A single-cell atlas of pig gastrulation as a resource for comparative embryology.

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

Zrsr2 and functional U12-dependent spliceosome are necessary for follicular development.

ZRSR2 is a splicing factor involved in recognition of 3'-intron splice sites that is frequently mutated in myeloid malignancies and several tumors; however, the role of mutations of Zrsr2 in other tissues has not been analyzed. To explore the biological role of ZRSR2, we generated three Zrsr2 mutant mouse lines. All Zrsr2 mutant lines exhibited blood cell anomalies, and in two lines, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2 mu secondary follicles showed aberrations in gene expression and showed altered alternative splicing (AS) events involving enrichment of U12-type intron retention (IR), supporting the functional Zrsr2 action in minor spliceosomes. IR events were preferentially associated with centriole replication, protein phosphorylation, and DNA damage checkpoint. Notably, we found alterations in AS events of 50 meiotic genes. These results indicate that ZRSR2 mutations alter splicing mainly in U12-type introns, which may affect peripheral blood cells, and impede oogenesis and female fertility.

Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species.

Despite four decades of effort, robust propagation of pluripotent stem cells from livestock animals remains challenging. The requirements for self-renewal are unclear and the relationship of cultured stem cells to pluripotent cells resident in the embryo uncertain. Here, we avoided using feeder cells or serum factors to provide a defined culture microenvironment. We show that the combination of activin A, fibroblast growth factor and the Wnt inhibitor XAV939 (AFX) supports establishment and continuous expansion of pluripotent stem cell lines from porcine, ovine and bovine embryos. Germ layer differentiation was evident in teratomas and readily induced in vitro. Global transcriptome analyses highlighted commonality in transcription factor expression across the three species, while global comparison with porcine embryo stages showed proximity to bilaminar disc epiblast. Clonal genetic manipulation and gene targeting were exemplified in porcine stem cells. We further demonstrated that genetically modified AFX stem cells gave rise to cloned porcine foetuses by nuclear transfer. In summary, for major livestock mammals, pluripotent stem cells related to the formative embryonic disc are reliably established using a common and defined signalling environment. This article has an associated 'The people behind the papers' interview.

Role of Alternative Splicing in Sex Determination in Vertebrates.

During the process of sex determination, a germ-cell-containing undifferentiated gonad is converted into either a male or a female reproductive organ. Both the composition of sex chromosomes and the environment determine sex in vertebrates. It is assumed that transcription level regulation drives this cascade of mechanisms; however, transcription factors can alter gene expression beyond transcription initiation by controlling pre-mRNA splicing and thereby mRNA isoform production. Using the key time window in sex determination and gonad development in mice, it has been reported that new non-transcriptional events, such as alternative splicing, could play a key role in sex determination in mammals. We know the role of key regulatory factors, like WT1(+/-KTS) or FGFR2(b/c) in pre-mRNA splicing and sex determination, indicating that important steps in the vertebrate sex determination process probably operate at a post-transcriptional level. Here, we discuss the role of pre-mRNA splicing regulators in sex determination in vertebrates, focusing on the new RNA-seq data reported from mice fetal gonadal transcriptome.

MicroRNAs in amniotic fluid and maternal blood plasma associated with sex determination and early gonad differentiation in cattle†.

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo-maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

Genome-wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline.

BACKGROUND: Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profiles of porcine germ cells during the time course of epigenetic reprogramming. RESULTS: Sows were artificially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic-activated cell sorting using an anti-SSEA-1 antibody, and recovered germ cells were subjected to whole-genome bisulphite sequencing. Methylation levels were quantified using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re-methylation. We identified a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transposable elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty-one percent of the introns located in these PMRs were found to be the first introns of a gene, suggesting their regulatory role in the expression of these genes. Interestingly, most of the identified PMRs were demethylated at the blastocyst stage. CONCLUSIONS: Our findings indicate that methylation reprogramming in pig germ cells follows the general dynamics shown in mice and human, unveiling genomic elements that behave differently between male and female germ cells.

Gene expression profiles of bovine genital ridges during sex determination and early differentiation of the gonads†.

Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.

Differential isoform expression and alternative splicing in sex determination in mice.

BACKGROUND: Alternative splicing (AS) may play an important role in gonadal sex determination (GSD) in mammals. The present study was designed to identify differentially expressed isoforms and AS modifications accompanying GSD in mice. RESULTS: Using deep RNA-sequencing, we performed a transcriptional analysis of XX and XY gonads during sex determination on embryonic days 11 (E11) and 12 (E12). Analysis of differentially expressed genes (DEG) identified hundreds of genes related to GSD and early sex differentiation that may represent good candidates for sex reversal. Expression at time point E11 in males was significantly enriched in RNA splicing and mRNA processing Gene Ontology terms. Differentially expressed isoform analysis identified hundreds of specific isoforms related to GSD, many of which showed no differences in the DEG analysis. Hundreds of AS events were identified as modified at E11 and E12. Female E11 gonads featured sex-biased upregulation of intron retention (in genes related to regulation of transcription, protein phosphorylation, protein transport and mRNA splicing) and exon skipping (in genes related to chromatin repression) suggesting AS as a post-transcription mechanism that controls sex determination of the bipotential fetal gonad. CONCLUSION: Our data suggests an important role of splicing regulatory mechanisms for sex determination in mice.

Embryo responses to stress induced by assisted reproductive technologies.

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long-term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.