Perspectives on folate with special reference to epigenetics and neural tube defects.

Folate is a micronutrient essential for DNA synthesis, cell division, fetal growth and development. Folate deficiency leads to genomic instability. Inadequate intake of folate during conception may lead to neural tube defects (NTDs) in the offspring. Folate influences the DNA methylation, histone methylation and homocysteine mediated gene methylation. DNA methylation influences the expression of microRNAs (miRNAs). Folate deficiency may be associated with miRNAs misregulation leading to NTDs. Mitochondrial epigenetics and folate metabolism has proved to be involved in embryogenesis and neural tube development. Folate related genetic variants also cause the occurrence of NTDs. Unmetabolized excessive folate may affect health adversely. Hence estimation of folate levels in the blood plays an important role in high-risk cases.

Arsenic disturbs neural tube closure involving AMPK/PKB-mTORC1-mediated autophagy in mice.

Arsenic exposure is a significant risk factor for folate-resistant neural tube defects (NTDs), but the potential mechanism is unclear. In this study, a mouse model of arsenic-induced NTDs was established to investigate how arsenic affects early neurogenesis leading to malformations. The results showed that in utero exposure to arsenic caused a decline in the normal embryos, an elevated embryo resorption, and a higher incidence of malformed embryos. Cranial and spinal deformities were the main malformation phenotypes observed. Meanwhile, arsenic-induced NTDs were accompanied by an oxidant/antioxidant imbalance manifested by elevated levels of reactive oxygen species (ROS) and decreased antioxidant activities. In addition, changes in the expression of autophagy-related genes and proteins (ULK1, Atg5, LC3B, p62) as well as an increase in autophagosomes were observed in arsenic-induced aberrant brain vesicles. Also, the components of the upstream pathway regulating autophagy (AMPK, PKB, mTOR, Raptor) were altered accordingly after arsenic exposure. Collectively, our findings propose a mechanism for arsenic-induced NTDs involving AMPK/PKB-mTORC1-mediated autophagy. Blocking autophagic cell death due to excessive autophagy provides a novel strategy for the prevention of folate-resistant NTDs, especially for arsenic-exposed populations.

Grainyhead-like 2 interacts with noggin to regulate tissue fusion in mouse.

Defective tissue fusion during mammalian embryogenesis results in congenital anomalies, such as exencephaly, spina bifida and cleft lip and/or palate. The highly conserved transcription factor grainyhead-like 2 (Grhl2) is a crucial regulator of tissue fusion, with mouse models lacking GRHL2 function presenting with a fully penetrant open cranial neural tube, facial and abdominal clefting (abdominoschisis), and an open posterior neuropore. Here, we show that GRHL2 interacts with the soluble morphogen protein and bone morphogenetic protein (BMP) inhibitor noggin (NOG) to impact tissue fusion during development. The maxillary prominence epithelium in embryos lacking Grhl2 shows substantial morphological abnormalities and significant upregulation of NOG expression, together with aberrantly distributed pSMAD5-positive cells within the neural crest cell-derived maxillary prominence mesenchyme, indicative of disrupted BMP signalling. Reducing this elevated NOG expression (by generating Grhl2-/-;Nog+/- embryos) results in delayed embryonic lethality, partial tissue fusion rescue, and restoration of tissue form within the craniofacial epithelia. These data suggest that aberrant epithelial maintenance, partially regulated by noggin-mediated regulation of BMP-SMAD pathways, may underpin tissue fusion defects in Grhl2-/- mice.

Noncanonical function of folate through folate receptor 1 during neural tube formation.

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation.

Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Cytoneme signaling provides essential contributions to mammalian tissue patterning.

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.

Prenatal cadmium exposure impairs neural tube closure via inducing excessive apoptosis in neuroepithelium.

Birth defects have become a public health concern. The hazardous environmental factors exposure to embryos could increase the risk of birth defects. Cadmium, a toxic environmental factor, can cross the placental barrier during pregnancy. Pregnant woman may be subjected to cadmium before taking precautionary protective actions. However, the link between birth defects and cadmium remains obscure. Cadmium exposure can induce excessive apoptosis in neuroepithelium during embryonic development progresses. Cadmium exposure activated the p53 via enhancing the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and reactive oxygen species' (ROS) level. And cadmium decreases the level of Paired box 3 (Pax3) and murine double minute 2 (Mdm2), disrupting the process of p53 ubiquitylation. And p53 accumulation induced excessive apoptosis in neuroepithelium during embryonic development progresses. Excessive apoptosis led to the failure of neural tube closure. The study emphasizes that environmental materials may increase the health risk for embryos. Cadmium caused the failure of neural tube closure during early embryotic day. Pregnant women may be exposed by cadmium before taking precautionary protective actions, because of cadmium concentration-containing foods and environmental tobacco smoking. This suggests that prenatal cadmium exposure is a threatening risk factor for birth defects.

Pax3 lineage-specific deletion of Gpr161 is associated with spinal neural tube and craniofacial malformations during embryonic development.

Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.

Stepwise modulation of apical orientational cell adhesions for vertebrate neurulation.

Neurulation transforms the neuroectoderm into the neural tube. This transformation relies on reorganising the configurational relationships between the orientations of intrinsic polarities of neighbouring cells. These orientational intercellular relationships are established, maintained, and modulated by orientational cell adhesions (OCAs). Here, using zebrafish (Danio rerio) neurulation as a major model, we propose a new perspective on how OCAs contribute to the parallel, antiparallel, and opposing intercellular relationships that underlie the neural plate-keel-rod-tube transformation, a stepwise process of cell aggregation followed by cord hollowing. We also discuss how OCAs in neurulation may be regulated by various adhesion molecules, including cadherins, Eph/Ephrins, Claudins, Occludins, Crumbs, Na+ /K+ -ATPase, and integrins. By comparing neurulation among species, we reveal that antiparallel OCAs represent a conserved mechanism for the fusion of the neural tube. Throughout, we highlight some outstanding questions regarding OCAs in neurulation. Answers to these questions will help us understand better the mechanisms of tubulogenesis of many tissues.

Maternal metabolism influences neural tube closure.

Changes in maternal nutrient availability due to diet or disease significantly increase the risk of neural tube defects (NTDs). Because the incidence of metabolic disease continues to rise, it is urgent that we better understand how altered maternal nutrient levels can influence embryonic neural tube development. Furthermore, primary neurulation occurs before placental function during a period of histiotrophic nutrient exchange. In this review we detail how maternal metabolites are transported by the yolk sac to the developing embryo. We discuss recent advances in understanding how altered maternal levels of essential nutrients disrupt development of the neuroepithelium, and identify points of intersection between metabolic pathways that are crucial for NTD prevention.

TMEM132A regulates mouse hindgut morphogenesis and caudal development.

Caudal developmental defects, including caudal regression, caudal dysgenesis and sirenomelia, are devastating conditions affecting the skeletal, nervous, digestive, reproductive and excretory systems. Defects in mesodermal migration and blood supply to the caudal region have been identified as possible causes of caudal developmental defects, but neither satisfactorily explains the structural malformations in all three germ layers. Here, we describe caudal developmental defects in transmembrane protein 132a (Tmem132a) mutant mice, including skeletal, posterior neural tube closure, genitourinary tract and hindgut defects. We show that, in Tmem132a mutant embryos, visceral endoderm fails to be excluded from the medial region of early hindgut, leading directly to the loss or malformation of cloaca-derived genitourinary and gastrointestinal structures, and indirectly to the neural tube and kidney/ureter defects. We find that TMEM132A mediates intercellular interaction, and physically interacts with planar cell polarity (PCP) regulators CELSR1 and FZD6. Genetically, Tmem132a regulates neural tube closure synergistically with another PCP regulator Vangl2. In summary, we have identified Tmem132a as a new regulator of PCP, and hindgut malformation as the underlying cause of developmental defects in multiple caudal structures.

Calcium dynamics at the neural cell primary cilium regulate Hedgehog signaling-dependent neurogenesis in the embryonic neural tube.

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube.

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Dual mechanism underlying failure of neural tube closure in the Zic2 mutant mouse.

Understanding the molecular mechanisms that lead to birth defects is an important step towards improved primary prevention. Mouse embryos homozygous for the Kumba (Ku) mutant allele of Zic2 develop severe spina bifida with complete lack of dorsolateral hinge points (DLHPs) in the neuroepithelium. Bone morphogenetic protein (BMP) signalling is overactivated in Zic2Ku/Ku embryos, and the BMP inhibitor dorsomorphin partially rescues neural tube closure in cultured embryos. RhoA signalling is also overactivated, with accumulation of actomyosin in the Zic2Ku/Ku neuroepithelium, and the myosin inhibitor Blebbistatin partially normalises neural tube closure. However, dorsomorphin and Blebbistatin differ in their effects at tissue and cellular levels: DLHP formation is rescued by dorsomorphin but not Blebbistatin, whereas abnormal accumulation of actomyosin is rescued by Blebbistatin but not dorsomorphin. These findings suggest a dual mechanism of spina bifida origin in Zic2Ku/Ku embryos: faulty BMP-dependent formation of DLHPs and RhoA-dependent F-actin accumulation in the neuroepithelium. Hence, we identify a multi-pathway origin of spina bifida in a mammalian system that may provide a developmental basis for understanding the corresponding multifactorial human defects.

Functional interaction between Vangl2 and N-cadherin regulates planar cell polarization of the developing neural tube and cochlear sensory epithelium.

Although the core constituents of the Wnt/planar cell polarity (PCP) signaling have been extensively studied, their downstream molecules and protein-protein interactions have not yet been fully elucidated. Here, we show genetic and molecular evidence that the PCP factor, Vangl2, functionally interacts with the cell-cell adhesion molecule, N-cadherin (also known as Cdh2), for typical PCP-dependent neural development. Vangl2 and N-cadherin physically interact in the neural plates undergoing convergent extension. Unlike monogenic heterozygotes, digenic heterozygous mice with Vangl2 and Cdh2 mutants exhibited defects in neural tube closure and cochlear hair cell orientation. Despite this genetic interaction, neuroepithelial cells derived from the digenic heterozygotes did not show additive changes from the monogenic heterozygotes of Vangl2 in the RhoA-ROCK-Mypt1 and c-Jun N-terminal kinase (JNK)-Jun pathways of Wnt/PCP signaling. Thus, cooperation between Vangl2 and N-cadherin is at least partly via direct molecular interaction; it is essential for the planar polarized development of neural tissues but not significantly associated with RhoA or JNK pathways.

Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.

Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD+; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis. Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation.

Two ways to skin a new cell fate.

To induce cell fate changes, do transcription factors engage open domains of chromatin or elicit chromatin opening in a pioneering fashion? In this issue of Developmental Cell, Delás et al. show that the same sonic hedgehog (Shh) inducing signal can yield different neural tube fates by either modality.

Short-term transcriptomic changes in the mouse neural tube induced by an acute alcohol exposure.

Alcohol exposure during the formation and closure of the neural tube, or neurulation (embryonic day [E] 8-10 in mice; ∼4th week of human pregnancy), perturbs development of midline brain structures and significantly disrupts gene expression in the rostroventral neural tube (RVNT). Previously, alcohol exposure during neurulation was found to alter gene pathways related to cell proliferation, p53 signaling, ribosome biogenesis, immune signaling, organogenesis, and cell migration 6 or 24 h after administration. Our current study expands upon this work by investigating short-term gene expression changes in the RVNT following a single binge-like alcohol exposure during neurulation. Female C57BL/6J mice were administered a single dose of 2.9 g/kg alcohol or vehicle on E9.0 to target mid-neurulation. The RVNTs of stage-matched embryos were collected 2 or 4 h after exposure and processed for RNA-seq. Functional profiling was performed with g:Profiler, as well as with the CiliaCarta and DisGeNet databases. Two hours following E9.0 alcohol exposure, 650 genes in the RVNT were differentially expressed. Functional enrichment analysis revealed that pathways related to cellular metabolism, gene expression, cell cycle, organogenesis, and Hedgehog signaling were down-regulated, and pathways related to cellular stress response, p53 signaling, and hypoxia were up-regulated by alcohol. Four hours after alcohol exposure, 225 genes were differentially expressed. Biological processes related to metabolism, RNA binding, ribosome biogenesis, and methylation were down-regulated, while protein localization and binding, autophagy, and intracellular signaling pathways were up-regulated. Two hours after alcohol exposure, the differentially expressed genes were associated with disease terms related to eye and craniofacial development and anoxia. These data provide further information regarding the biological functions targeted by alcohol exposure during neurulation in regions of the neural tube that give rise to alcohol-sensitive midline brain structures. Disruption of these gene pathways contributes to the craniofacial and brain malformations associated with prenatal alcohol exposure.

MicroRNA-Mediated Regulation of BMP Signaling in the Developing Neural Tube.

BACKGROUND: Neural tube (NT) morphogenesis is reliant on the proper temporospatial expression of numerous genes and synchronized crosstalk between diverse signaling cascades and gene regulatory networks governing key cellular processes. MicroRNAs (miRNAs), a group of small non-coding regulatory RNAs, execute defining roles in directing key canonical pathways during embryogenesis. OBJECTIVE: In order to comprehend the mechanistic underpinnings of miRNA regulation of NT morphogenesis, we have identified in the current study various miRNAs and their target mRNAs associated with BMP signaling during critical stages of neurulation. METHODS: We previously demonstrated the expression of several miRNAs during the critical stages of neurulation (gestational days (GD) 8.5, 9.0, and 9.5) employing high-sensitivity, high-coverage microarrays. In the present study, bioinformatic analyses were used to identify miRNAs differentially expressed (DE) in the embryonic NT that target messenger RNAs (mRNAs) associated with the bone morphogenetic protein (BMP) signaling pathway. RNAs extracted from the developing NT were hybridized to both miRNA and mRNA arrays to evaluate miRNA-mRNA interactions. RESULTS: Bioinformatic analysis identified several DE miRNAs that targeted mRNAs encoding members of (and proteins associated with) the BMP signaling pathway - a signaling cascade central to normal NT development. CONCLUSION: Identification of the miRNAs and their mRNA targets associated with BMP signaling facilitates a better understanding of the crucial epigenetic mechanisms underlying normal NT development as well as the pathogenesis of NT defects. The current study supports the notion that miRNAs function as key regulators of neural tube morphogenesis via modulation of the BMP signaling cascade. Altered expression of these miRNAs during neurulation may therefore result in NT defects.

OLIG2 is an in vivo bookmarking transcription factor in the developing neural tube in mouse.

Cells possess intrinsic features that are inheritable via epigenetic regulation, such as DNA methylation and histone modification. These inheritable features maintain a unique gene expression pattern, underlying cellular memory. Because of the degradation or displacement of mitotic chromosomes, most transcription factors do not contribute to cellular memory. However, accumulating in vitro evidence indicates that some transcription factors can be retained in mitotic chromosomes called as bookmarking. Such transcription factors may contribute to a novel third mechanism of cellular memory. Since most findings of transcription factor bookmarking have been reported in vitro, little is currently known in vivo. In the neural tube of mouse embryos, we discovered that OLIG2, a basic helix loop helix (bHLH) transcription factor that regulates proliferation of neural progenitors and the cell fate of motoneurons and oligodendrocytes, binds to chromatin through every cell cycle including M-phase. OLIG2 chromosomal localization coincides with mitotic cell features such as the phosphorylation of histone H3, KI67, and nuclear membrane breakdown. Chromosomal localization of OLIG2 is regulated by an N-terminus triple serine motif. Photobleaching analysis revealed slow OLIG2 mobility, suggesting a high affinity of OLIG2 to DNA. In Olig2 N-terminal deletion mutant mice, motoneurons and oligodendrocyte progenitor numbers are reduced in the neural tube, suggesting that the bookmarking regulatory domain is important for OLIG2 function. We conclude that OLIG2 is a de novo in vivo bookmarking transcription factor. Our results demonstrate the presence of in vivo bookmarking in a living organism and illustrate a novel function of transcription factors.