In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such "cranial-like" placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that forms from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here we show the involvement of protocadherin-mediated cell-cell interactions in the proper morphogenesis of the OSP. Protocadherin.e (Pcdhe.e) is expressed specifically in the OSP but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in the failure of the OSP to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in similar loss of a physically separate and distinct OSP territory. This effect is likely mediated by homophilic adhesion in trans, as Pcdh.e with scrambled extracellular domains failed to recapitulate the phenotype. Finally, we show that Pcdh.e expression in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP morphogenesis requires precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates.

Polymorphisms in congenital heart disease and extracardiac disorders.

The effectiveness of mandatory folic acid fortification compared with pre-fortification periods on reducing neural tube defects (NTDs): a systematic review and meta-analysis.

Endocytosis constitutes a fundamental cellular process governing development through coordinated regulation of plasma membrane remodeling and ciliogenesis, processes essential for cell shape changes and tissue development. Although Twist1 null embryos display complete cranial neural tube (NT) closure defects and conditional knockout in neuroectoderm disrupts cranial neural crest cell fate determination and delamination, the function of TWIST1 in NT morphogenesis remains unknown. We investigated the basis underlying neuroectodermal morphological abnormalities in TWIST1 mutant embryos, specifically the formation of ectopic lateral bending points and cellular disorganization, by examining Twist1's role in cilia formation, adherens junction integrity, and endocytic vesicle dynamics. Immunofluorescence analysis revealed that cytosolic TWIST1 colocalizes with β-catenin and endocytic regulators LRP2 and RAB11B along the apical surface of cranial neuroectoderm. Twist1 knockout resulted in reduced ciliary length and number. Quantitative polymerase chain reaction (PCR) and Western blot analyses demonstrated upregulation of RAB11B and β-catenin at mRNA and protein levels in Twist1 mutants. This molecular dysregulation coincided with increased accumulation of apical endocytic vesicles and altered expression profiles of endocytic component genes, ultimately modifying the apical neuroectodermal cell-cell junctions. Our findings establish TWIST1 as a crucial factor for neuroectodermal morphology, demonstrating its importance in ciliogenesis, endocytic vesicle dynamics, and cell-cell integrity.

Population-level research focused on major congenital abnormalities (MCAs) in sub-Saharan Africa is needed. The Tsepamo study estimated the prevalence of MCAs evaluable on neonatal surface examinations in Botswana. Data were collected from up to 18 government hospitals throughout Botswana. Surface examinations were conducted by trained nurses after birth, and photographs and/or descriptions were used to identify and classify MCAs. Birth defects were categorised as MCAs if they were of clinical, surgical or cosmetic importance. We estimated MCA prevalence for all live and stillborn infants who were not exposed to HIV in utero. Variation by site, calendar year and season of MCAs (and, separately, of neural tube defects (NTDs)) was evaluated with the χ2 and Benjamini-Hochberg methods. There were 1066 newborns with at least one MCA (59.3 (95% CI 55.8 to 62.9) per 10 000 births). NTDs accounted for 112 (10.5%) MCAs (6.2 (95% CI 5.2 to 7.5) per 10 000 births). Limb defects accounted for the majority of the birth defects (32.4 (95% CI 29.9 to 35.1) per 10 000 births), followed by major nervous system defects (16.7 (95% CI 14.9 to 18.7) per 10 000 births). The most common individual MCA was talipes (clubfoot) (18.5 (95% CI 16.6 to 20.6) per 10 000 births). While there was no meaningful variability of total MCAs by calendar year or season of conception, there was notable variation of NTDs by these factors. The estimated prevalence of MCAs was 59.3 (95% CI 55.8 to 62.9) per 10 000 births. While NTD prevalence varied by calendar year and season of conception, overall MCA prevalence did not. Ongoing surveillance of MCAs is needed to monitor the occurrence of birth defects and support preventive interventions in Botswana and the region.

Folate-mediated one-carbon metabolism is implicated in several pathologies including neural tube defects (NTDs), cancer, and neurodegenerative disorders, whereas diabetes is associated with NTDs and peripheral neuropathy (PN). The development of peripheral neuropathy was assessed in Shmt1+/- and Shmt1-/- mice, which are models of human folic acid-responsive NTDs, and diabetic (Leprdb) mice to determine whether NTDs and PN have a shared etiology. From 6 wk of age, male and female mice with reduced Shmt1 expression exhibited PN, with greater severity in females compared to males. The neuropathic progression was distinct from diabetic peripheral neuropathy (DPN) observed in Leprdb mice. Excess dietary folic acid prevented PN in both Shmt1-/- and Leprdb/db mice, whereas dietary uridine caused demyelinating PN in mice independent of genotype and folate status. The transcriptome from L3-L5 dorsal root ganglia (DRG) exhibited distinct sex-specific differences in glial cell gene expression when comparing Shmt1+/+ and Shmt1-/- mice. DRG sensory neurons exhibited changes in the expression of solute carriers and ion channels involved in nociception, neurotransmission, and structural support. We conclude that reduced thymidylate synthesis causes folic acid-responsive NTDs and PN in mice and that diabetes sensitizes mice to folic acid-responsive PN. Diabetes induces a special nutritional requirement for high intake of folic acid to prevent PN.

Occipital encephalocele is a rare neural tube defect that presents significant challenges. Despite advances in neurosurgery and prenatal imaging, it remains associated with substantial morbidity and mortality. This study aimed to define the neurodevelopmental outcomes of patients with occipital encephalocele, highlighting the incidence of psychomotor delay and visual agnosia, and identifying key prognostic factors that influence these outcomes. We conducted a retrospective descriptive study including all patients treated for occipital encephalocele at the Mongi Ben Hmida National Institute of Neurology in Tunis over a 23-year period. Demographic, clinical, radiological, surgical, and outcome data were collected from medical records and analyzed to identify postoperative complications and prognostic indicators. A total of 40 patients were treated, with a median age at surgery of 11.8 months and a female predominance (sex ratio 0.38). Hydrocephalus was the most frequent associated anomaly (40%). Postoperative complications included meningitis (12.5%) and secondary hydrocephalus (37.5%), with a mortality rate of 10%. Psychomotor delay was observed in 67.6% of cases and visual agnosia in 10%. Univariate analysis identified preoperative hydrocephalus, herniation of functional brain tissue, and delayed surgery as significant adverse prognostic factors. Occipital encephalocele remains a severe congenital anomaly with considerable neurodevelopmental impact. Delayed surgical intervention, the presence of herniated functional brain tissue, and postoperative hydrocephalus negatively impact outcomes. A multidisciplinary management approach and early neurodevelopmental follow-up are essential to improve long-term prognosis and quality of life for these patients.

Animal models of Chiari malformation types 1 and 2: Mechanistic insights and translational challenges.

A rapid LC-MS/MS method for the simultaneous quantification of 10 key folate cycle intermediates in human plasma.

Algorithm validation studies of health outcomes using real-world data are used to generate real-world evidence. We developed the Algorithm CErtaInty Tool (ACE-IT) to enable users to appraise whether a given real-world data-based algorithm is fit for purpose for a specific decision context, using the outcome of ischemic stroke as an exemplar. Our objective was to refine, develop, and tailor the ACE-IT to address whether validated algorithms for pregnancy and birth outcomes are fit for purpose for a specific decision context. We also tested the feasibility, acceptability, appropriateness, and content validity of the Pregnancy ACE-IT for the evaluation of an algorithm on pregnancy/birth outcomes. We engaged an expert panel of pharmacoepidemiologists and regulators to guide the development of the Pregnancy ACE-IT. The panel contributed to crafting a vignette on the selected pregnancy outcome of neural tube defects. Twenty-seven external raters used the Pregnancy ACE-IT to appraise whether a selected peer-reviewed algorithm to identify neural tube defects was fit for purpose and generalized as a target data source. Most raters found the Pregnancy ACE-IT acceptable and appropriate, although some did not find it easy to use. The Pregnancy ACE-IT represents a suitable, flexible, acceptable, and appropriate tool with relevant content for determining whether algorithms for pregnancy outcomes are fit for purpose for a specific decision context. Future studies to encourage broader acceptance and improve ease of use will require familiarizing users with the tool's operational characteristics and evaluating its suitability in the context of additional pregnancy/birth outcomes in different therapeutic areas.

Fumonisins are mycotoxins produced primarily by Fusarium species that frequently contaminate maize and other cereal crops, posing significant food safety and public health concerns worldwide. Chronic exposure to fumonisins, particularly fumonisin B₁ (FB₁), has been associated with adverse human health outcomes, including disruption of sphingolipid metabolism, oxidative stress, immunotoxicity, neural tube defects, and increased risk of oesophageal cancer in high-exposure populations. This review synthesizes scientific knowledge on the sources, global occurrence, and epidemiology of fumonisin contamination, highlighting environmental and storage factors that influence toxin production. The key molecular mechanisms of fumonisin toxicity, especially inhibition of ceramide synthase and consequent alterations in membrane lipid homeostasis, are discussed alongside evidence from epidemiological and experimental studies. Advances in fumonisin detection methods and international regulatory guidelines established by WHO and EFSA are also reviewed. The paper emphasizes the need for continued surveillance, improved food safety practices, and effective mitigation strategies, particularly in regions where maize-based diets predominate.

Genetic background influences susceptibility to exencephaly in Scavenger receptor Class B type 1-deficient mouse embryos.

Maternal deficiency of vitamin B12 (B12) is associated with neural tube defects, fetal growth restriction, and future risk of non-communicable disease in the offspring. Little is known about the molecular basis of these associations. We hypothesized that B12 regulates the expression of fetal genes, thereby influencing fetal growth and fetal programming. We investigated the association of B12 and other micronutrient concentrations in the cord blood with gene expression in the cord blood mononuclear cells. We performed a Weighted Gene Co-expression Network Analysis (WGCNA) on cord blood transcriptome of babies born in a pre-conception trial Pune Rural Intervention in Young Adolescents of B12 and multi-micronutrients (MMN). The gene modules (clusters) in WGCNA that showed a significant correlation with cord blood B12 and MMN were subjected to gene ontology (GO) analysis. WGCNA generated 23 different modules. Cord blood B12 concentrations were strongly correlated with modules of genes involved in methylation reactions and gene regulation. Cord B2 concentrations correlated with gene modules associated with demethylation reactions. Vitamins B6 and B9 did not show a unique association either with gene modules or specific GO terms. Our results demonstrate that maternal B12 may regulate expression of fetal genes involved in methylation reaction. This is a novel suggestion for the role of B12 in fetal growth, development, and the Developmental Origins of Health and Disease paradigm.

It has been proven that periconceptional folic acid (FA) supplementation could prevent neural tube defects. However, FA supplementation during different stages of pregnancy and its association with the neurobehavioral development of offspring remains unclear, particularly the effects of continued FA supplementation during the second and third trimesters. This study aimed to investigate the relationship between maternal FA supplementation at various stages of pregnancy and infant neurobehavioral development. In a prospective birth cohort study involving 3246 parent-child pairs, 2905 infants completed neurobehavioral development assessments at 6 months and 3005 infants at 18 months. Information regarding micronutrient supplementation at various stages of pregnancy was recorded. Multivariable logistic regression was used to evaluate the association between FA supplementation and infant neurobehavioral developmental delays. In addition, propensity score analysis was performed to correct the potential imbalances in the distribution of related factors between the groups. During the periconceptional period, maternal standardized FA supplementation (taking 0.4 mg FA daily before and in early pregnancy, as officially recommended) was associated with a reduced risk of possible development delays in 18-month-old infants in the communication domain (RR = 0.48, 95% CI: 0.26-0.86; P = 0.015). After the 12th gestational week, maternal continuous FA supplementation in the second and third trimesters was significantly associated with a decreased risk of possible neurobehavioral development delay in 6-month-old infants in fine motor domain (RR = 0.31, 95% CI: 0.12-0.82; P = 0.019) and the problem-solving domain (RR = 0.20, 95% CI: 0.05-0.79; P = 0.022). These associations remained significant after adjusting for confounders and propensity scores. FA supplementation at different stages of pregnancy may enhance neurobehavioral development in offspring. To confirm these findings, additional investigations or trials with larger sample sizes and consistent tracking of folate status throughout pregnancy are recommended.

Background/Objectives: Emerging evidence has suggested that choline is an effective treatment for at least some of the neurobehavioral deficits associated with Fetal Alcohol Spectrum Disorders (FASD). However, the mechanism of how choline works to ameliorate ethanol’s teratogenic effects, and whether it acts directly on the fetus or indirectly by altering the uterine environment, remains unknown. Previous work from our lab demonstrated that 4 BXD mouse strains that show high levels of ethanol-induced cell death on embryonic day 9.5 (E9.5) have differential responses to choline supplementation. This differential response in mouse strains highlights a need to further understand the role of genetics in choline metabolism. Because the liver is the central organ for choline metabolism, and the embryonic liver of mice is not functional this early in gestation, we focused on choline metabolism in the liver of the dam. Methods: Using a bioinformatics approach, the goals were to assess whether (1) genetic differences in liver choline metabolism in the dam could affect ethanol-induced cell death in a genotype-specific manner and (2) any of these candidate genes in the liver of the dam could be linked to differential response to choline amongst the strains. By performing a literature review, haplotype analysis among the 4 BXD strains, and liver protein expression analysis among 3 strains, we show that there are genetic differences in choline metabolic genes that are consistent with the hypothesis that maternal choline metabolism could mediate differential sensitivity. Results: While we identified two genes as promising candidates for the variable responses to choline supplementation among the four previously identified BXD strains choline/ethanolamine phosphotransferase 1 (cept1) and choline transporter gene solute carrier family 44 member 1 (slc44a1), the wealth of data on slc44a1 makes it the stronger candidate and suggests that it should be further explored. Conclusions: Genetic differences in maternal choline metabolism are present and may underlie variable therapeutic responses to choline, warranting a hypothesis that requires further investigation across animal models and human populations.

Maternal nutrition is a cornerstone of a healthy pregnancy and birth outcomes, influencing everything from fetal development to neonatal survival. Ensuring adequate and balanced nutrition during pregnancy is not just a personal health choice – it is a public health imperative. Maternal nutrition plays a pivotal role in shaping the health trajectory of both mother and child. The “first 1000 days” – from conception to a child’s second birthday – are widely recognized as a critical window for growth and development. During this period, nutritional adequacy directly impacts fetal growth, birth weight, and long-term cognitive and physical outcomes. Undernutrition in pregnant women has been linked to increased risks of anemia, preterm labor, low birth weight, and cesarean delivery. A recent study found that neonates born to undernourished mothers were significantly more likely to have lower Appearance, Pulse, Grimace, Activity, and Respiration (APGAR) scores and delayed breastfeeding initiation. These outcomes not only affect immediate survival but also predispose children to chronic diseases later in life. Micronutrient deficiencies – particularly in iron, folate, iodine, and Vitamin D – are common in low-resource settings and contribute to poor maternal and neonatal health. For instance, iron deficiency anemia is associated with increased maternal mortality and impaired fetal oxygenation, whereas folate deficiency raises the risk of neural tube defects. Conversely, adequate maternal nutrition supports optimal placental function, gestational weight gain, and fetal tissue accretion. Balanced diets rich in fruits, vegetables, whole grains, lean proteins, and essential fats are universally recommended. Supplementation programs and nutritional counseling during antenatal care have shown promising results in improving outcomes, especially in vulnerable populations. In conclusion, maternal nutrition is not merely a biological necessity – it is a determinant of equity, survival, and human potential. Governments and healthcare systems must prioritize nutritional education, access to supplements, and food security for pregnant women. The health of future generations begins with the nourishment of mothers.

Objective: To investigate the molecular mechanism by which fibroblast growth factor 1 (FGF1) participates in neural tube defects (NTD) through regulating cell proliferation in a retinoic acid (RA)-induced mouse model, and to elucidate the role of FGF1 during neural tube closure in mice. Methods: An NTD mouse model was established using RA induction, with mice divided into a treatment group and a control group (n=9 each). Pregnant mice in the treatment group received 28 mg/kg RA via gavage at embryonic day 7.5 (E7.5), while the control group received sesame oil. Embryonic brain vesicle tissues were collected at E8.5, E9.5, and E10.5 for RNA and protein extraction to assess gene expression. Embryonic brain sections from E9.5 and E10.5 were prepared for pathological examination and gene expression analysis. At the mouse embryonic brain tissue level, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate FGF1 mRNA expression, and immunohistochemistry (IHC) was performed to detect FGF1 protein expression. An FGF1 knockdown plasmid was constructed, and cell proliferation was assessed in vitro. Western blot was used to measure the expression of the proliferation marker proliferating cell nuclear antigen (PCNA), 5-Ethynyl-2'-deoxyuridine (EdU) staining was employed to evaluate cell proliferation, and flow cytometry was conducted to analyze apoptosis. Results: The RA treatment group of mice showed delayed embryonic development, abnormal brain structure, and unclosed neural tubes, inducing the formation of NTD mouse models. At the animal level, both the mRNA and protein expressions of FGF1 were abnormally highly expressed in the brain tissues of NTD mouse embryos at embryonic day 9.5 (E9.5) and E10.5 (P<0.001). At the cellular level, FGF1 was significantly highly expressed in HT-22 cells treated with RA (P<0.001), while its expression was significantly downregulated after FGF1 knockdown (P<0.001); the protein expression of PCNA (a cellular proliferation marker) in RA-treated cells was significantly increased (P<0.01), and this expression was significantly decreased after FGF1 gene knockdown in the RA-treated group (P<0.01). EdU staining experiments also showed that excessive cell proliferation occurred after RA treatment (P<0.001), whereas cell proliferation was reduced in RA-treated cells with FGF1 knockdown (P<0.001); flow cytometry analysis indicated that the proportion of apoptotic cells increased significantly after RA intervention (P<0.001), and this proportion continued to increase after FGF1 knockdown (P<0.05). Conclusions: FGF1 was significantly upregulated in the RA-induced NTD model. Overexpression of FGF1 inhibited cell proliferation and induced apoptosis. Knockdown of FGF1 restored the reduced cell proliferation caused by RA intervention and further increased RA-induced apoptosis. It provides a new perspective on the mechanism of NTD and may serve as a potential biomarker for clinical diagnosis.

ABSTRACTHomozygous or compound heterozygous mutations in the ITGB4 gene are associated with the pathogenesis of junctional epidermolysis bullosa, characterized by increased fragility of the skin and mucous membranes. The prenatal diagnosis of this condition continues to represent a rare occurrence and a significant clinical challenge. A pregnant woman in her third decade underwent second‐trimester screening, which revealed elevated alpha‐fetoprotein levels, suggestive of a high risk for open neural tube defects. Comprehensive ultrasonographic evaluation revealed multiple fetal anomalies, including bilateral agenesis of the auricular structures, a shortened nasal bone, a flattened nasal tip, cutaneous disruptions in the abdominal and gluteal regions, and cystic fluid accumulation around the external genitalia. Amniocentesis was performed at 20 weeks of gestation to obtain amniotic fluid for genetic analysis. Family based whole exome sequencing identified compound heterozygous mutations in the ITGB4 gene (c.794dupC, c.1882delG) inherited parents, with the c.1882delG variant being a novel mutation not previously documented. Elevated maternal serum alpha‐fetoprotein levels may represent a promising biomarker for the prenatal screening of junctional epidermolysis bullosa. This study reports a novel frameshift mutation in the ITGB4 gene, thereby expanding the known mutational spectrum of this disorder.

Maternal-effect genes (MEGs) play a crucial role in early mammalian development, and their dysfunction can lead to severe embryonic and extra-embryonic abnormalities. NLRP2, a MEG that encodes a subcortical maternal complex (SCMC) protein, has been implicated in preimplantation development, but its role after implantation remains underexplored. In this study, we investigated the developmental consequences of maternal Nlrp2 loss-of-function in a maternal knockout (KO) mouse model at embryonic day 11.5. Embryos derived from Nlrp2-KO females have abnormal yolk sac vasculature, increased embryonic resorption, craniofacial abnormalities, neural tube defects, and congenital heart defects. Placental architecture is disrupted with an altered junctional zone and labyrinth structure. Transcriptome profiling of maternal decidua and placenta demonstrated dysregulation of genes involved in trophoblast differentiation and extra-embryonic development. Bisulfite sequencing of these tissues revealed persistence at E11.5 of previously observed locus-specific disruption in DNA methylation at four imprinted loci following maternal Nlrp2 loss. We further describe pregnancy outcomes and offspring phenotypes for two unrelated women with bi-allelic maternal NLRP2 variants. The first carried homozygous NLRP2 nonsense variants and experienced recurrent pregnancy loss and fetuses with multiple structural anomalies, including omphalocele, craniofacial dysmorphism, and cardiac defects. The second carried compound heterozygous frameshift and missense NLRP2 variants and had a child with neurodevelopmental impairment of uncertain etiology. These findings indicate a conserved role for maternal NLRP2 in embryonic viability and placental development, and support further studies in humans into the contribution of NLRP2 and other similar MEGs to offspring congenital anomalies and adverse pregnancy outcomes.

Congenital heart disease involving outflow tract (OFT) malformations remains a major clinical challenge, particularly in 22q11.2 deletion syndrome. Although folic acid (FA) reduces the incidence of neural tube defects, its mechanistic role in cardiac OFT development is not fully understood. Using Tbx1neo/neo hypomorphic mice as a model of 22q11.2 deletion syndrome, we investigated the effects of maternal FA supplementation on cardiac development. Pregnant dams received FA through diet or intraperitoneal injection and embryonic cardiac morphology was assessed at E15.5 and E18.5. Maternal FA administration significantly improved the persistent truncus arteriosus (PTA) phenotype, with 60% of Tbx1neo/neo embryos exhibiting a partially septated PTA (Van Praagh type A1) vs. complete PTA (type A2) in controls. Neural crest cell (NCC) migration from the neural tube into the OFT was enhanced. GFP lineage tracing confirmed the presence of increased NCCs in the OFT and reduced ectopic neuronal differentiation. Single-cell RNA-sequencing and immunohistochemistry revealed activation of the Notch and Midkine signaling pathways in NCCs following FA treatment. Maternal FA supplementation improved cardiac OFT malformations in Tbx1neo/neo embryos by enhancing NCC migration and fate specification, possibly mediated by Notch and Midkine signaling activation. Our findings provide mechanistic insights into the observed reduction in congenital heart defects with FA and suggest its potential as a minimally invasive prenatal intervention.