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Purpose: The etiopathogenesis of coronal nonsyndromic craniosynostosis (cNCS), a congenital condition defined by premature fusion of 1 or both coronal sutures, remains largely unknown., Methods: We conducted the largest genome-wide association study of cNCS followed by replication, fine mapping, and functional validation of the most significant region using zebrafish animal model., Results: Genome-wide association study identified 6 independent genome-wide-significant risk alleles, 4 on chromosome 7q21.3 SEM1-DLX5-DLX6 locus, and their combination conferred over 7-fold increased risk of cNCS. The top variants were replicated in an independent cohort and showed pleiotropic effects on brain and facial morphology and bone mineral density. Fine mapping of 7q21.3 identified a craniofacial transcriptional enhancer (eDlx36) within the linkage region of the top variant (rs4727341; odds ratio [95% confidence interval], 0.48[0.39-0.59]; P = 1.2E-12) that was located in SEM1 intron and enriched in 4 rare risk variants. In zebrafish, the activity of the transfected human eDlx36 enhancer was observed in the frontonasal prominence and calvaria during skull development and was reduced when the 4 rare risk variants were introduced into the sequence., Conclusion: Our findings support a polygenic nature of cNCS risk and functional role of craniofacial enhancers in cNCS susceptibility with potential broader implications for bone health., Competing Interests: Conflict of Interest The authors declare no competing interests in relation to the work described.

Folate receptor delta (FRδ) has been used as a biomarker for regulatory T cells (Tregs), because its expression is limited to Tregs and ovum. Although FRδ is unable to bind folate, we have used molecular docking software to identify a folate congener that binds FRδ with high affinity and have exploited this FRδ-specific ligand to target attached drugs (imaging agents, immune activators, and immune suppressors) specifically to Tregs in murine tumor xenografts. Analysis of treated tumors demonstrates that targeting of a Toll-like receptor 7 agonist inhibits Treg expression of FOXP3, PD-1, CTLA4, and HELIOS, resulting in 40-80% reduction in tumor growth and repolarization of other tumor-infiltrating immune cells to more inflammatory phenotypes. Targeting of the immunosuppressive drug dexamethasone, in contrast, promotes enhanced tumor growth and shifts the tumor-infiltrating immune cells to more anti-inflammatory phenotypes. Since Tregs comprise <1% of cells in the tumor masses examined, and since the targeted drugs are not internalized by cancer cells, these data demonstrate that Tregs exert a disproportionately large effect on tumor growth. Because the targeted drug did not bind to Tregs or other immune cells in healthy tissues, the data demonstrate that the immunosuppressive properties of Tregs in tumors can be manipulated without causing systemic toxicities associated with global reprogramming of the immune system., Competing Interests: RA and PSL report a patent application covering the above technology and also PSL reports funding from Morphimmune Inc. in which he owns stock. RF was formerly in a leadership position with TeratOmic Consulting which has since been dissolved. He also receives travel funds to attend quarterly editorial board meetings for the Journal of Reproductive and Developmental Medicine. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alfar, Napoleon, Shahriar, Finnell, Walchle, Johnson and Low.)

The formation of the brain and spinal cord is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Convergent and extension movements transforms a flat sheet of ectodermal cells into a narrow and elongated line of neuroepithelia, while a major source of Sonic Hedgehog signaling from the notochord induces the overlying neuroepithelial cells to form two apposed neural folds. Afterward, neural tube closure occurs by synchronized coordination of the surface ectoderm and adjacent neuroepithelial walls at specific axial regions known as neuropores. Environmental or genetic interferences can impair neurulation resulting in neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, which is a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. In this work, we demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent loss of ventral neuroepithelial stiffness, in a notochord adjacent area at the level of the rhombomere 5, preceding the development of exencephaly in Fuz ablated mutants. The formation of cranial and paravertebral ganglia is also impaired in these embryos, indicating that Fuz has a critical function sustaining normal neural tube development and neuronal differentiation., Significance Statement: Neural tube defects (NTDs) are a common cause of disability in children, representing the second most common congenital structural malformation in humans following only congenital cardiovascular malformations. NTDs affect approximately 1 to 2 pregnancies per 1000 births every year worldwide, when the mechanical forces folding the neural plate fails to close at specific neuropores located anteriorly (cranial) or posteriorly (caudal) along the neural tube, in a process known as neurulation, which happens throughout the third and fourth weeks of human pregnancy.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Wnt signaling pathways, including the canonical Wnt/β-catenin pathway, planar cell polarity pathway, and Wnt/Ca 2+ signaling pathway, play important roles in neural development during embryonic stages. The DVL genes encode the hub proteins for Wnt signaling pathways. The mutations in DVL2 and DVL3 were identified from patients with neural tube defects (NTDs), but their functions in the pathogenesis of human neural diseases remain elusive. Here, we sequenced the coding regions of three DVL genes in 176 stillborn or miscarried fetuses with NTDs or Dandy-Walker malformation (DWM) and 480 adult controls from a Han Chinese population. Four rare mutations were identified: DVL1 p.R558H, DVL1 p.R606C, DVL2 p.R633W, and DVL3 p.R222Q. To assess the effect of these mutations on NTDs and DWM, various functional analyses such as luciferase reporter assay, stress fiber formation, and in vivo teratogenic assay were performed. The results showed that the DVL2 p.R633W mutation destabilized DVL2 protein and upregulated activities for all three Wnt signalings (Wnt/β-catenin signaling, Wnt/planar cell polarity signaling, and Wnt/Ca 2 + signaling) in mammalian cells. In contrast, DVL1 mutants (DVL1 p.R558H and DVL1 p.R606C) decreased canonical Wnt/β-catenin signaling but increased the activity of Wnt/Ca 2 + signaling, and DVL3 p.R222Q only decreased the activity of Wnt/Ca 2 + signaling. We also found that only the DVL2 p.R633W mutant displayed more severe teratogenicity in zebrafish embryos than wild-type DVL2. Our study demonstrates that these four rare DVL mutations, especially DVL2 p.R633W, may contribute to human neural diseases such as NTDs and DWM by obstructing Wnt signaling pathways., (Copyright © 2020 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Objective: To examine occurrence of severe adverse fetal outcomes (SAO), including fetal loss and major congenital malformations (MCMs), in pregnant women with epilepsy (PWWE) vs healthy pregnant women (HPW)., Methods: The Maternal Outcomes and Neurodevelopmental Effects of Antiepileptic Drugs (MONEAD) study is an NIH-funded, prospective, observational, multicenter investigation of pregnancy outcomes for both mother and child, which enrolled women December 2012 through January 2016., Results: The 351 PWWE had 365 conceptions, and 105 HPW had 109 conceptions. SAOs occurred more often in PWWE (7.9%) vs HPW (1.9%) ( p = 0.025) with odds ratio (OR) 4.45 (95% confidence intervals [CI] 1.04-19.01). There were no significant differences for fetal loss (2.8% vs 0%, p = 0.126) or MCMs (5.2% vs 1.9%, p = 0.185; OR 2.86, 95% CI 0.65-12.53) individually. No fetal losses in PWWE appeared to be related to acute seizures. Outcomes were not affected by periconceptional folate, unplanned/unwanted pregnancies, prior maternal pregnancy history, or antiepileptic drug (AED) blood levels, except for an AED level effect for fetal loss that appeared to be due to polytherapy. Combined maternal or paternal family history of MCM was marginally associated with increased SAOs ( p = 0.046)., Conclusions: The findings provide additional information on risks of SAOs in PWWE, assessing effects of both AED levels and periconceptional folate. Group differences in average enrollment gestational age could have affected fetal loss results. Analyses are limited by small sample sizes as the MONEAD study was not powered for these secondary outcomes. The large majority of pregnancies in women with epilepsy do not have SOAs., (© 2019 American Academy of Neurology.)