, this study offers valuable insights into the widespread presence of CNVs in the horse genome. The findings contribute to mapping horse CNVs and advance research on unique miniature traits observed in the Debao horse.

Ring1a protects against colitis through regulating mucosal immune system and colonic microbial ecology.

Inflammatory bowel disease (IBD) represents a prominent chronic immune-mediated inflammatory disorder, yet its etiology remains poorly comprehended, encompassing intricate interactions between genetics, immunity, and the gut microbiome. This study uncovers a novel colitis-associated risk gene, namely Ring1a, which regulates the mucosal immune response and intestinal microbiota. Ring1a deficiency exacerbates colitis by impairing the immune system. Concomitantly, Ring1a deficiency led to a Prevotella genus-dominated pathogenic microenvironment, which can be horizontally transmitted to co-housed wild type (WT) mice, consequently intensifying dextran sodium sulfate (DSS)-induced colitis. Furthermore, we identified a potential mechanism linking the altered microbiota in Ring1aKO mice to decreased levels of IgA, and we demonstrated that metronidazole administration could ameliorate colitis progression in Ring1aKO mice, likely by reducing the abundance of the Prevotella genus. We also elucidated the immune landscape of DSS colitis and revealed the disruption of intestinal immune homeostasis associated with Ring1a deficiency. Collectively, these findings highlight Ring1a as a prospective candidate risk gene for colitis and suggest metronidazole as a potential therapeutic option for clinically managing Prevotella genus-dominated colitis.

We found that PcG protein Ring1a could be a new risk gene for colitis. Ring1a deficiency causes aggravated colitis by regulating the mucosal immune system and colonic microbial ecology.

Simultaneous Detection and Distribution of Five Juvenile Hormones in 58 Insect Species and the Absolute Configuration in 32 Insect Species.

Juvenile hormone (JH) plays an important role in regulating various insect physiological processes. Herein, a novel method (chiral and achiral) for the simultaneous detection of five JHs was established by processing a whole insect without complicated hemolymph extraction. The proposed method was used to determine the distribution of JHs in 58 insect species and the absolute configuration of JHs in 32 species. The results showed that JHSB3 was uniquely synthesized in Hemiptera, JHB3 was unique to Diptera, and JH I and JH II were unique to Lepidoptera. JH III was present in most insect species surveyed, with social insects having generally higher JH III titers. Interestingly, JHSB3 and JHB3, both double epoxidation JHs, were found in insects with sucking mouthparts. The absolute conformation of JH III and the 10C of the detected JHs were all R stereoisomers.

FMO3 deficiency of duck leads to decreased lipid deposition and increased antibacterial activity.

BACKGROUND: Most duck eggs possess a fishy odor, indicating that ducks generally exhibit impaired trimethylamine (TMA) metabolism. TMA accumulation is responsible for this unpleasant odor, and TMA metabolism plays an essential role in trimethylaminuria (TMAU), also known as fish odor syndrome. In this study, we focused on the unusual TMA metabolism mechanism in ducks, and further explored the unclear reasons leading to the debilitating TMA metabolism. METHODS: To achieve this, transcriptome, proteome, and metagenome analyses were first integrated based on the constructed duck populations with high and low TMA metabolism abilities. Additionally, further experiments were conducted to validate the hypothesis regarding the limited flavin-containing monooxygenase 3 (FMO3) metabolism ability of ducks. RESULTS: The study demonstrated that liver FMO3 and cecal microbes, including Akkermansia and Mucispirillum, participated in TMA metabolism in ducks. The limited oxidation ability of FMO3 explains the weakening of TMA metabolism in ducks. Nevertheless, it decreases lipid deposition and increases antibacterial activity, contributing to its survival and reproduction during the evolutionary adaptation process. CONCLUSIONS: This study demonstrated the function of FMO3 and intestinal microbes in regulating TMA metabolism and illustrated the biological significance of FMO3 impairment in ducks.

Identification of circRNAs Associated with Adipogenesis Based on RNA-seq Data in Pigs.

Adipocytes or fat cells play a vital role in the storage and release of energy in pigs, and many circular RNAs (circRNAs) have emerged as important regulators in various tissues and cell types in pigs. However, the spatio-temporal expression pattern of circRNAs between different adipose deposition breeds remains elusive. In this study, RNA sequencing (RNA-seq) produced transcriptome profiles of Western Landrace (lean-type) and Chinese Songliao black pigs (obese-type) with different thicknesses of subcutaneous fat tissues and were used to identify circRNAs involved in the regulation of adipogenesis. Gene expression analysis revealed 883 circRNAs, among which 26 and 11 circRNAs were differentially expressed between Landrace vs. Songliao pigs and high- vs. low-thickness groups, respectively. We also analyzed the interaction between circRNAs and microRNAs (miRNAs) and constructed their interaction network in adipogenesis; gene ontology classification and pathway analysis revealed two vital circRNAs, with the majority of their target genes enriched in biological functions such as fatty acids biosynthesis, fatty acid metabolism, and Wnt/TGF-ß signaling pathways. These candidate circRNAs can be taken as potential targets for further experimental studies. Our results show that circRNAs are dynamically expressed and provide a valuable basis for understanding the molecular mechanism of circRNAs in pig adipose biology.

Integration of multi-omics data reveals cis-regulatory variants that are associated with phenotypic differentiation of eastern from western pigs.

BACKGROUND: The genetic mechanisms that underlie phenotypic differentiation in breeding animals have important implications in evolutionary biology and agriculture. However, the contribution of cis-regulatory variants to pig phenotypes is poorly understood. Therefore, our aim was to elucidate the molecular mechanisms by which non-coding variants cause phenotypic differences in pigs by combining evolutionary biology analyses and functional genomics. RESULTS: We obtained a high-resolution phased chromosome-scale reference genome with a contig N50 of 18.03 Mb for the Luchuan pig breed (a representative eastern breed) and profiled potential selective sweeps in eastern and western pigs by resequencing the genomes of 234 pigs. Multi-tissue transcriptome and chromatin accessibility analyses of these regions suggest that tissue-specific selection pressure is mediated by promoters and distal cis-regulatory elements. Promoter variants that are associated with increased expression of the lysozyme (LYZ) gene in the small intestine might enhance the immunity of the gastrointestinal tract and roughage tolerance in pigs. In skeletal muscle, an enhancer-modulating single-nucleotide polymorphism that is associated with up-regulation of the expression of the troponin C1, slow skeletal and cardiac type (TNNC1) gene might increase the proportion of slow muscle fibers and affect meat quality. CONCLUSIONS: Our work sheds light on the molecular mechanisms by which non-coding variants shape phenotypic differences in pigs and provides valuable resources and novel perspectives to dissect the role of gene regulatory evolution in animal domestication and breeding.

MiR-743a-5p regulates differentiation of myoblast by targeting Mob1b in skeletal muscle development and regeneration.

The microRNAs (miRNAs) play an important role in regulating myogenesis by targeting mRNA. However, the understanding of miRNAs in skeletal muscle development and diseases is unclear. In this study, we firstly performed the transcriptome profiling in differentiating C2C12 myoblast cells. Totally, we identified 187 miRNAs and 4260 mRNAs significantly differentially expressed that were involved in myoblast differentiation. We carried out validation of microarray data based on 5 mRNAs and 5 miRNAs differentially expressed and got a consistent result. Then we constructed and validated the significantly up- and down-regulated mRNA-miRNA interaction networks. Four interaction pairs (miR-145a-5p-Fscn1, miR-200c-5p-Tmigd1, miR-27a-5p-Sln and miR-743a-5p-Mob1b) with targeted relationships in differentiated myoblast cells were demonstrated. They are all closely related to myoblast development. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated cell cycle signals important for exploring skeletal muscle development and disease. Functionally, we discovered that miR-743a targeting gene Mps One Binder Kinase Activator-Like 1B (Mob1b) gene in differentiated C2C12. The up-regulated miR-743a can promote the differentiation of C2C12 myoblast. While the down-regulated Mob1b plays a negative role in differentiation. In addition, the expression profile of miR-743a and Mob1b are consistent with skeletal muscle recovery after Cardiotoxin (CTX) injury. Our study revealed that miR-743a-5p regulates myoblast differentiation by targeting Mob1b involved in skeletal muscle development and regeneration. Our findings made a further exploration for mechanisms in myogenesis and might provide potential possible miRNA-based target therapies for skeletal muscle regeneration and disease in the near future.

Juvenile Hormone III R Stereoisomer Is Specifically Synthesized by Honeybees (Apis mellifera ligustica) and Shows a Higher Biological Activity in Regulating Their Social Behavior.

The juvenile hormone (JH) plays a key role in the development of honeybee larvae and the alteration of adult behavior. Verification methods of types and stereoisomers of JHs in honeybees were established in this study. The regulatory modes of different stereoisomers of JH III on the social behaviors of honeybees were revealed by the disparity of interaction and RNA-seq. This result represented the first assessment of the effects of R-JH III and S-JH III in honeybee interactions; the former (367 times in total) was significantly higher than the latter (235 times in total); honeybees with high JH titers are always welcome in the colony because the effect of JH III on bees involves the sensing and signaling of hormones, and R-JH III is much more active than S-JH III in this regulation. Efficient R-JH III may be the insurance for bees to establish their social system advantages.

The m6A methylation regulates gonadal sex differentiation in chicken embryo.

BACKGROUND: As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. RESULTS: The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. CONCLUSIONS: This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Adipokinetic hormone signaling regulates cytochrome P450-mediated chlorantraniliprole sensitivity in Spodoptera frugiperda (Lepidoptera: Noctuidae).

BACKGROUND: Fall armyworm (FAW, Spodoptera frugiperda) is one of the most destructive and invasive pests worldwide and causes significant economic losses. Intensive and frequent use of insecticides has led to the development of resistance in FAW. Adipokinetic hormone (AKH) have been proven to be involved in insecticide resistance in insects. However, the molecular mechanism underlying chlorantraniliprole resistance mediated by AKH signaling in FAW remains unclear. RESULTS: The expression of SpfAKHR was highest in male adults and lowest in 1st instar larvae. SpfAKH was expressed the highest in eggs and the lowest in 6th instar larvae. AKH signaling was involved in the sensitivity of FAW to chlorantraniliprole through a toxicological bioassay, and the combination of chlorantraniliprole and bithionol (an inhibitor of key enzymes in the AKH pathway) significantly increased the mortality of FAW. Chlorantraniliprole significantly induced the expression of ten P450s, SpfAKH and SpfAKHR in FAW. RNA interference against SpfAKHR significantly decreased the P450 content, downregulated the expression of three P450 genes (SpfCYP6B50, SpfCYP321A9 and SpfCYP9A58) and inhibited the resistance of FAW to chlorantraniliprole. The topical application of AKH peptide significantly increased the P450 content, upregulated the expression of five P450 genes (SpfCYP321A9, SpfCY321A8, SpfCYP321A10, SpfCYP321A7 and SpfCYP6AB12), and enhanced the survival of FAW against chlorantraniliprole. CONCLUSIONS: AKH plays an important role in enhancing chlorantraniliprole resistance in FAW by exerting a positive influence on P450 gene expression and P450 content. These results provide valuable insights into insecticide resistance regulation and FAW control strategies. © 2022 Society of Chemical Industry.

Time-Course Transcriptional and Chromatin Accessibility Profiling Reveals Genes Associated With Asymmetrical Gonadal Development in Chicken Embryos.

In birds, male gonads form on both sides whereas most females develop asymmetric gonads. Multiple early lines of evidence suggested that the right gonad fails to develop into a functional ovary, mainly due to differential expression of PITX2 in the gonadal epithelium. Despite some advances in recent years, the molecular mechanisms underlying asymmetric gonadal development remain unclear. Here, using bulk analysis of whole gonads, we established a relatively detailed profile of four representative stages of chicken gonadal development at the transcriptional and chromatin levels. We revealed that many candidate genes were significantly enriched in morphogenesis, meiosis and subcellular structure formation, which may be responsible for asymmetric gonadal development. Further chromatin accessibility analysis suggested that the transcriptional activities of the candidate genes might be regulated by nearby open chromatin regions, which may act as transcription factor (TF) binding sites and potential cis-regulatory elements. We found that LHX9 was a promising TF that bound to the left-biased peaks of many cell cycle-related genes. In summary, this study provides distinctive insights into the potential molecular basis underlying the asymmetric development of chicken gonads.

Presence of mutant p53 increases stem cell frequency and is associated with reduced binding to classic TP53 binding sites in cell lines and primary AMLs.

With an overall 5%-10% incidence rate in acute myeloid leukemia (AML), the occurrence of TP53 mutations is low compared with that in solid tumors. However, when focusing on high-risk groups including secondary AML (sAML) and therapy-related AMLs, the frequency of mutations reaches up to 35%. Mutations may include loss of heterozygosity (LOH) or deletion of the 17p allele, but are mostly missense substitutions that are located in the DNA-binding domain. Despite elaborate research on the effects of TP53 mutations in solid tumors, in hematological malignancies, the effects of TP53 mutations versus loss of TP53 remain unclear and under debate. Here, we compared the cellular effects of a TP53 mutant and loss of TP53 in human hematopoietic stem and progenitor cells (HSPCs). We found that when expressing TP53 mutant or loss of TP53 using siRNA, CD34+/CD38- cells have a significantly enhanced replating potential, which could not be demonstrated for the CD34+/CD38+ population. Using RNA-sequencing analysis, we found a loss of expression of p53 target genes in cells with TP53 knockdown. In contrast, an increased expression of a large number of genes was observed when expressing TP53 mutant, resulting in an increase in expression of genes involved in megakaryocytic differentiation, plasma membrane binding, and extracellular structure organization. When binding of p53 wild type and p53 mutant was compared in cell lines, we found that mutant p53 binds to a large number of binding sites genomewide, contrary to wild-type p53, for which binding is restricted to genes with a p53 binding motif. These findings were verified in primary AMLs with and without mutated TP53. In conclusion, in our models, we identified overlapping effects of TP53 mutant and loss of TP53 on in vitro stem cell properties but distinct effects on DNA binding and gene expression.

The genome variation and developmental transcriptome maps reveal genetic differentiation of skeletal muscle in pigs.

Natural and artificial directional selections have resulted in significantly genetic and phenotypic differences across breeds in domestic animals. However, the molecular regulation of skeletal muscle diversity remains largely unknown. Here, we conducted transcriptome profiling of skeletal muscle across 27 time points, and performed whole-genome re-sequencing in Landrace (lean-type) and Tongcheng (obese-type) pigs. The transcription activity decreased with development, and the high-resolution transcriptome precisely captured the characterizations of skeletal muscle with distinct biological events in four developmental phases: Embryonic, Fetal, Neonatal, and Adult. A divergence in the developmental timing and asynchronous development between the two breeds was observed; Landrace showed a developmental lag and stronger abilities of myoblast proliferation and cell migration, whereas Tongcheng had higher ATP synthase activity in postnatal periods. The miR-24-3p driven network targeting insulin signaling pathway regulated glucose metabolism. Notably, integrated analysis suggested SATB2 and XLOC_036765 contributed to skeletal muscle diversity via regulating the myoblast migration and proliferation, respectively. Overall, our results provide insights into the molecular regulation of skeletal muscle development and diversity in mammals.