Stages Of Postnatal Development Research Articles

Intrauterine exposure to chronic hypoxia in the rat leads to progressive diastolic function and increased aortic stiffness from early postnatal developmental stages.

AimWe sought to explore whether fetal hypoxia exposure, an insult of placental insufficiency, is associated with left ventricular dysfunction and increased aortic stiffness at early postnatal ages.MethodsPregnant Sprague Dawley rats were exposed to hypoxic conditions (11.5% FiO2) from embryonic day E15‐21 or normoxic conditions (controls). After delivery, left ventricular function and aortic pulse wave velocity (measure of aortic stiffness) were assessed longitudinally by echocardiography from day 1 through week 8. A mixed ANOVA with repeated measures was performed to compare findings between groups across time. Myocardial hematoxylin and eosin and picro‐sirius staining were performed to evaluate myocyte nuclear shape and collagen fiber characteristics, respectively.ResultsSystolic function parameters transiently increased following hypoxia exposure primarily at week 2 (p < .008). In contrast, diastolic dysfunction progressed following fetal hypoxia exposure beginning weeks 1–2 with lower early inflow Doppler velocities, and less of an increase in early to late inflow velocity ratios and annular and septal E’/A’ tissue velocities compared to controls (p < .008). As further evidence of altered diastolic function, isovolumetric relaxation time was significantly shorter relative to the cardiac cycle following hypoxia exposure from week 1 onward (p < .008). Aortic stiffness was greater following hypoxia from day 1 through week 8 (p < .008, except week 4). Hypoxia exposure was also associated with altered nuclear shape at week 2 and increased collagen fiber thickness at week 4.ConclusionChronic fetal hypoxia is associated with progressive LV diastolic dysfunction, which corresponds with changes in nuclear shape and collagen fiber thickness, and increased aortic stiffness from early postnatal stages.

Expression and localization of endogenous phospholipase Cβ3 confined to basal cells in situ of immature ducts and adult excretory ducts of submandibular gland of mice

Our previous study demonstrated that, different from the parotid and sublingual glands, the submandibular glands of adult mice did not show an immunoblot band for PLCβ3 which is critical in the secretion mechanism by muscarinic cholinergic signaling. Therefore, the submandibular glands of mice at various stages of postnatal development were examined for this enzyme molecule in immunoblot and immunohistochemistry. In immunoblot, a weak band for PLCβ3-expression was detected only at early postnatal stages. In immunohistochemistry, PLCβ3-immunoreactivity was distinctly found in most basally located cells of immature ducts, while the immunoreactivity was weakly seen in terminal tubule cells without significant immunoreactivity in adjacent acinar cells. In contrast, the immunoreactivity was distinctly found in some basal cells of adult excretory ducts, and it was ultrastructurally localized densely in close association with bundles of tonofilaments in the cells. The present finding suggests the possibility that Ca2+ signaling governed by phospholipase Cβ3 is involved in the differentiation of ductal basal cells into apical cells through control of keratin molecule(s) in the cells.

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF MICROGLIA IN THE POSTNATAL RAT STRIATUM

The aim of this study was to evaluate the morphological and functional characteristics of microglial cells in the rat striatum at different stages of postnatal development. Using immunocytochemical methods and fluorescent microscopy, brain sections were analysed at each of the following postnatal ages: day 7 (n = 4), day 14 (n = 3), 4 to 6 months (n = 3). Different morphological and functional types of microglial cells were identified and characterized in details. It was noted that microglia activated during normal brain development do not contain vimentin, which is characteristic of stroke-activated microglia.

Hippocampal Lnx1\u2013NMDAR multiprotein complex mediates initial social memory

Social interaction and communication are evolutionary conserved behaviours that are developed in mammals to establish partner cognition. Deficit in sociability has been represented in human patients and animal models of neurodevelopmental disorders, which are connected with genetic variants of synaptic glutamate receptors and associated PDZ-binding proteins. However, it remains elusive how these key proteins are specialized in the cellular level for the initial social behaviour during postnatal developmental stage. Here we identify a hippocampal CA3 specifically expressed PDZ scaffold protein Lnx1 required for initial social behaviour. Through gene targeting we find that Lnx1 deficiency led to a hippocampal subregional disorder in neuronal activity and social memory impairments for partner discrimination observed in juvenile mice which also show cognitive defects in adult stage. We further demonstrate that Lnx1 deletion causes NMDA receptor (NMDAR) hypofunction and this is attributable to decreased GluN2B expression in PSD compartment and disruption of the Lnx1–NMDAR–EphB2 complex. Specific restoration of Lnx1 or EphB2 protein in the CA3 area of Lnx1−/− mice rescues the defective synaptic function and social memory. These findings thus reveal crucial roles of postsynaptic NMDAR multiprotein complex that regulates the formation of initial social memory during the adolescent period.

ALK4 coordinates extracellular and intrinsic signals to regulate development of cortical somatostatin interneurons.

Although the role of transcription factors in fate specification of cortical interneurons is well established, how these interact with extracellular signals to regulate interneuron development is poorly understood. Here we show that the activin receptor ALK4 is a key regulator of the specification of somatostatin interneurons. Mice lacking ALK4 in GABAergic neurons of the medial ganglionic eminence (MGE) showed marked deficits in distinct subpopulations of somatostatin interneurons from early postnatal stages of cortical development. Specific losses were observed among distinct subtypes of somatostatin+/Reelin+ double-positive cells, including Hpse+ layer IV cells targeting parvalbumin+ interneurons, leading to quantitative alterations in the inhibitory circuitry of this layer. Activin-mediated ALK4 signaling in MGE cells induced interaction of Smad2 with SATB1, a transcription factor critical for somatostatin interneuron development, and promoted SATB1 nuclear translocation and repositioning within the somatostatin gene promoter. These results indicate that intrinsic transcriptional programs interact with extracellular signals present in the environment of MGE cells to regulate cortical interneuron specification.

Selective demethylation of two CpG sites causes postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum

BackgroundProgrammed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as d-serine and d-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of d-amino acids are found. We recently demonstrated that Ddo, the d-aspartate degradation gene, is actively demethylated to ultimately reduce d-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate d-ser levels during brain development has been poorly investigated to date.MethodsWe performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based d-amino acids level analyses of genes controlling the mammalian brain levels of d-serine and d-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis.ResultsWe report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar d-serine a few days after mouse birth. High amount of 5′-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process.ConclusionThe present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

Rat caval vein myocardium undergoes changes in conduction characteristics during postnatal ontogenesis.

The electrophysiological properties of the superior vena cava (SVC) myocardium, which is considered a minor source of atrial arrhythmias, were studied in this study during postnatal development. Conduction properties were investigated in spontaneously active and electrically paced SVC preparations obtained from 7-60-day-old male Wistar rats using optical mapping and microelectrode techniques. The presence of high-conductance connexin 43 (Cx43) was evaluated in SVC cross-sections using immunofluorescence. It was found that SVC myocardium is excitable, electrically coupled with the atrial tissue, and conducts excitation waves at all stages of postnatal development. However, the conduction velocity (CV) of excitation and action potential (AP) upstroke velocity in SVC were significantly lower in neonatal than in adult animals and increased with postnatal maturation. Connexins Cx43 were identified in both neonatal and adult rat SVC myocardium; however, the abundance of Cx43 was significantly less in neonates. The gap junction uncoupler octanol affected conduction more profound in the neonatal than in adult SVC. We demonstrated for the first time that the conduction characteristics of SVC myocardium change from a slow-conduction (nodal) to a high-conduction (working) phenotype during postnatal ontogenesis. An age-related CV increase may occur due to changes of AP characteristics, electrical coupling, and Cx43 presence in SVC cardiomyocyte membranes. Observed changes may contribute to the low proarrhythmicity of adult caval vein cardiac tissue, while pre- or postnatal developmental abnormalities that delay the establishment of the working conduction phenotype may facilitate SVC proarrhythmia.

Inter- and intraspecific diversity of ontogeny and fecundity patterns in relation to reproductive strategy choice in Myomorpha (Rodentia: Calomyscidae, Cricetidae, Muridae)

Studying the life history of various organisms is essential for ecological and evolutionary inferences. However, publications regarding models of evolutionary shifts to either r- or K-reproductive strategies are scarce. Here, the combined original and previously published data on reproduction and development of Goodwin’s brush-tailed mouse (Calomyscus elburzensis), golden hamster (Mesocricetus auratus), and house mouse (Mus musculus) were compared. An adult male and female in estrous state (from each species) were coupled with each other for a night and embryos were harvested at embryonic days (E) 10–17 from pregnant females. The mean gestation period in Goodwin’s brush-tailed mouse was 31.5 ± 2.1 days; growth was rapid at the first half of this period and after that it was reduced by half. During the postnatal period, growth was rapid up to weaning, but after day 35, the rate of growth decreased abruptly. Growth rate of head and body length was very rapid prior to weaning (PN35) in comparison with weight but the rates of the two characters were inversed following weaning. Totally, weight gain was more dynamic in rate, as compared with head and body length, up to adultness. For golden hamster and house mouse, gestation period was 15.6 ± 0.8 and 20.8 ± 0.8 days, respectively. Growth was slow during approximately the first half of the gestation period in both species but increased during the second half. During postnatal period in golden hamster, growth rate of head and body length was rapid both before and after weaning as compared with that of weight. However, head and body length showed a stable rate of growth before and after weaning (around PN15), but for body weight, the growth rate was slightly faster after weaning. Similarly, changes in head and body length in house mouse were rapid both before and after weaning (PN16) as compared with that of weight. Since approximately two-fold increases were observed in the growth rates of weight and head and body length in the second stage of postnatal development (PN16-adult) compared with the rates during the first stage (PN0-PN16), both showed dynamic changes during the postnatal development in this species. Typically, r-strategic house mouse appeared to be more similar in early morphological development to comparatively K-strategic Goodwin’s brush-tailed mouse, than to comparatively r-strategic golden hamster. Nevertheless, growth rate and pattern were different in the three species after birth. Hence, results showed that peculiarities of association between an ontogenetic pattern and a tendency in reproductive strategy can differ in diverse groups of myomorph rodents.

Maternal Protein Restriction Modulates Angiogenesis and AQP9 Expression Leading to a Delay in Postnatal Epididymal Development in Rat.

The maternal nutritional status is essential to the health and well-being of the fetus. Maternal protein restriction during the perinatal stage causes sperm alterations in the offspring that are associated with epididymal dysfunctions. Vascular endothelial growth factor (VEGF) and its receptor, VEGFr-2, as well as aquaporins (AQPs) are important regulators of angiogenesis and the epididymal microenvironment and are associated with male fertility. We investigated the effects of maternal protein restriction on epididymal angiogenesis and AQP expression in the early stages of postnatal epididymal development. Pregnant rats were divided into two experimental groups that received either a normoprotein (17% protein) or low-protein diet (6% protein) during gestation and lactation. At postnatal day (PND)7 and PND14, male offspring were euthanized, the epididymides were subjected to morphometric and microvascular density analyses and to VEGF-A, VEGF-r2, AQP1 and AQP9 expression analyses. The maternal low-protein diet decreased AQP9 and VEGFr-2 expression, decreased epididymal microvascularity and altered the morphometric features of the epididymal epithelium; no changes in AQP1 expression were observed at the beginning of postnatal epididymal development. Maternal protein restriction alters microvascularization and affects molecules involved in the epidydimal microenvironment, resulting in morphometric alterations related to a delay in the beginning of epididymis postnatal development.

Investigation of Postnatal Craniofacial Bone Development with Tissue Clearing-Based Three-Dimensional Imaging.

Traditional two-dimensional histological sections and microcomputed tomography remain to be the major tools for studying craniofacial bones despite the complicated spatial organization of craniofacial organs. Recently, our laboratory developed the Poly(Ethylene Glycol) Associated Solvent System (PEGASOS) tissue clearing method, which can efficiently render hard tissues, including bones and teeth fully transparent without losing endogenous fluorescent signals. Complete tissue transparency enables us to acquire three-dimensional (3D) images of craniofacial bone vasculature, osteogenesis utilizing various labeling strategies, thus to investigate the spatial relationship among different tissues during postnatal craniofacial development. We found out that during the early stage of postnatal development, craniofacial osteogenesis occurs throughout the entire craniofacial bones, including the periosteum, dura, bone marrow, and suture. After 3-4 weeks, craniofacial osteogenesis is gradually restricted to the suture region and remaining bone marrow space. Similarly, craniofacial bone vasculature gradually restricts to the suture region. Osteogenesis is spatially associated with vasculature during the entire postnatal development. Importantly, we demonstrated that in adult calvarial bones, Gli1+ mesenchymal stem cells were also spatially associated with the vasculature. These findings indicate that craniofacial bones share similar osteogenesis mechanism as the long bone despite their distinct osteogenic mechanisms. In addition, the PEGASOS tissue clearing method-based 3D imaging technique is a useful new tool for craniofacial research.

Molecular profiling of failed endochondral ossification in mucopolysaccharidosis VII.

Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient activity of β-glucuronidase, leading to progressive accumulation of incompletely degraded heparan, dermatan, and chondroitin sulfate glycosaminoglycans (GAGs). Patients with MPS VII exhibit progressive skeletal deformity including kyphoscoliosis and joint dysplasia, which decrease quality of life and increase mortality. Previously, using the naturally-occurring canine model, we demonstrated that one of the earliest skeletal abnormalities to manifest in MPS VII is failed initiation of secondary ossification in vertebrae and long bones at the requisite postnatal developmental stage. The objective of this study was to obtain global insights into the molecular mechanisms underlying this failed initiation of secondary ossification. Epiphyseal tissue was isolated postmortem from the vertebrae of control and MPS VII-affected dogs at 9 and 14 days-of-age (n = 5 for each group). Differences in global gene expression across this developmental window for both cohorts were measured using whole-transcriptome sequencing (RNA-Seq). Principal Component Analysis revealed clustering of samples within each group, indicating clear effects of both age and disease state. At 9 days-of-age, 1375 genes were significantly differentially expressed between MPS VII and control, and by 14 days-of-age, this increased to 4719 genes. A targeted analysis focused on signaling pathways important in the regulation of endochondral ossification was performed, and a subset of gene expression differences were validated using qPCR. Osteoactivin (GPNMB) was the top upregulated gene in MPS VII at both ages. In control samples, temporal changes in gene expression from 9 to 14 days-of-age were consistent with chondrocyte maturation, cartilage resorption, and osteogenesis. In MPS VII samples, however, elements of key osteogenic pathways such as Wnt/β-catenin and BMP signaling were not upregulated during this same developmental window suggesting that important bone formation pathways are not activated. In conclusion, this study represents an important step towards identifying therapeutic targets and biomarkers for bone disease in MPS VII patients during postnatal growth.

Dynamic changes in DNA methylation during embryonic and postnatal development of an altricial wild bird.

DNA methylation could shape phenotypic responses to environmental cues and underlie developmental plasticity. Environmentally induced changes in DNA methylation during development can give rise to stable phenotypic traits and thus affect fitness. In the laboratory, it has been shown that the vertebrate methylome undergoes dynamic reprogramming during development, creating a critical window for environmentally induced epigenetic modifications. Studies of DNA methylation in the wild are lacking, yet are essential for understanding how genes and the environment interact to affect phenotypic development and ultimately fitness. Furthermore, our knowledge of the establishment of methylation patterns during development in birds is limited. We quantified genome‐wide DNA methylation at various stages of embryonic and postnatal development in an altricial passerine bird, the great tit Parus major. While, there was no change in global DNA methylation in embryonic tissue during the second half of embryonic development, a twofold increase in DNA methylation in blood occurred between 6 and 15 days posthatch. Though not directly comparable, DNA methylation levels were higher in the blood of nestlings compared with embryonic tissue at any stage of prenatal development. This provides the first evidence that DNA methylation undergoes global change during development in a wild bird, supporting the hypothesis that methylation mediates phenotypic development. Furthermore, the plasticity of DNA methylation demonstrated during late postnatal development, in the present study, suggests a wide window during which DNA methylation could be sensitive to environmental influences. This is particularly important for our understanding of the mechanisms by which early‐life conditions influence later‐life performance. While, we found no evidence for differences in genome‐wide methylation in relation to habitat of origin, environmental variation is likely to be an important driver of variation in methylation at specific loci.

115 Negative impacts of early weaning on lifelong gut health in the pig

Abstract Of all the stressors a pig encounters in production, early weaning is the most stressful. Under current early weaning practices, the young pig is exposed to multiple concurrent stressors including maternal and littermate separation, transportation, changes in social hierarchy, and a change in environment and food source. Moreover, the stress of weaning causes significant intestinal barrier loss and inflammation during critical stages of postnatal gut development. The immediate, negative impacts of early weaning on gut health and performance are well-known, but the long-term consequences are poorly understand. However, recent evidence from field investigations and basic science research have demonstrated that early weaning has negative consequences on performance and health from wean-to-finish. A fundamental understanding of the biological mechanisms by which current early weaning practices alter gut develop and predispose pigs to performance inefficiencies and disease throughout the production lifespan is lacking. This represents a significant gap in knowledge for the U.S. swine industry, as biological targets for interventions and management are currently lacking. This presentation will provide an overview of postnatal gut development in the pig and how early life stressors, such as early weaning, alter the normal development of critical gut health parameters, including intestinal epithelial barrier function, nutrient transport and immune function, and the link to performance and disease resistance throughout the production lifespan.

Structural Changes in the Elements of the Blood-Brain Barrier of the Hippocampus in Rats after Exposure to Perinatal Hypoxia and the Potential for Their Pharmacological Correction

Objectives. To study the dynamics of structural changes in elements of the blood-brain barrier of the hippocampus during the early postnatal period and subsequent ontogeny after exposure to perinatal hypoxia and to investigate the potential for pharmacological correction of these changes. Materials and methods. Experiments were performed on Wistar laboratory rats with a model of the encephalopathy of premature human infants using electron microscopy. Results. Perinatal hypoxia was followed by detection of structural damage to capillary walls: there were increases in the size of endotheliocytes, increases in the numbers of cytoplasmic processes, changes in the diameter of capillary lumens, and delays in the formation of the basal membrane. A marked endothelium-protecting effect was seen with the formulation Salifen at all study time points. Salifen was found to decrease the intensity of the reactions of elements of the blood-brain barrier to the harmful actions of perinatal hypoxia. Conclusions. Perinatal hypoxia has marked damaging effects on all elements of the blood-brain barrier of the hippocampus, the induced lesions being detected at both the early stages of postnatal development and in animals reaching adulthood. Use of Salifen immediately after exposure to perinatal hypoxia eliminated much of the structural damage to endotheliocytes and the basal membrane by the juvenile period. This study provides grounds for believing that the use of Salifen in ischemic brain injury in neonates has potential and our series of further preclinical studies will be continued.

An aromatic substance, eugenol induces distinct depressant effects on respiratory activity in different postnatal developmental stages of the rat

Eugenol modulates neuronal activity through actions on voltage-gated ionic channels and/or transient receptor potential channels. We previously suggested that eugenol inhibited cellular (and/or network) mechanisms essential for the maintenance of the respiratory burst activity in a brainstem-spinal cord preparation from newborn rat (postnatal day 0–3). Study of the distinct effects of eugenol in neonatal and later developmental stage rats may offer new information about postnatal developmental changes of respiratory neuron networks. In the present study, therefore, we compared effects of eugenol in an in vitro newborn rat preparation with those in an arterially perfused in situ preparation from juvenile rat (postnatal day 12–15). In the former preparation, application of 1 mM eugenol decreased respiratory rate and inspiratory burst duration. In contrast, in the latter preparation, 1 mM eugenol induced a gradual decrease in the amplitude of integrated phrenic nerve activity. Phrenic nerve activity gradually recovered at 25–30 min after washout with a burst duration similar to control values. We hypothesized that the depressant effects of eugenol were caused by inhibition of cell excitability in the neonatal rat in vitro preparation but by a reduction of synaptic interactions in the juvenile rat in situ preparation.

Post-Natal Developmental Stages Of Lesser Whistling Duck At Paleik In (Lake), Sinkaing Township, Mandalay Division, Myanmar

Post-Natal Developmental Stages Of Lesser Whistling Duck At Paleik In (Lake), Sinkaing Township, Mandalay Division, Myanmar

Pediatric regional anesthesia: A review of the relevance of surface anatomy and landmarks used for peripheral nerve blockades in infants and children.

In regional anesthesia, accurate knowledge of surface anatomy and landmarks are essential for successful nerve blockades with least possible complications. The long-established anatomical landmarks used for peripheral nerve blockades in adult patients have also gained a place in pediatric anesthesia. Despite the continuing benefits and need for more effective peripheral techniques in pediatrics, there is a paucity of data on pediatric landmarks, and hence the absence of more appropriate techniques for the different age groups of children. Recent advancement in imaging has shown that there is a greater awareness for the different surface projections at various stages of postnatal development, as compared with that of adults. Thus, if adult landmarks are not appropriately adapted for use in children, complication rates may rise due to injuries to anatomical structures. This review evaluated the existing evidence of the pediatric nerve block landmarks in the reported literature, in order to highlight the limitations in our current understanding of pediatric surface anatomy and landmarks. A literature search was done using five databases to obtain 141 references. Only a few of these studies provided detailed reports of the relations of relevant nerves to anatomical landmarks. A few clinical imaging studies have provided new anatomical landmarks in children with reference to their age-related growth and anatomy. Most landmarks need to be standardized in children of various ages, and furthermore, inconsistencies in the current descriptions of the surface landmarks need to be validated and standardized for use in pediatric clinical practice. Clin. Anat. 32:803-823, 2019. © 2019 Wiley Periodicals, Inc.

Relationship between haematological profile and progression or spontaneous regression of melanoma in the Melanoma-bearing Libechov Minipigs

Haematological parameters, plasma iron concentration, and bodyweight were monitored in Melanoma-bearing Libechov Minipigs (MeLiM) from 5 to 18 weeks old. Animals with melanoma progression (P group) and spontaneous regression (SR group) were compared.The P group showed the lowest median values of red blood cell counts (RBC), haematocrit (HCT), haemoglobin concentration (HGB), and bodyweight, whereas the control white (tumour-free) pigs (C group) revealed the highest mean values of these parameters. The mean values of pigs with SR fell between the P and C groups. In addition, a stable concentration of plasma iron was found in the C group, while iron deficiency that increases with age was observed in the MeLiM groups. These results indicate that MeLiM are affected by cancer-related microcytic hypochromic anaemia. The lowest values of HGB, RBC, and HCT, together with the highest number of platelets (PLT) in the P group correspond to melanoma progression. Higher values of these parameters and lower PLT in the MeLiM pigs with SR reflected health improvement due to the destruction of melanoma cells during spontaneous regression. Monitoring of these haematological parameters can help distinguish MeLiM piglets with progression and spontaneous regression of melanoma in the early stages of postnatal development. The findings of this study correspond to findings in human patients in which cancer-related anaemia, thrombocytosis, and iron deficiency are often diagnosed.

Methylation status and expression patterns of myomaker gene play important roles in postnatal development in the Japanese flounder (Paralichthys olivaceus)

Myomaker is a membrane protein that plays a crucial role in the fusion of myoblasts during muscle growth. DNA methylation, a significant factor, regulates gene expression. The aim of this study was to examine the methylation and mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (L: 7 days post hatch (dph); M1: 21 dph; M2: 28 dph; M3: 35 dph; J1: 90 dph; J2: 180 dph; A1: 24 months; A2: 36 months). Muscle tissue samples were taken from Japanese flounder at different postnatal development stages to measure the extent of DNA methylation and gene expression. Methylation level in the promoter and exon 1 of myomaker was measured using bisulfite sequencing, and the relative expression of myomaker during each developmental stage was measured by quantitative PCR. The relative expression levels of myomaker were up-regulated from stages L to M2, M3 to J2, and methylation of myomaker was negatively correlated with mRNA expression. Furthermore, the CpG site located at −26 bp in the promoter was the lowest methylated region in all developmental stages. These results offer a basis for understanding the mechanism by which myomaker regulates muscle formation during postnatal development.

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma.

The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.