A roadmap for the Human Developmental Cell Atlas.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice.

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the distal spine. We have previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here, we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling, and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH- and FOXA2-positive ventral NT domains, resulting in OLIG2 labelling of the ventral-most NT. The OLIG2 domain is also subsequently lost, eventually producing a NT that is entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Association of embryonic inositol status with susceptibility to neural tube defects, metabolite profile, and maternal inositol intake.

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry.

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry.

AAV-mediated expression of mouse or human GLDC normalises metabolic biomarkers in a GLDC-deficient mouse model of Non-Ketotic Hyperglycinemia.

Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH have limited effect and are not curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using a ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one­carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one­carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.

Hindbrain neuropore tissue geometry determines asymmetric cell-mediated closure dynamics in mouse embryos.

Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse strings at their leading edge and establish the initial contacts across the embryonic midline. Fibronectin and laminin are present, and tensin 1 accumulates in focal adhesion-like puncta at this leading edge. The HNP gap closes asymmetrically, faster from its rostral than caudal end, while maintaining an elongated aspect ratio. Cell-based physical modeling identifies two closure mechanisms sufficient to account for tissue-level HNP closure dynamics: purse-string contraction and directional cell motion implemented through active crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by asymmetric embryo tissue geometry, namely a narrower rostral gap apex, whereas biomechanical tension inferred from laser ablation is equivalent at the gaps' rostral and caudal closure points. At the cellular level, the physical model predicts rearrangements of cells at the HNP rostral and caudal extremes as the gap shortens. These behaviors are reproducibly live imaged in mouse embryos. Thus, mammalian embryos coordinate cellular- and tissue-level mechanics to achieve this critical gap closure event.

A topographical analysis of encephalocele locations: generation of a standardised atlas and cluster analysis.

OBJECTIVE: Encephaloceles are considered to result from defects in the developing skull through which meninges, and potentially brain tissue, herniate. The pathological mechanism underlying this process is incompletely understood. We aimed to describe the location of encephaloceles through the generation of a group atlas to determine whether they occur at random sites or clusters within distinct anatomical regions. METHODS: Patients diagnosed with cranial encephaloceles or meningoceles were identified from a prospectively maintained database between 1984 and 2021. Images were transformed to atlas space using non-linear registration. The bone defect, encephalocele and herniated brain contents were manually segmented allowing for a 3-dimensional heat map of encephalocele locations to be generated. The centroids of the bone defects were clustered utilising a K-mean clustering machine learning algorithm in which the elbow method was used to identify the optimal number of clusters. RESULTS: Of the 124 patients identified, 55 had volumetric imaging in the form of MRI (48/55) or CT (7/55) that could be used for atlas generation. Median encephalocele volume was 14,704 (IQR 3655-86,746) mm3 and the median surface area of the skull defect was 679 (IQR 374-765) mm2. Brain herniation into the encephalocele was found in 45% (25/55) with a median volume of 7433 (IQR 3123-14,237) mm3. Application of the elbow method revealed 3 discrete clusters: (1) anterior skull base (22%; 12/55), (2) parieto-occipital junction (45%; 25/55) and (3) peri-torcular (33%; 18/55). Cluster analysis revealed no correlation between the location of the encephalocele with gender (χ2 (2, n = 91) = 3.86, p = 0.15). Compared to expected population frequencies, encephaloceles were relatively more common in Black, Asian and Other compared to White ethnicities. A falcine sinus was identified in 51% (28/55) of cases. Falcine sinuses were more common (χ2 (2, n = 55) = 6.09, p = 0.05) whilst brain herniation was less common (χ2 (2, n = 55) = .16.24, p < 0.0003) in the parieto-occipital location. CONCLUSION: This analysis revealed three predominant clusters for the location of encephaloceles, with the parieto-occipital junction being the most common. The stereotypic location of encephaloceles into anatomically distinct clusters and the coexistence of distinct venous malformations at certain sites suggests that their location is not random and raises the possibility of distinct pathogenic mechanisms unique to each of these regions.

Neural tube defects.

Neural tube defects (NTDs), including spina bifida and anencephaly, are severe birth defects of the central nervous system that originate during embryonic development when the neural tube fails to close completely. Human NTDs are multifactorial, with contributions from both genetic and environmental factors. The genetic basis is not yet well understood, but several nongenetic risk factors have been identified as have possibilities for prevention by maternal folic acid supplementation. Mechanisms underlying neural tube closure and NTDs may be informed by experimental models, which have revealed numerous genes whose abnormal function causes NTDs and have provided details of critical cellular and morphological events whose regulation is essential for closure. Such models also provide an opportunity to investigate potential risk factors and to develop novel preventive therapies.

Investigating Genetic Determinants of Plasma Inositol Status in Adult Humans.

BACKGROUND: Myo-inositol (MI) is incorporated into numerous biomolecules, including phosphoinositides and inositol phosphates. Disturbance of inositol availability or metabolism is associated with various disorders, including neurological conditions and cancers, whereas supplemental MI has therapeutic potential in conditions such as depression, polycystic ovary syndrome, and congenital anomalies. Inositol status can be influenced by diet, synthesis, transport, utilization, and catabolism. OBJECTIVES: We aimed to investigate potential genetic regulation of circulating MI status and to evaluate correlation of MI concentration with other metabolites. METHODS: GC-MS was used to determine plasma MI concentration of >2000 healthy, young adults (aged 18-28 y) from the Trinity Student Study. Genotyping data were used to test association of plasma MI with single nucleotide polymorphisms (SNPs) in candidate genes, encoding inositol transporters and synthesizing enzymes, and test for genome-wide association. We evaluated potential correlation of plasma MI with d-chiro-inositol (DCI), glucose, and other metabolites by Spearman rank correlation. RESULTS: Mean plasma MI showed a small but significant difference between males and females (28.5 and 26.9 µM, respectively). Candidate gene analysis revealed several nominally significant associations with plasma MI, most notably for SLC5A11 (solute carrier family 5 member 11), encoding a sodium-coupled inositol transporter, also known as SMIT2 (sodium-dependent myo-inositol transporter 2). However, these did not survive correction for multiple testing. Subsequent testing for genome-wide association with plasma MI did not identify associations of genome-wide significance (P < 5 × 10-8). However, 8 SNPs exceeded the threshold for suggestive significant association with plasma MI concentration (P < 1 × 10-5), 3 of which were located within or close to genes: MTDH (metadherin), LAPTM4B (lysosomal protein transmembrane 4 ß), and ZP2 (zona pellucida 2). We found significant positive correlation of plasma MI concentration with concentration of dci and several other biochemicals including glucose, methionine, betaine, sarcosine, and tryptophan. CONCLUSIONS: Our findings suggest potential for modulation of plasma MI in young adults by variation in SLC5A11, which is worthy of further investigation.