Neurulation of the cynomolgus monkey embryo achieved from 3D blastocyst culture.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure.

Neural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage. Convergent extension takes place during the first stage and is spatially restricted at the posterior tissue, whereas apical constriction occurs during the second stage throughout the neural plate. We also show that the surface ectoderm is mechanically coupled with the neural plate and its movement during NTC is driven by neural plate morphogenesis. Finally, we show that an increase in surface ectoderm resistive forces is detrimental for neural plate morphogenesis.

A journey in the world of craniofacial development: From 1968 to the future.

This article is based on my talk at the meeting "3rd Advances in Craniosynostosis: Basic Science to Clinical Practice", held at University College, London, on 25 August 2023. It describes my contribution, together with that of my research team and external collaborators, to the field of craniofacial development. This began with my PhD research on the effects of excess vitamin A in rat embryos, which led to a study of normal as well as abnormal formation of the cranial neural tube. Many techniques for analysing morphogenetic change became available to me over the years: whole embryo culture, scanning and transmission electron microscopy, cell division analysis, immunohistochemistry and biochemical analysis of the extracellular matrix. The molecular revolution of the 1980s, and key collaborations with international research teams, enabled functional interpretation of some of the earlier morphological observations and required a change of experimental species to the mouse. Interactions between the molecular and experimental analysis of craniofacial morphogenesis in my laboratory with specialists in molecular genetics and clinicians brought my research journey near to my original aim: to contribute to a better understanding of the causes of human congenital anomalies.

Associations between exposure to extreme ambient heat and neural tube defects in Georgia, USA: A population-based case-control study.

INTRODUCTION: The association between extreme ambient heat exposures during pregnancy and neural tube defects (NTDs) in offspring remains unclear. This study sought to estimate the association between exposure to extreme ambient heat during periconception and NTDs. METHODS: This population-based case-control study in Georgia, USA (1994-2017) included 825 isolated NTD cases (473 anencephaly, 352 spina bifida) and 3,300 controls matched 1:4 on county of residence and time period of delivery. Daily ambient temperature data were linked to fetal death and birth records by county of residence. Extreme ambient heat exposure was defined as the number of consecutive days the daily apparent temperature exceeded the county-specific 95th percentile (derived over 1980-2010) during an eight-week periconception period. We calculated adjusted odds ratios (aORs) and 95% confidence intervals (CI) using conditional logistic regression models adjusted for maternal age, education, and ethnicity and month and year of last menstrual period. RESULTS: The aORs for NTDs were 1.09 (95% CI 1.01, 1.17), 1.18 (95% CI 1.03, 1.36), and 1.29 (95% CI 1.04, 1.58) for exposure to 1-2, 3-5, and 6 or more consecutive days with apparent ambient temperatures exceeding the county-specific 95th percentile during periconception, respectively, compared to no days of extreme ambient heat exposure. Weekly analysis of extreme heat exposure indicated consistently elevated odds of offspring NTDs during periconception. These results were largely driven by spina bifida cases. CONCLUSIONS: Our results highlight potential health threats posed by increasing global average temperatures for pregnant people with implications for increased risk of neural tube defects in their offspring.

Fibrous hamartoma of infancy of the spinal cord resembling conus and filum, with a coexisting sacral dimple.

Fibrous hamartoma of infancy (FHI) is a rare benign soft tissue lesion of infants and young children. It usually occurs within the first 2 years of life at the superficial layer of the axilla, trunk, upper arm, and external genitalia. FHI in the central nervous system (CNS) is extremely rare. So far, only two spinal cord FHI cases have been reported. We present a case of a 1-month-old girl who presented with a skin dimple in the coccygeal area. Her MRI showed a substantial intramedullary mass in the thoracolumbar area with a sacral soft tissue mass and a track between the skin lesion to the coccygeal tip. Her normal neurological status halted immediate surgical resection. A skin lesion biopsy was first performed, revealing limited information with no malignant cells. A short-term follow-up was performed until the intramedullary mass had enlarged on the 5-month follow-up MRI. Based on the frozen biopsy result of benign to low-grade spindle cell mesenchymal tumor, subtotal resection of the mass was done, minimizing damage to the functioning neural tissue. Both the skin lesion and the intramedullary mass were diagnosed as FHI. Postoperative 5.5-year follow-up MRI revealed minimal size change of the residual mass. Despite being diagnosed with a neurogenic bladder, the patient maintained her ability to void spontaneously, managed infrequent UTIs, and continued toilet training, all while demonstrating good mobility and no motor weakness. This case is unique because the lesion resembled the secondary neurulation structures, such as the conus and the filum, along with a related congenital anomaly of the dimple.

Clinical presentation and outcomes of neonates born with neural tube defects- an experience from a level III B NICU in Western India.

PURPOSE: Neural tube defects (NTDs) are one of the most common congenital anomalies and a cause of chronic disability. The study was done to study outcomes of neural tube defects admitted at a tertiary level neonatal intensive care unit (NICU) from 2018 to 2022, a period of 4 years that also coincided with the COVID pandemic. The secondary outcome was to study the clinical presentation, associated anomalies and epidemiological features. METHODS: It was a retrospective observational study; data of infants was obtained from medical records and analysis was done. RESULTS: Thirty-four neonates were enrolled, of which there were 16 (47%) males and 18 (53%) females. History of pre-pregnancy maternal folate intake was present in 4 (11.7%) cases. 33 (97%) babies were diagnosed with meningomyelocele (MMC) and one each had anencephaly, iniencephaly and encephalocele, of which one had frontal and two had occipital encephalocele. The median age of surgery was 16 days of life with primary repair being the most common procedure followed by MMC repair with VP shunt. Twenty babies (58.8%) were discharged successfully, while 9 (26.5%) expired and 5 (14.7%) were discharged against medical advice; which can be attributed to the financial problems of the patients in a developing country. The overall deaths in our series were four (26.5%) which is slightly higher than other studies which may be due to the fact that this study was conducted during the COVID era with lesser rates of folate supplementation, reduced access to prenatal diagnosis coupled with poor follow-up and compliance of patients post-surgical repair. CONCLUSION: This study emphasizes the importance of periconceptional folic acid supplementation, prenatal diagnosis, early surgery and meticulous follow-up as being pivotal to improving outcomes in children with NTDs.

Association of spinal cord abnormalities with vertebral anomalies: an embryological perspective.

PURPOSE: To analyze the relationship between spinal cord and vertebral abnormalities from the point of view of embryology. METHODS: We analyzed the clinical and radiological data of 260 children with different types of spinal cord malformations in combination with vertebral abnormalities. RESULTS: Among 260 individuals, approximately 109 presented with open neural tube defects (ONTDs), 83 with split cord malformations (SCMs), and 83 with different types of spinal lipomas. Pathological spina bifida emerged as the most frequent vertebral anomaly, affecting 232 patients, with a higher prevalence in ONTD. Vertebral segmentation disorders, including unsegmented bars, butterfly vertebrae, and hemivertebrae, were present in 124 cases, with a higher prevalence in SCM. The third most common spinal anomaly group consisted of various forms of sacral agenesis (58 cases), notably associated with blunt conus medullaris, spinal lipomas, and sacral myelomeningocele. Segmental aplasia of the spinal cord had a typical association with segmental spinal absence (N = 17). CONCLUSION: The association between SCM and neuroenteric cyst/canal and vertebral segmentation disorders is strong. High ONTDs often coincide with pathological spina bifida posterior. Type 1 spinal lipomas and focal spinal nondisjunction also correlate with pathologic spina bifida. Segmental spinal absence or dysgenesis involves localized spinal and spinal cord aplasia, sometimes with secondary filar lipoma.

Canonical and Non-Canonical Localization of Tight Junction Proteins during Early Murine Cranial Development.

This study investigates the intricate composition and spatial distribution of tight junction complex proteins during early mouse neurulation. The analyses focused on the cranial neural tube, which gives rise to all head structures. Neurulation brings about significant changes in the neuronal and non-neuronal ectoderm at a cellular and tissue level. During this process, precise coordination of both epithelial integrity and epithelial dynamics is essential for accurate tissue morphogenesis. Tight junctions are pivotal for epithelial integrity, yet their complex composition in this context remains poorly understood. Our examination of various tight junction proteins in the forebrain region of mouse embryos revealed distinct patterns in the neuronal and non-neuronal ectoderm, as well as mesoderm-derived mesenchymal cells. While claudin-4 exhibited exclusive expression in the non-neuronal ectoderm, we demonstrated a neuronal ectoderm specific localization for claudin-12 in the developing cranial neural tube. Claudin-5 was uniquely present in mesenchymal cells. Regarding the subcellular localization, canonical tight junction localization in the apical junctions was predominant for most tight junction complex proteins. ZO-1 (zona occludens protein-1), claudin-1, claudin-4, claudin-12, and occludin were detected at the apical junction. However, claudin-1 and occludin also appeared in basolateral domains. Intriguingly, claudin-3 displayed a non-canonical localization, overlapping with a nuclear lamina marker. These findings highlight the diverse tissue and subcellular distribution of tight junction proteins and emphasize the need for their precise regulation during the dynamic processes of forebrain development. The study can thereby contribute to a better understanding of the role of tight junction complex proteins in forebrain development.

Composite morphogenesis during embryo development.

Morphogenesis drives the formation of functional living shapes. Gene expression patterns and signaling pathways define the body plans of the animal and control the morphogenetic processes shaping the embryonic tissues. During embryogenesis, a tissue can undergo composite morphogenesis resulting from multiple concomitant shape changes. While previous studies have unraveled the mechanisms that drive simple morphogenetic processes, how a tissue can undergo multiple and simultaneous changes in shape is still not known and not much explored. In this chapter, we focus on the process of concomitant tissue folding and extension that is vital for the animal since it is key for embryo gastrulation and neurulation. Recent pioneering studies focus on this problem highlighting the roles of different spatially coordinated cell mechanisms or of the synergy between different patterns of gene expression to drive composite morphogenesis.

Neural Differentiation of Induced Pluripotent Stem Cells for a Xenogeneic Material-Free 3D Neurological Disease Model Neurulation from Pluripotent Cells Using a Human Hydrogel.

Alzheimer's Disease (AD) is characterized by synapse and neuronal loss and the accumulation of neurofibrillary tangles and Amyloid ß plaques. Despite significant research efforts to understand the late stages of the disease, its etiology remains largely unknown. This is in part because of the imprecise AD models in current use. In addition, little attention has been paid to neural stem cells (NSC), which are the cells responsible for the development and maintenance of brain tissue during an individual's lifespan. Thus, an in vitro 3D human brain tissue model using induced pluripotent stem (iPS) cell-derived neural cells in human physiological conditions may be an excellent alternative to standard models to investigate AD pathology. Following the differentiation process mimicking development, iPS cells can be turned into NSCs and, ultimately, neural cells. During differentiation, the traditionally used xenogeneic products may alter the cells' physiology and prevent accurate disease pathology modeling. Hence, establishing a xenogeneic material-free cell culture and differentiation protocol is essential. This study investigated the differentiation of iPS cells to neural cells using a novel extracellular matrix derived from human platelet lysates (PL Matrix). We compared the stemness properties and differentiation efficacies of iPS cells in a PL matrix against those in a conventional 3D scaffold made of an oncogenic murine-matrix. Using well-defined conditions without xenogeneic material, we successfully expanded and differentiated iPS cells into NSCs via dual-SMAD inhibition, which regulates the BMP and TGF signaling cascades in a manner closer to human conditions. This in vitro, 3D, xenogeneic-free scaffold will enhance the quality of disease modeling for neurodegenerative disease research, and the knowledge produced could be used in developing more effective translational medicine.

Junctional Neural Tube Defect (JNTD): A Rare and Relatively New Spinal Dysraphic Malformation.

Junctional neurulation completes the sequential embryological processes of primary and secondary neurulation as the intermediary step linking the end of primary neurulation and the beginning of secondary neurulation. Its exact molecular process is a matter of ongoing scientific debate. Abnormality of junctional neurulation-junctional neural tube defect (JNTD)-was first described in 2017 based on a series of three patients who displayed a well-formed secondary neural tube, the conus, that is physically separated by a fair distance from its companion primary neural tube and functionally disconnected from rostral corticospinal control. Several other cases conforming to this bizarre neural tube arrangement have since appeared in the literature, reinforcing the validity of this entity. The clinical, neuroimaging, and electrophysiological features of JNTD, as well as the hypothesis of its embryogenetic mechanism, will be described in this chapter.

Secondary Neurulation Defect: Terminal Myelocystocele, a Biological Leviathan.

Terminal myelocystocele (TMC) has been a puzzling entity of spinal dysraphism. It is found in the sacrococcygeal region usually forming a subcutaneous hump of various sizes. The wide variation of its morphology has been clarified by defining the essential and nonessential features as described in this chapter. Although it is not a common entity, TMC is attractive in that a highly plausible hypothesis on its pathoembryogenesis has been proposed based on observations on the secondary neurulation of the chick embryo. In this chapter, the embryology will be described, followed by the surgical strategy in accordance with the embryogenesis. The clinical features and prognosis will also be presented in detail.

Secondary Neurulation Defects: Retained Medullary Cord.

Retained medullary cord (RMC) is a defect resulting from impaired secondary neurulation. Intraoperatively, RMC is recognizable as an elongated cord-like structure caudal to the conus, that contains histologically confirmed neuroglial components and a lumen with an ependymal lining. It characteristically does not possess neurological function. This chapter aims to summarize (1) the mechanisms that lead to the occurrence of RMC; (2) the various forms of RMC, such as cystic RMC and 'possible RMC', and (3) the treatment strategies, especially untethering through limited exposure.

Surgery for spina bifida occulta: spinal lipoma and tethered spinal cord.

The technical evolution of the surgery for spina bifida occulta (SBO) over the course of a half-century was reviewed with special foci placed on the spinal lipoma and tethered spinal cord. Looking back through history, SBO had been included in spina bifida (SB). Since the first surgery for spinal lipoma in the mid-nineteenth century, SBO has come to be recognized as an independent pathology in the early twentieth century. A half-century ago, the only option available for SB diagnosis was the plain X-ray, and pioneers of the time persevered in the field of surgery. The classification of spinal lipoma was first described in the early 1970s, and the concept of tethered spinal cord (TSC) was proposed in 1976. Surgical management of spinal lipoma with partial resection was the most widely practiced approach and was indicated only for symptomatic patients. After understanding TSC and tethered cord syndrome (TCS), more aggressive approaches became preferred. A PubMed search suggested that there was a dramatic increase of publications on the topic beginning around 1980. There have been immense academic achievements and technical evolutions since then. From the authors' viewpoint, landmark achievements in this field are listed as follows: (1) establishment of the concept of TSC and the understanding of TCS; (2) unraveling the process of secondary and junctional neurulation; (3) introduction of modern intraoperative neurophysiological mapping and monitoring (IONM) for surgery of spinal lipomas, especially the introduction of bulbocavernosus reflex (BCR) monitoring; (4) introduction of radical resection as a surgical technique; and (5) proposal of a new classification system of spinal lipomas based on embryonic stage. Understanding the embryonic background seems critical because different embryonic stages bring different clinical features and of course different spinal lipomas. Surgical indications and selection of surgical technique should be judged based on the background embryonic stage of the spinal lipoma. As time flows forward, technology continues to advance. Further accumulation of clinical experience and research will open the new horizon in the management of spinal lipomas and other SBO in the next half-century.

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.

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

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

Three-dimensional visualization of secondary neurulation in chick embryos using microCT.

BACKGROUND: Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body formation mainly by microscopic observations and molecular experiments. Although conventional histology is a powerful tool for observing the details of morphology, it has limitations in the presentation of gross three-dimensional (3D) configurations of small embryos. The goal of this study was to visualize secondary neurulation and related structures in chick embryos in Hamburger and Hamilton (HH) stages 10-22 using microCT. RESULTS: The gross morphology of the chick embryo of various developmental stages was well visualized using microCT. Also, the detailed structures of the caudal cell mass (CCM) were presented starting from HH stage 12 to stage 16. The spatiotemporal relationship of CCM with the floor plate of the neural tube and notochord was shown. The dynamic changes of the chordoneural hinge, the cavitation of the secondary neural tube, and the primitive streak were described throughout the early stages of secondary neurulation. CONCLUSIONS: By utilizing the advantages of the microCT technique, our study shed light on the secondary neurulation in early-stage chick embryos and this can be the 3D reference for related structures.

Synthetic by design: Exploiting tissue self-organization to explore early human embryology.

Recent advances in synthetic human embryology has provided a previously inexistent molecular portrait of human development. Models of synthetic human embryonic tissues capitalize on the self-organizing capabilities of human embryonic stem cells when they are cultured on biomimetic conditions that simulate in vivo human development. In this Review, we discuss these models and how they have shed light on the early stages of human development including amniotic sac development, gastrulation and neurulation. We discuss the mechanisms underlying the molecular logic of embryonic tissue self-organization that have been dissected using synthetic models of human embryology and explore future challenges in the field. Geared with technological advances in bioengineering, high resolution gene expression and imaging tools, these models are set to transform our understanding of the mechanistic basis of embryonic tissue self-organization during human development and how they may go awry in disease.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.