A systems biology approach reveals neuronal and muscle developmental defects after chronic exposure to ionising radiation in zebrafish.

Contamination of the environment after the Chernobyl and Fukushima Daiichi nuclear power plant (NPP) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120 hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to IR. Whole-mount RNA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of IR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.

CCAR-1 affects hemidesmosome biogenesis by regulating unc-52/perlecan alternative splicing in the C. elegans epidermis.

Hemidesmosomes are epithelial-specific attachment structures that maintain tissue integrity and resist tension. Despite their importance, how hemidesmosomes are regulated at the post-transcriptional level is poorly understood. Caenorhabditiselegans hemidesmosomes (CeHDs) have a similar structure and composition to their mammalian counterparts, making C. elegans an ideal model for studying hemidesmosomes. Here, we focus on the transcription regulator CCAR-1, identified in a previous genetic screen searching for enhancers of mutations in the conserved hemidesmosome component VAB-10A (known as plectin in mammals). Loss of CCAR-1 function in a vab-10(e698) background results in CeHD disruption and muscle detachment from the epidermis. CCAR-1 regulates CeHD biogenesis, not by controlling the transcription of CeHD-related genes, but by affecting the alternative splicing of unc-52 (known as perlecan or HSPG2 in mammals), the predicted basement extracellular matrix (ECM) ligand of CeHDs. CCAR-1 physically interacts with HRP-2 (hnRNPR in mammals), a splicing factor known to mediate unc-52 alternative splicing to control the proportions of different UNC-52 isoforms and stabilize CeHDs. Our discovery underlines the importance of post-transcriptional regulation in hemidesmosome reorganization. It also uncovers previously unappreciated roles of CCAR-1 in alternative splicing and hemidesmosome biogenesis, shedding new light on the mechanisms through which mammalian CCAR1 functions in tumorigenesis.

General principles of spinal motor circuit development: early contributions from research on avian embryos.

Birds and mammals, both being amniotes, share many common aspects of development. Thus our understanding of how limb-innervating mammalian spinal motor circuits develop was greatly influenced by the use of the avian embryo (chick/quail) to bring about experimental perturbations to identify basic underlying mechanisms. These included embryonic surgery, the application of drugs to influence activity or molecular interactions, and the ability to observe motor behavior and make physiological recordings in intact developing embryos. This article will review some of these contributions, highlighting several areas including the acquisition of motoneuron subtype identity and target selection, as well as the role of spontaneous rhythmic activity in circuit development.

Building functional units of movement-generation and movement-sensation in the embryo.

The musculoskeletal and proprioceptive sensory systems exhibit intricate crosstalk between force generation, force sensation and force transmission, all of which are critical for coordinated animal locomotion. Recent developmental studies of the musculoskeletal and proprioceptive units of the invertebrate Drosophila embryo, have revealed several common molecular and structural principles mediating the formation of each of these systems. These common principles, as well as the differences between the developmental programs of the two systems, are discussed. Interestingly, a molecular pathway triggered by the Neuregulin/Vein ligand-dependent activation of the epidermal growth factor receptor (EGFR) pathway, which upregulates the early growth response (EGR)-like transcription factor Stripe, is utilized not only by the Drosophila muscle-tendon and proprioceptive organ-ectoderm attachment, but also by their vertebrate counterparts. An additional theme that has been observed during the development of the musculoskeletal system in both invertebrates and vertebrates is the functional importance of the extracellular matrix and its adhesion receptors. The contribution of mechanical forces to proper junction formation between muscles and tendons and between the sensory cap/ligament cells and their epidermal attachment cells is discussed. The structural and genetic similarities between the musculoskeletal and the proprioceptive systems offer new perspectives as to their common developmental nature.

Development of cranial muscles in the actinopterygian fish Senegal bichir, Polypterus senegalus Cuvier, 1829.

Polypterus senegalus Cuvier, 1829 is one of the most basal living actinopterygian fish and a member of the Actinopterygii. We analyzed the spatial and temporal pattern of cranial muscle development of P. senegalus using whole-mount immunostaining and serial sectioning. We described the detailed structure of the external gill muscles which divided into dorsal and ventral parts after yolk exhaustion. The pattern of the division is similar to that of urodeles. We suggest that, the external gill muscles of P. senegalus are involved in spreading and folding of the external gill stem and the branches. The fibers of the external gill muscles appear postero-lateral to the auditory capsule. In addition, the facial nerve passes through the external gills. Therefore, the external gill muscles are probably derived from the m. constrictor hyoideus dorsalis. In contrast to previous studies, we described the mm. interhyoideus and hyohyoideus fibers as independent components in the yolk-sac larvae. The m. hyohyoideus fibers appear lateral to the edge of the ventral portion of the external gill muscles, which are probably derived from the m. constrictor hyoideus dorsalis. These findings suggest that the m. hyohyoidues is derived from the m. constrictor hyoideus dorsalis in P. senegalus. In other actinopterygians, the m. hyohyoideus is derived from the m. constrictor hyoideus ventralis; therefore, the homology of the m. hyohyoidues of P. senegalus and other actinopterygians remains unclear. J. Morphol. 278:450-463, 2017. © 2017 Wiley Periodicals, Inc.

DNA Methylation Variation Trends during the Embryonic Development of Chicken.

The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation and gene body hypermethylation.

Analysis of expression profiles of genes involved in F(o)F(1)-ATP synthase biogenesis during perinatal development in rat liver and skeletal muscle.

During the process of intra-uterine mammalian fetal development, the oxygen supply in growing fetus is low. A rapid switch from glycolysis-based metabolism to oxidative phosphorylation (OXPHOS) must proceed during early postnatal adaptation to extra-uterine conditions. Mitochondrial biogenesis and mammalian mitochondrial F(o)F(1)-ATP synthase assembly (complex V, EC 3.6.3.14, ATPase) are complex processes regulated by multiple transcription regulators and assembly factors. Using RNA expression analysis of rat liver and skeletal tissue (Rattus norvegicus, Berkenhout, 1769), we describe the expression profiles of 20 genes involved in mitochondrial maturation and ATP synthase biogenesis in detail between the 16th and 22nd day of gestation and the first 4 days of life. We observed that the most important expression shift occurred in the liver between the 20th and 22nd day of gestation, indicating that the fetus prepares for birth about two days before parturition. The detailed mechanism regulating the perinatal adaptation process is not yet known. Deeper insights in perinatal physiological development will help to assess mitochondrial dysfunction in the broader context of cell metabolism in preterm newborns or neonates with poor adaptation to extra-uterine life.

E2F function in muscle growth is necessary and sufficient for viability in Drosophila.

The E2F transcription factor is a key cell cycle regulator. However, the inactivation of the entire E2F family in Drosophila is permissive throughout most of animal development until pupation when lethality occurs. Here we show that E2F function in the adult skeletal muscle is essential for animal viability since providing E2F function in muscles rescues the lethality of the whole-body E2F-deficient animals. Muscle-specific loss of E2F results in a significant reduction in muscle mass and thinner myofibrils. We demonstrate that E2F is dispensable for proliferation of muscle progenitor cells, but is required during late myogenesis to directly control the expression of a set of muscle-specific genes. Interestingly, E2f1 provides a major contribution to the regulation of myogenic function, while E2f2 appears to be less important. These findings identify a key function of E2F in skeletal muscle required for animal viability, and illustrate how the cell cycle regulator is repurposed in post-mitotic cells.

Cleaved Slit directs embryonic muscles.

The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis.

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

Transmembrane 4 L Six Family Member 5 (TM4SF5)-Mediated Epithelial-Mesenchymal Transition in Liver Diseases.

The membrane protein TM4SF5, a member of the transmembrane 4L six family, forms a tetraspanin-enriched microdomain (TEM) on the cell surface, where many different membrane proteins and receptors form a massive protein-protein complex to regulate cellular functions including transdifferentiation, migration, and invasion. We recently reported that TM4SF5 causes epithelial-mesenchymal transition (EMT), eventually contributing to aberrant multilayer cellular growth, drug resistance, enhanced migration, invasion, its circulation in the blood, tumor initiation for successful metastasis, and muscle development in zebrafish. In this review, I summarize the information on the role of TM4SF5 in EMT-related functions at TM4SF5-enriched microdomain (T5EM) on cell surface, where proteins such as TM4SF5, CD151, CD44, integrins, and epidermal growth factor receptor (EGFR) can form numerous protein complexes. TM4SF5-mediated EMT contributes to diverse cellular functions, leading to fibrotic phenotypes and initiating and maintaining tumors in primary and/or metastatic regions, in addition to its role in muscle development in zebrafish. Anti-TM4SF5 strategies for addressing the protein networks can lead to regulation of the fibrotic, tumorigenic, and tumor-maintaining functions of TM4SF5-positive hepatic cells. This review is for us to (re)consider the antifibrotic or antitumorigenic (i.e., anti-EMT-related diseases) strategies of dealing with protein networks that would be involved in cross-talks to regulate various cellular functions during TM4SF5-dependent progression from fibrotic to cancerous hepatic cells.

Kank Is an EB1 interacting protein that localises to muscle-tendon attachment sites in Drosophila.

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells.

The PDZ-containing unconventional myosin XVIIIA regulates embryonic muscle integrity in zebrafish.

Myosin XVIIIA, or MYO18A, is a unique PDZ domain-containing unconventional myosin and is evolutionarily conserved from Drosophila to vertebrates. Although there is evidence indicating its expression in the somites, whether it regulates muscle function remains unclear. We show that the two zebrafish myo18a genes (myo18aa and myo18ab) are predominantly expressed at somite borders during early developmental stages. Knockdown of these genes or overexpression of the MYO18A PDZ domain disrupts myofiber integrity, induces myofiber lesions, and compromises the localization of dystrophin, α-dystroglycan (α-DG) and laminin at the myotome boundaries. Cell transplantation experiments indicate that myo18a morphant myoblasts fail to form elongated myofibers in the myotomes of wild-type embryos, which can be rescued by the full-length MYO18A protein. These results suggest that MYO18A likely functions in the adhesion process that maintains the stable attachment of myofibers to ECM (extracellular matrix) and muscle integrity during early development.

Extensive nonmuscle expression and epithelial apicobasal localization of the Drosophila ALP/Enigma family protein, Zasp52.

This study describes the broad tissue distribution and subcellular localization of Drosophila Zasp52, which is related to the large family of ALP (α-actinin associated protein)/Enigma PDLIM (PDZ and LIM domain) proteins of vertebrates. Results demonstrate that ZCL423 is a protein trap insertion in the Zasp52 locus tagging multiple endogenous splice isoforms with GFP. While Zasp52 has been previously characterized in muscle tissues primarily, visualization of GFP fluorescence in Zasp52 protein trap lines revealed expression in many nonmuscle tissues including the central nervous system, secretory glands, and epithelial tissues constituting the embryonic epidermis, the somatic follicle cell layer encapsulating the germline during oogenesis, and imaginal disc precursors to the adult body. In epithelial cells, Zasp52 typically accumulated basally, adjacent to integrin adhesion sites, and apically along adherens junctions, particularly enriched near junctional vertices of multicellular interfaces. Also Zasp52 showed polarized accumulation at the leading edge of migrating cell populations and morphogenetic boundaries similarly enriched for myosin. As such, Zasp52 GFP protein traps may be useful molecular markers for dynamic epithelial rearrangements. Moreover, the pattern of Zasp52 expression within nonmuscle tissues reveals potential functional roles in cell-cell and cell-matrix adhesion, specifically at sites of increased actomyosin contractile tension. In these contexts, the investigation of Zasp52 may provide insights into the functions of numerous PDLIM proteins of the metazoan lineages.

An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight.

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.

Translocating myonuclei have distinct leading and lagging edges that require kinesin and dynein.

Nuclei are precisely positioned within all cells, and mispositioned nuclei are a hallmark of many muscle diseases. Myonuclear positioning is dependent on Kinesin and Dynein, but interactions between these motor proteins and their mechanisms of action are unclear. We find that in developing Drosophila muscles, Dynein and Kinesin work together to move nuclei in a single direction by two separate mechanisms that are spatially segregated. First, the two motors work together in a sequential pathway that acts from the cell cortex at the muscle poles. This mechanism requires Kinesin-dependent localization of Dynein to cell cortex near the muscle pole. From this location Dynein can pull microtubule minus-ends and the attached myonuclei toward the muscle pole. Second, the motors exert forces directly on individual nuclei independently of the cortical pathway. However, the activities of the two motors on the nucleus are polarized relative to the direction of myonuclear translocation: Kinesin acts at the leading edge of the nucleus, whereas Dynein acts at the lagging edge of the nucleus. Consistent with the activities of Kinesin and Dynein being polarized on the nucleus, nuclei rarely change direction, and those that do, reorient to maintain the same leading edge. Conversely, nuclei in both Kinesin and Dynein mutant embryos change direction more often and do not maintain the same leading edge when changing directions. These data implicate Kinesin and Dynein in two distinct and independently regulated mechanisms of moving myonuclei, which together maximize the ability of myonuclei to achieve their proper localizations within the constraints imposed by embryonic development.

A chimeric RNA characteristic of rhabdomyosarcoma in normal myogenesis process.

UNLABELLED: Gene fusions and their chimeric products are common features of neoplasia. Given that many cancers arise by the dysregulated recapitulation of processes in normal development, we hypothesized that comparable chimeric gene products may exist in normal cells. Here, we show that a chimeric RNA, PAX3-FOXO1, identical to that found in alveolar rhabdomyosarcoma, is transiently present in cells undergoing differentiation from pluripotent cells into skeletal muscle. Unlike cells of rhabdomyosarcoma, these cells do not seem to harbor the t(2;13) chromosomal translocation. Importantly, both PAX3-FOXO1 RNA and protein could be detected in the samples of normal fetal muscle. Overexpression of the chimera led to continuous expression of MYOD and MYOG-two myogenic markers that are overexpressed in rhabdomyosarcoma cells. Our results are consistent with a developmental role of a specific chimeric RNA generated in normal cells without the corresponding chromosomal rearrangement at the DNA level seen in neoplastic cells presumably of the same lineage. SIGNIFICANCE: A chimeric fusion RNA, PAX3-FOXO1, associated with alveolar rhabdomyosarcoma, is also present in normal non-cancer cells and tissues. Its transient expression nature and the absence of t(2;13) chromosomal translocation are consistent with a posttranscriptional mechanism. When constantly expressed, PAX3-FOXO1 interfered with the muscle differentiation process, which presumably contributes to tumorigenesis.

The polycomb group protein ring1b/rnf2 is specifically required for craniofacial development.

Polycomb group (PcG) genes are chromatin modifiers that mediate epigenetic silencing of target genes. PcG-mediated epigenetic silencing is implicated in embryonic development, stem cell plasticity, cell fate maintenance, cellular differentiation and cancer. However, analysis of the roles of PcG proteins in maintaining differentiation programs during vertebrate embryogenesis has been hampered due to the early embryonic lethality of several PcG knock-outs in the mouse. Here, we show that zebrafish Ring1b/Rnf2, the single E3 ubiquitin ligase in the Polycomb Repressive Complex 1, critically regulates the developmental program of craniofacial cell lineages. Zebrafish ring1b mutants display a severe craniofacial phenotype, which includes an almost complete absence of all cranial cartilage, bone and musculature. We show that Cranial Neural Crest (CNC)-derived cartilage precursors migrate correctly into the pharyngeal arches, but fail to differentiate into chondrocytes. This phenotype is specific for cartilage precursors, since other neural crest-derived cell lineages, including glia, neurons and chromatophores, are formed normally in ring1b mutants. Our results therefore reveal a critical and specific role for Ring1b in promoting the differentiation of cranial neural crest cells into chondrocytes. The molecular mechanisms underlying the pathogenesis of craniofacial abnormalities, which are among the most common genetic birth defects in humans, remain poorly understood. The zebrafish ring1b mutant provides a molecular model for investigating these mechanisms and may lead to the discovery of new treatments or preventions of craniofacial abnormalities.

Noncanonical transforming growth factor ß (TGFß) signaling in cranial neural crest cells causes tongue muscle developmental defects.

Microglossia is a congenital birth defect in humans and adversely impacts quality of life. In vertebrates, tongue muscle derives from the cranial mesoderm, whereas tendons and connective tissues in the craniofacial region originate from cranial neural crest (CNC) cells. Loss of transforming growth factor ß (TGFß) type II receptor in CNC cells in mice (Tgfbr2(fl/fl);Wnt1-Cre) causes microglossia due to a failure of cell-cell communication between cranial mesoderm and CNC cells during tongue development. However, it is still unclear how TGFß signaling in CNC cells regulates the fate of mesoderm-derived myoblasts during tongue development. Here we show that activation of the cytoplasmic and nuclear tyrosine kinase 1 (ABL1) cascade in Tgfbr2(fl/fl);Wnt1-Cre mice results in a failure of CNC-derived cell differentiation followed by a disruption of TGFß-mediated induction of growth factors and reduction of myogenic cell proliferation and differentiation activities. Among the affected growth factors, the addition of fibroblast growth factor 4 (FGF4) and neutralizing antibody for follistatin (FST; an antagonist of bone morphogenetic protein (BMP)) could most efficiently restore cell proliferation, differentiation, and organization of muscle cells in the tongue of Tgfbr2(fl/fl);Wnt1-Cre mice. Thus, our data indicate that CNC-derived fibroblasts regulate the fate of mesoderm-derived myoblasts through TGFß-mediated regulation of FGF and BMP signaling during tongue development.

Aberrant DNA methylation of imprinted loci in human spontaneous abortions after assisted reproduction techniques and natural conception.

STUDY QUESTION: Do assisted reproduction techniques (ARTs) affect DNA methylation of imprinted genes and does aberrant methylation of imprinted genes account for the incidence of human spontaneous abortion (SA)? SUMMARY ANSWER: Our results show that imprinting errors of imprinted genes may contribute to human SA, and the occurrence of aberrant methylation of imprinted genes in ART pregnancies was comparable with that in natural pregnancies. WHAT IS KNOWN ALREADY: Animal data and human studies demonstrated that in vitro culture of embryos can cause methylation defects in individual genes, which might affect subsequent embryonic development and contribute to SA. However, our previous studies showed an abnormal methylation pattern of PEG1 in human aborted chrionic villus samples (CVS) but an increased occurrence of aberrant methylation in CVS from ART-derived pregnancies was not observed. STUDY DESIGN, SIZE AND DURATION: CVS were collected from women who underwent abortion procedures in the Department of Gynecology and Obstetrics in Nanfang Hospital from May 2008 to July 2011. Muscle samples (MS) were obtained from aborted fetuses and stillbirths. The samples were divided into four experimental groups: (A) SA/stillbirth after ART (n = 75), (B) multi-fetal reduction after ART (n = 73), (C) SA/stillbirth of natural pregnancies (n = 90) and (D) induced abortion (IA) of natural pregnancies (n = 82). PARTICIPANTS/MATERIALS, SETTING AND METHODS: The mean ± SD age of patients was 31.0 ± 4.1 (range: 18-45 years). The DNA methylation patterns of one paternally methylated (H19) and two maternally methylated (LIT1 and SNRPN) genes were analyzed in CVS and MS using pyrosequencing and bisulfite sequencing PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Clear hypo-methylation (<10%) or hyper-methylation (>90%) were not detected in LIT1 and SNRPN but two regions of hyper-methylation (91.7 and 91.4%) were observed in H19. The mean percentage of methylation in the SA samples (groups A and C) was higher than that in the IA samples (groups B and D; P<0.05). Box plot analyses showed that in the 165 SA samples, methylation values for 40/495 (8.1%) differentially methylated regions of the three genes represented outliers. The incidence of outlier was highest for LIT1 (13.3%, 22/165). In contrast, no outliers were found in the 155 IA samples. The receiver operating characteristic curve analyses showed a positive correlation between percentage methylation of all three genes and incidence of SA (P<0.05). In addition, the conception modes (natural versus ART) and the fertilization methods used in ART (IVF and ICSI) did not affect the methylation patterns of the imprinted genes. No increase in the rate of abnormal methylation was found in the ART samples. LIMITATIONS AND REASONS FOR CAUTION: The studied loci represent only a small fraction of developmentally important genes. Further studies are needed to evaluate changes in the expression and the methylation status of other genes that may lead to SA. WIDER IMPLICATIONS OF THE FINDINGS: The findings provide new insights into the etiology of human SA. The possibility that the abnormal methylation seen is a consequence of the defect that led to the SA cannot be excluded. STUDY FUNDING/COMPETING INTEREST(S): None of the authors has any competing interest. This study was supported by National Natural Science Foundation of China (81170574), The National Key Basic Research Development Plan of China (973 Program) (2007CB948104), Comprehensive strategic sciences cooperation projects of Guangdong Province and Chinese Academy (04020416) and Guangzhou Science and Technology Program key projects (11C22120737).