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Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within ECM, cilia and actin networks, but the genetic architecture and underlying mechanisms remains unresolved. Here, we used whole exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (MAF C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of a second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7(co63/co63) zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7(co63/co63) embryos, although alterations were seen in Hh pathway gene expression. This research suggests defects in KIF7-dependent Hh signaling may drive pathogenesis in a subset of individuals with IS.

Neural tube closure (NTC) is crucial for proper development of the brain and spinal cord and requires precise morphogenesis from a sheet of cells to an intact three-dimensional structure. NTC is dependent on successful regulation of hundreds of genes, a myriad of signaling pathways, concentration gradients, and is influenced by epigenetic and environmental cues. Failure of NTC is termed a neural tube defect (NTD) and is a leading class of congenital defects in the United States and worldwide. Though NTDs are all defined as incomplete closure of the neural tube, the pathogenesis of an NTD determines the type, severity, positioning, and accompanying phenotypes. In this review, we survey pathogenesis of NTDs relating to disruption of cellular processes arising from genetic mutations, altered epigenetic regulation, and environmental influences by micronutrients and maternal condition. This article is categorized under: Congenital DiseasesGenetics/Genomics/Epigenetics Neurological DiseasesGenetics/Genomics/Epigenetics Neurological DiseasesStem Cells and Development.

Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N=10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase (www.facebase.org, doi.org/10.25550/3-HXMC) and GitHub (https://github.com/jaydevine/MusMorph).

Neural tube defects are among the most common birth defects, with a prevalence of close to 19 per 10,000 births worldwide. The etiology of neural tube defects (NTD) is complex involving the interplay of genetic and environmental factors. Since nutrient deficiency is a risk factor and dietary changes are the major preventative measure to reduce the risk of NTDs, a more detailed understanding of how common micronutrient imbalances contribute to NTDs is crucial. While folic acid has been the most discussed environmental factor due to the success that population-wide fortification has had on prevention of NTDs, folic acid supplementation does not prevent all NTDs. The imbalance of several other micronutrients have been implicated as risks for NTDs by epidemiological studies and in vivo studies in animal models. In this review, we highlight recent literature deciphering the multifactorial mechanisms underlying NTDs with an emphasis on mouse and human data. Specifically, we focus on advances in our understanding of how too much or too little retinoic acid, zinc, and iron alter gene expression and cellular processes contributing to the pathobiology of NTDs. Synthesis of the discussed literature reveals common cellular phenotypes found in embryos with NTDs resulting from several micronutrient imbalances. The goal is to combine knowledge of these common cellular phenotypes with mechanisms underlying micronutrient imbalances to provide insights into possible new targets for preventative measures against NTDs.

Background Myelomeningocele (MMC) results in lifelong neurologic and functional deficits. Currently, prenatal repair of MMC closes the defect, resulting in a 50% reduction in postnatal ventriculoperitoneal shunting. However, this invasive fetal surgery is associated with significant morbidities to mother and baby. We have pioneered a novel reverse thermal gel (RTG) to cover MMC defects in a minimally invasive manner. Here, we test in-vitro RTG long-term stability in amniotic fluid and in vivo application in the Grainy head-like 3 (Grhl3) mouse MMC model. Materials and methods RTG stability in amniotic fluid (in-vitro) was monitored for 6 mo and measured using gel permeation chromatography and solution–gel transition temperature (lower critical solution temperature). E16.5 Grhl3 mouse fetuses were injected with the RTG or saline and harvested on E19.5. Tissue was assessed for RTG coverage of the gross defect and inflammatory response by immunohistochemistry for macrophages. Results Polymer backbone molecular weight and lower critical solution temperature remain stable in amniotic fluid after 6 mo. Needle injection over the MMC of Grhl3 fetuses successfully forms a stable gel that covers the entire defect. On harvest, some animals demonstrate >50% RTG coverage. RTG injection is not associated with inflammation. Conclusions Our results demonstrate that the RTG is a promising candidate for a minimally invasive approach to patch MMC. We are now poised to test our RTG patch in the large preclinical ovine model used to evaluate prenatal repair of MMC.

Disruptions in neural tube (NT) closure result in neural tube defects (NTDs). To understand the molecular processes required for mammalian NT closure, we investigated the role of Snx3, a sorting nexin gene. Snx3−/− mutant mouse embryos display a fully-penetrant cranial NTD. In vivo, we observed decreased canonical WNT target gene expression in the cranial neural epithelium of the Snx3−/− embryos and a defect in convergent extension of the neural epithelium. Snx3−/− cells show decreased WNT secretion, and live cell imaging reveals aberrant recycling of the WNT ligand-binding protein WLS and mis-trafficking to the lysosome for degradation. The importance of SNX3 in WNT signaling regulation is demonstrated by rescue of NT closure in Snx3−/− embryos with a WNT agonist. The potential for SNX3 to function in human neurulation is revealed by a point mutation identified in an NTD-affected individual that results in functionally impaired SNX3 that does not colocalize with WLS and the degradation of WLS in the lysosome. These data indicate that Snx3 is crucial for NT closure via its role in recycling WLS in order to control levels of WNT signaling.

Wnt signaling pathway plays indispensable roles in embryonic development and adult tissue homeostasis. However, the regulatory mechanisms involved in Wnt ligand trafficking within and secretion from the signal sending cells is still relatively uncharacterized. Here, we discover a novel regulator of Wnt signaling pathway called transmembrane protein 132A (TMEM132A). Our evidence shows a physical and functional interaction of TMEM132A with the Wnt ligand transporting protein Wntless (WLS). We show that TMEM132A stabilizes Wnt ligand, enhances WLS–Wnt ligand interaction, and activates the Wnt signaling pathway. Our results shed new light on the cellular mechanism underlying the fundamental aspect of WNT secretion from Wnt signal sending cells.

Development of the craniofacial structures requires the precise differentiation of cranial neural crest cells into osteoblasts or chondrocytes. Here, we explore the epigenetic and non-epigenetic mechanisms that are required for the development of craniofacial chondrocytes. We previously demonstrated that the acetyltransferase activity of the highly conserved acetyltransferase GCN5, or KAT2A, is required for murine craniofacial development. To further test the potential cell autonomous function, we hypothesize that GCN5 is required for chondrocyte development following the arrival of the cranial neural crest within the pharyngeal arches. Here, we show that Gcn5 is required cell autonomously in the cranial neural crest. Using a combination of in vivo and in vitro inhibition of GCN5 acetyltransferase activity, we demonstrate that GCN5 is a potent activator of chondrocyte maturation, acting to control chondrocyte maturation and size increase during pre-hypertrophic maturation to hypertrophic chondrocytes. Rather than acting as an epigenetic regulator of histone H3K9 acetylation, our findings suggest GCN5 primarily acts as a non-histone acetyltransferase to regulate chondrocyte development. Here, we investigate the contribution of GCN5 acetylation to the activity of the mTORC1 pathway. Our findings indicate that GCN5 acetylation is required for activation of this pathway, either via direct activation of mTORC1 or through indirect mechanisms. We also investigate one possibility of how mTORC1 activity is regulated through RAPTOR acetylation, which is hypothesized to enhance mTORC1 downstream phosphorylation. This study contributes to our understanding of the specificity of acetyltransferases, and the cell type specific roles in which these enzymes function.

Idiopathic scoliosis (IS) is a structural lateral spinal curvature of ≥10° that affects up to 3% of otherwise healthy children and can lead to life-long problems in severe cases. It is well-established that IS is a genetic disorder. Previous studies have identified genes that may contribute to the IS phenotype, but the overall genetic etiology of IS is not well understood. We used exome sequencing to study five multigenerational families with IS. Bioinformatic analyses identified unique and low frequency variants (minor allele frequency ≤5%) that were present in all sequenced members of the family. Across the five families, we identified a total of 270 variants with predicted functional consequences in 246 genes, and found that eight genes were shared by two families. We performed GO term enrichment analyses, with the hypothesis that certain functional annotations or pathways would be enriched in the 246 genes identified in our IS families. Using three complementary programs to complete these analyses, we identified enriched categories that include stereocilia and other actin-based cellular projections, cilia and other microtubule-based cellular projections, and the extracellular matrix (ECM). Our results suggest that there are multiple paths to IS and provide a foundation for future studies of IS pathogenesis.

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat)), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused byin vitroinhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENTChanges in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 – a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Micronutrition is essential for neural tube closure, and zinc deficiency is associated with human neural tube defects. Here, we modeled zinc deficiency in mouse embryos, and used live imaging and molecular studies to determine how zinc deficiency affects neural tube closure. Embryos cultured with the zinc chelator TPEN failed to close the neural tube and showed excess apoptosis. TPEN-induced p53 protein stabilization in vivo and in neuroepithelial cell cultures and apoptosis was dependent on p53. Mechanistically, zinc deficiency resulted in disrupted interaction between p53 and the zinc-dependent E3 ubiquitin ligase Mdm2, and greatly reduced p53 ubiquitylation. Overexpression of human CHIP, a zinc-independent E3 ubiquitin ligase that targets p53, relieved TPEN-induced p53 stabilization and reduced apoptosis. Expression of p53 pro-apoptotic target genes was upregulated by zinc deficiency. Correspondingly, embryos cultured with p53 transcriptional activity inhibitor pifithrin-α could overcome TPEN-induced apoptosis and failure of neural tube closure. Our studies indicate that zinc deficiency disrupts neural tube closure through decreased p53 ubiquitylation, increased p53 stabilization and excess apoptosis.

Intratumoral genetic heterogeneity is a widely accepted characteristic of human cancer, including the most common primary malignant brain tumor, glioblastoma. However, the variability in biological behaviors amongst cells within individual tumors is not well described. Invasion into unaffected brain parenchyma is one such behavior, and a leading mechanism of tumor recurrence unaddressed by the current therapeutic armamentarium. Further, providing insight into variability of tumor cell migration within individual tumors may inform discovery of novel anti-invasive therapeutics. In this study, ex vivo organotypic slice cultures from EGFR-wild type and EGFR-amplified patient tumors were treated with the EGFR inhibitor gefitinib to evaluate potential sub-population restricted intratumoral drug-specific responses. High-resolution time-lapse microscopy and quantitative path tracking demonstrated migration of individual cells are punctuated by intermittent bursts of movement. Elevation of population aggregate mean speeds were driven by subpopulations of cells exhibiting frequent high-amplitude bursts, enriched within EGFR-amplified tumors. Treatment with gefitinib specifically targeted high-burst cell subpopulations only in EGFR-amplified tumors, decreasing bursting frequency and amplitude. We provide evidence of intratumoral subpopulations of cells with enhanced migratory behavior in human glioblastoma, selectively targeted via EGFR inhibition. These data justify use of direct human tumor slice cultures to investigate patient-specific therapies designed to limit tumor invasion.

Cranial neural crest cells undergo cellular growth, patterning, and differentiation within the branchial arches to form cartilage and bone, resulting in a precise pattern of skeletal elements forming the craniofacial skeleton. However, it is unclear how cranial neural crest cells are regulated to give rise to the different shapes and sizes of the bone and cartilage. Epigenetic regulators are good candidates to be involved in this regulation, since they can exert both broad as well as precise control on pattern formation. Here, we investigated the role of the histone acetyltransferases Kat2a and Kat2b in craniofacial development using TALEN/CRISPR/Cas9 mutagenesis in zebrafish and the Kat2ahat/hat (also called Gcn5) allele in mice. kat2a and kat2b are broadly expressed during embryogenesis within the central nervous system and craniofacial region. Single and double kat2a and kat2b zebrafish mutants have an overall shortening and hypoplastic nature of the cartilage elements and disruption of the posterior ceratobranchial cartilages, likely due to smaller domains of expression of both cartilage- and bone-specific markers, including sox9a and col2a1, and runx2a and runx2b, respectively. Similarly, in mice we observe defects in the craniofacial skeleton, including hypoplastic bone and cartilage and altered expression of Runx2 and cartilage markers (Sox9, Col2a1). In addition, we determined that following the loss of Kat2a activity, overall histone 3 lysine 9 (H3K9) acetylation, the main epigenetic target of Kat2a/Kat2b, was decreased. These results suggest that Kat2a and Kat2b are required for growth and differentiation of craniofacial cartilage and bone in both zebrafish and mice by regulating H3K9 acetylation.

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.

Locomotion and respiration require motor axon connectivity and activation of the neuromuscular junction (NMJ). Through a forward genetic screen for muscle weakness, we recently reported an allele of ryanodine receptor type 1 (Ryr1AG). Here we reveal a role for functional RyR1 during acetylcholine receptor (AChR) cluster formation and embryonic synaptic transmission. Ryr1AG homozygous embryos are non-motile. Motor axons extend past AChR clusters and enlarged AChR clusters are found under fasciculated nerves. Using physiological and pharmacological methods, we show that contractility can be resumed through the masking of a potassium leak, and evoked vesicular release can be resumed via bypassing the defect in RyR1 induced calcium release. Moreover, we show the involvement of ryanodine receptors in presynaptic release at the NMJ. This data provides evidence of a role for RyR1 on both the pre- and postsynaptic sides of the NMJ.

Background: Cilia are important for Hedgehog signaling in vertebrates and many genes that encode proteins involved in ciliogenesis have been studied for their roles in embryonic development. Null mutations in many of these genes cause early embryonic lethality, hence an understanding of their roles in postnatal development is limited. Results: The Inturned (Intu) gene is required for ciliogenesis and here we report a recessive hypomorphic mutation, resulting in substitution of a conserved hydrophobic residue (I813N) near the C-terminus, that sheds light on later functions of Intu. Mice homozygous for this Double-thumb (IntuDtm) allele exhibit polydactyly, retarded growth, and reduced survival. There is a moderate loss of cilia in IntuDtm/Dtm mutants, and IntuI813N exhibits compromised ability to increase ciliogenesis in cultured Intu null mutant cells. IntuDtm mutants show rib defects and delay of endochondral ossification in long bones, digits, vertebrae, and the sternum. These skeletal defects correlate with a decrease in Hh signaling. However, patterning of the neural tube and planar cell polarity appear to be normal. Conclusions: This hypomorphic Intu allele highlights an important role of Intu in mouse skeletal development. Developmental Dynamics 244:736–747, 2015. © 2015 Wiley Periodicals, Inc.

Neural progenitor cells (NPCs) have distinct proliferation capacities at different stages of brain development. Lin28 is an RNA-binding protein with two homologs in mice: Lin28a and Lin28b. Here we show that Lin28a/b are enriched in early NPCs and their expression declines during neural differentiation. Lin28a single-knockout mice show reduced NPC proliferation, enhanced cell cycle exit and a smaller brain, whereas mice lacking both Lin28a alleles and one Lin28b allele display similar but more severe phenotypes. Ectopic expression of Lin28a in mice results in increased NPC proliferation, NPC numbers and brain size. Mechanistically, Lin28a physically and functionally interacts with Imp1 (Igf2bp1) and regulates Igf2-mTOR signaling. The function of Lin28a/b in NPCs could be attributed, at least in part, to the regulation of their mRNA targets that encode Igf1r and Hmga2. Thus, Lin28a and Lin28b have overlapping functions in temporally regulating NPC proliferation during early brain development.

Rare variants are considered underlying causes of complex diseases. The complex and severe group of disorders called neural tube defects (NTDs) results from failure of the neural tube to close during early embryogenesis. Neural tube closure requires the coordination of numerous signaling pathways, including the precise regulation of retinoic acid (RA) concentration, which is controlled by enzymes involved in RA synthesis and degradation. Here, we used a case-control mutation screen study to reveal rare variants in retinoid-related genes in a Han Chinese NTD population by sequencing six genes in 355 NTD cases and 225 controls. More specific rare variants were found in exonic and upstream regions in NTD cases. The RA-responsive genes CYP26A1, CRABP1, and ALDH1A2 harbored NTD-specific rare variants in their upstream regions. Unexpectedly, the majority of missense variants in NTD cases were found in CYP26B1, which encodes a RA degradation enzyme, whereas no missense variants in this gene were found in controls. Functional analysis indicated that the CYP26B1 NTD variants were inefficient in the degradation of RA using assays of RA-induced transcription and RA-initiated neuronal differentiation. Our study supports the contribution of rare variants in RA-related genes to the etiology of human NTDs.

Signals from nerve and muscle regulate the formation of synapses. Transgenic mouse models and muscle cell cultures have elucidated the molecular mechanisms required for aggregation and stabilization of synaptic structures. However, far less is known about the molecular pathways involved in redistribution of muscle synaptic components. Here we established a physiologically viable whole-muscle embryonic explant system, in the presence or absence of the nerve, which demonstrates the synaptic landscape is dynamic and malleable. Manipulations of factors intrinsic to the muscle or extrinsically provided by the nerve illustrate vital functions during formation, redistribution and elimination of acetylcholine receptor (AChR) clusters. In particular, RyR1 activity is an important mediator of these functions. This physiologically relevant and readily accessible explant system provides a new approach to genetically uncouple nerve-derived signals and for manipulation via signaling molecules, drugs, and electrical stimulation to examine early formation of the neuromuscular circuit.

The formation of the embryonic brain and spinal cord begins as the neural plate bends to form the neural folds, which meet and adhere to close the neural tube. The neural ectoderm and surrounding tissues also coordinate proliferation, differentiation, and patterning. This highly orchestrated process is susceptible to disruption, leading to neural tube defects (NTDs), a common birth defect. Here, we highlight genetic and epigenetic contributions to neural tube closure. We describe an online database we created as a resource for researchers, geneticists, and clinicians. Neural tube closure is sensitive to environmental influences, and we discuss disruptive causes, preventative measures, and possible mechanisms. New technologies will move beyond candidate genes in small cohort studies toward unbiased discoveries in sporadic NTD cases. This will uncover the genetic complexity of NTDs and critical gene-gene interactions. Animal models can reveal the causative nature of genetic variants, the genetic interrelationships, and the mechanisms underlying environmental influences.

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155msp3, a unique ENU-induced allele in mice. Homozygous Baf155mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA-Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014

Mouse models provide valuable opportunities for probing the underlying pathology of human birth defects. By using an N-ethyl-N-nitrosourea-based screen for recessive mutations affecting craniofacial anatomy, we isolated a mouse strain, Dogface-like (DL), with abnormal skull and snout morphology. Examination of the skull indicated that these mice developed craniosynostosis of the lambdoid suture. Further analysis revealed skeletal defects related to the pathology of basal cell nevus syndrome (BCNS) including defects in development of the limbs, scapula, ribcage, secondary palate, cranial base, and cranial vault. In humans, BCNS is often associated with mutations in the Hedgehog receptor PTCH1 and genetic mapping in DL identified a point mutation at a splice donor site in Ptch1. By using genetic complementation analysis we determined that DL is a hypomorphic allele of Ptch1, leading to increased Hedgehog signaling. Two aberrant transcripts are generated by the mutated Ptch1(DL) gene, which would be predicted to reduce significantly the levels of functional Patched1 protein. This new Ptch1 allele broadens the mouse genetic reagents available to study the Hedgehog pathway and provides a valuable means to study the underlying skeletal abnormalities in BCNS. In addition, these results strengthen the connection between elevated Hedgehog signaling and craniosynostosis.

The neural tube (NT), the embryonic precursor of the vertebrate brain and spinal cord, is generated by a complex and highly dynamic morphological process. In mammals, the initially flat neural plate bends and lifts bilaterally to generate the neural folds followed by fusion of the folds at the midline during the process of neural tube closure (NTC). Failures in any step of this process can lead to neural tube defects (NTDs), a common class of birth defects that occur in approximately 1 in 1000 live births. These severe birth abnormalities include spina bifida, a failure of closure at the spinal level; craniorachischisis, a failure of NTC along the entire body axis; and exencephaly, a failure of the cranial neural folds to close which leads to degeneration of the exposed brain tissue termed anencephaly. The mouse embryo presents excellent opportunities to explore the genetic basis of NTC in mammals; however, its in utero development has also presented great challenges in generating a deeper understanding of how gene function regulates the cell and tissue behaviors that drive this highly dynamic process. Recent technological advances are now allowing researchers to address these questions through visualization of NTC dynamics in the mouse embryo in real time, thus offering new insights into the morphogenesis of mammalian NTC.

Background: Lung-branching morphogenesis is a fundamental developmental process, yet the cellular dynamics that occur during lung development and the molecular mechanisms underlying recent postulated branching modes are poorly understood. Results: Here, we implemented a time-lapse video microscopy method to study the cellular behavior and molecular mechanisms of planar bifurcation and domain branching in lung explant- and organotypic cultures. Our analysis revealed morphologically distinct stages that are shaped at least in part by a combination of localized and orientated cell divisions and by local mechanical forces. We also identified myosin light-chain kinase as an important regulator of bud bifurcation, but not domain branching in lung explants. Conclusions: This live imaging approach provides a method to study cellular behavior during lung-branching morphogenesis and suggests the importance of a mechanism primarily based on oriented cell proliferation and mechanical forces in forming and shaping the developing lung airways. Developmental Dynamics 242:622–637, 2013. © 2013 Wiley Periodicals, Inc.

In the field of developmental biology, live imaging is a powerful tool for studying, in real time, the dynamic behaviors of tissues and cells during organ formation. Mammals, which develop in utero, have presented a challenge for live imaging. Here, we offer a novel, prolonged and robust live imaging system for visualizing the development of a variety of embryonic tissues in the midgestation mouse embryo. We demonstrate the advantages of this imaging system by following the dynamics of neural tube closure during mouse embryogenesis and reveal extensive movements of the cranial neural tissue that are independent of neural fold zipping.

Harlequin Ichthyosis is a severe skin disease caused by mutations in the human gene encoding ABCA12. Here, we characterize a novel mutation in intron 29 of the mouse Abca12 gene that leads to the loss of a 5' splice donor site and truncation of the Abca12 RNA transcript. Homozygous mutants of this smooth skin or smsk allele die perinatally with shiny translucent skin, typical of animal models of Harlequin Ichthyosis. Characterization of smsk mutant skin showed that the delivery of glucosylceramides and CORNEODESMOSIN was defective, while ultrastructural analysis revealed abnormal lamellar bodies and the absence of lipid lamellae in smsk epidermis. Unexpectedly, mutant stratum corneum remained intact when subjected to harsh chemical dissociation procedures. Moreover, both KALLIKREIN 5 and -7 were drastically decreased, with retention of desmoplakin in mutant SC. In cultured wild type keratinocytes, both KALLIKREIN 5 and -7 colocalized with ceramide metabolites following calcium-induced differentiation. Reducing the intracellular levels of glucosylceramide with a glucosylceramide synthase inhibitor resulted in decreased secretion of KALLIKREIN proteases by wild type keratinocytes, but not by smsk mutant keratinocytes. Together, these findings suggest an essential role for ABCA12 in transferring not only lipids, which are required for the formation of multilamellar structures in the stratum corneum, but also proteolytic enzymes that are required for normal desquamation. Smsk mutant mice recapitulate many of the pathological features of HI and can be used to explore novel topical therapies against a potentially lethal and debilitating neonatal disease.

The embryonic brain and spinal cord initially form through the process of neural tube closure (NTC). NTC is thought to be highly similar between rodents and humans, and studies of mouse genetic mutants have greatly increased our understanding of the molecular basis of NTC with relevance for human neural tube defects. In addition, studies using amphibian and chick embryos have shed light into the cellular and tissue dynamics underlying NTC. However, the dynamics of mammalian NTC has been difficult to study due to in utero development until recently when advances in mouse embryo ex vivo culture techniques along with confocal microscopy have allowed for imaging of mouse NTC in real time. Here, we have performed live imaging of mouse embryos with a particular focus on the non-neural ectoderm (NNE). Previous studies in multiple model systems have found that the NNE is important for proper NTC, but little is known about the behavior of these cells during mammalian NTC. Here we utilized a NNE-specific genetic labeling system to assess NNE dynamics during murine NTC and identified different NNE cell behaviors as the cranial region undergoes NTC. These results bring valuable new insight into regional differences in cellular behavior during NTC that may be driven by different molecular regulators and which may underlie the various positional disruptions of NTC observed in humans with neural tube defects.

Tissue fusion events during embryonic development are crucial for the correct formation and function of many organs and tissues, including the heart, neural tube, eyes, face and body wall. During tissue fusion, two opposing tissue components approach one another and integrate to form a continuous tissue; disruption of this process leads to a variety of human birth defects. Genetic studies, together with recent advances in the ability to culture developing tissues, have greatly enriched our knowledge of the mechanisms involved in tissue fusion. This review aims to bring together what is currently known about tissue fusion in several developing mammalian organs and highlights some of the questions that remain to be addressed.

Hirschsprung disease (HSCR) is caused by a reduction of enteric neural crest cells (ENCCs) in the gut and gastrointestinal blockage. Knowledge of the genetics underlying HSCR is incomplete, particularly genes that control cellular behaviors of ENCC migration. Here we report a novel regulator of ENCC migration in mice. Disruption of the Phactr4 gene causes an embryonic gastrointestinal defect due to colon hypoganglionosis, which resembles human HSCR. Time-lapse imaging of ENCCs within the embryonic gut demonstrates a collective cell migration defect. Mutant ENCCs show undirected cellular protrusions and disrupted directional and chain migration. Phactr4 acts cell-autonomously in ENCCs and colocalizes with integrin and cofilin at cell protrusions. Mechanistically, we show that Phactr4 negatively regulates integrin signaling through the RHO/ROCK pathway and coordinates protein phosphatase 1 (PP1) with cofilin activity to regulate cytoskeletal dynamics. Strikingly, lamellipodia formation and in vivo ENCC chain migration defects are rescued by inhibition of ROCK or integrin function. Our results demonstrate a previously unknown pathway in ENCC collective migration in vivo and provide new candidate genes for human genetic studies of HSCR.

Neural tube defects (NTDs), a common birth defect in humans, result from the failure of the embryonic neural tube (NT) to close properly. NT closure is a complex, poorly understood morphogenetic process influenced by genes and environment. The most effective environmental influence in decreasing the risk for NTDs is folic acid (FA) fortification and supplementation, and these findings led to the recommendation of periconceptual FA intake and mandatory fortification of the US grain supply in 1998. To explore the relationship between genetics and responsiveness to FA supplementation, we used five mouse NTDs models-Zic2, Shroom3, Frem2, Grhl2 (Grainyhead-like 2) and L3P (Line3P)-and a long-term generational FA supplementation scheme. Contrary to expectations, we find that three genetic mutants respond adversely to FA supplementation with increased incidence of NTDs in homozygous mutants, occurrence of NTDs in heterozygous embryos and embryonic lethality prior to NT closure. Because of these unexpected responses, we examined NTD risk after short-term FA supplementation. Our results indicate that, for the same genetic allele, NTD risk can depend on the length of FA exposure. Our data indicate that, depending on the gene mutation, FA supplementation may adversely influence embryonic development and NT closure.

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with reduced ranges of motility. We further show that CCDC40 mutations in humans result in a variant of PCD characterized by misplacement of the central pair of microtubules and defective assembly of inner dynein arms and dynein regulatory complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is required for axonemal recruitment of CCDC39, disruption of which underlies a similar variant of PCD.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1ffe/ffe mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1ffe/KI mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Neurulation, the process of neural tube formation, is a complex morphogenetic event. In the mammalian embryo, an understanding of the dynamic nature of neurulation has been hampered due to its in utero development. Here we use laser point scanning confocal microscopy of a membrane expressed fluorescent protein to visualize the dynamic cell behaviors comprising neural tube closure in the cultured mouse embryo. In particular, we have focused on the final step wherein the neural folds approach one another and seal to form the closed neural tube. Our unexpected findings reveal a mechanism of closure in the midbrain different from the zipper-like process thought to occur more generally. Individual non-neural ectoderm cells on opposing sides of the neural folds undergo a dramatic change in shape to protrude from the epithelial layer and then form intermediate closure points to “button-up” the folds. Cells from the juxtaposed neural folds extend long and short flexible extensions and form bridges across the physical gap of the closing folds. Thus, the combination of live embryo culture with dynamic imaging provides intriguing insight into the cell biological processes that mold embryonic tissues in mammals.

The lungs are generated by branching morphogenesis as a result of reciprocal signalling interactions between the epithelium and mesenchyme during development. Mutations that disrupt formation of either the correct number or shape of epithelial branches affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signalling pathways which drive epithelial tube formation will likely shed light on both congenital and adult lung disease. Here we show that mutations in the planar cell polarity (PCP) genes Celsr1 and Vangl2 lead to disrupted lung development and defects in lung architecture. Lungs from Celsr1(Crsh) and Vangl2(Lp) mouse mutants are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. We observe a recapitulation of these branching defects following inhibition of Rho kinase, an important downstream effector of the PCP signalling pathway. Moreover, epithelial integrity is disrupted, cytoskeletal remodelling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. We further show that Celsr1 and Vangl2 proteins are present in restricted spatial domains within lung epithelium. Our data show that the PCP genes Celsr1 and Vangl2 are required for foetal lung development thereby revealing a novel signalling pathway critical for this process that will enhance our understanding of congenital and adult lung diseases and may in future lead to novel therapeutic strategies.

The transcription factor Grainyhead-like 2 (GRHL2) is expressed in non-neural ectoderm (NNE) and Grhl2 loss results in fully penetrant cranial neural tube defects (NTDs) in mice. GRHL2 activates expression of several epithelial genes, however additional molecular targets and functional processes regulated by GRHL2 in the NNE remain to be determined, as well as the underlying cause of the NTDs in Grhl2 mutants. Here, we find that Grhl2 loss results in abnormal mesenchymal phenotypes in the NNE, including aberrant Vimentin expression and increased cellular dynamics that affects the NNE and neural crest cells. The resulting loss of NNE integrity contributes to an inability of the cranial neural folds to move toward the midline and results in NTD. Further, we identified Esrp1, Sostdc1, Fermt1, Tmprss2 and Lamc2, novel NNE-expressed genes that are downregulated in Grhl2 mutants. Our in vitro assays show they act as suppressors of the epithelial-to-mesenchymal transition (EMT). Thus, GRHL2 promotes the epithelial nature of the NNE during the dynamic events of neural tube formation by both activating key epithelial genes and actively suppressing EMT through novel downstream EMT suppressors.