Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Molecular architecture of the developing mouse brain.

The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1-8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system.

Construction of a Mex3c Gene-Deficient Mouse Model to Study C-FOS Expression in Hypothalamic Nuclei and Observe Morphological Differences in Embryonic Neural Tube Development.

BACKGROUND This study utilized CRISPR/Cas9 gene editing technology to construct a Mex3c gene-deficient mouse model, and studied C-FOS expression in hypothalamic nuclei. MATERIAL AND METHODS Thirty Mex3c-/+ mice, 30 mice in the normal group, and 30 Mex3c-/+ mice were randomly divided into control, leptin, and ghrelin groups according to different intraperitoneal injections. HE and Nissl staining were performed to observe the morphology of hypothalamic nerve cells. The C-FOS expression in hypothalamic nuclei of each group was analyzed by immunohistochemical techniques. HE staining was used to observe neural tube morphology, and LFB staining was used to observe nerve myelin sheath morphology. TEM was used to observe neuronal ultrastructure and immunohistochemical techniques were utilized to analyze nestin expression. RESULTS C-FOS expression was lower in the normal control group than in the leptin and ghrelin groups. The Mex3c control group and the leptin group had higher C-FOS expression than the ghrelin group. In neural tube studies, no significant differences were found in the neural tube pathological sections of E14.5-day embryos in each group. Nestin results demonstrated lower expression in the normal group and there was little difference between the HD and Mex3c groups. CONCLUSIONS Mex3c appears to participate in the regulation of energy metabolism by inducing C-FOS expression in the hypothalamus. The neural tubes of the offspring of Mex3c-/+ mice had defects during development.

OCT-Detected Optic Nerve Head Neural Canal Direction, Obliqueness, and Minimum Cross-Sectional Area in Healthy Eyes.

PURPOSE: To assess anterior scleral canal opening (ASCO) offset relative to Bruch's membrane opening (BMO) (ASCO/BMO offset) so as to determine neural canal direction, obliqueness, and minimum cross-sectional area (NCMCA) in 362 healthy eyes. DESIGN: Cross-sectional study. METHODS: After optical coherence tomography optic nerve head and retinal nerve fiber layer thickness (RNFLT) imaging, BMO and ASCO were manually segmented. Planes, centroids, size, and shape were calculated. Neural canal direction was defined by projecting the neural canal axis vector (connecting BMO and ASCO centroids) onto the BMO plane. Neural canal obliqueness was defined by the angle between the neural canal axis and the BMO plane perpendicular vector. NCMCA was defined by projecting BMO and ASCO points onto a neural canal axis perpendicular plane and measuring the area of overlap. The angular distance between superior and inferior peak RNFLT was measured, and correlations between RFNLT, BMO, ASCO, ASCO/BMO offset, and NCMCA were assessed. RESULTS: Mean (SD) NCMCA was significantly smaller than either the BMO or ASCO area (1.33 (0.42), 1.82 (0.38), 2.22 (0.43) mm2, respectively), and most closely correlated to RNFLT (P < .001, R2 = 0.158). Neural canal direction was most commonly superior-nasal (55%). Mean neural canal obliqueness was 39.4° (17.3°). The angular distance between superior and inferior peak RNFLT correlated to neural canal direction (P ≤ .008, R2 = 0.093). CONCLUSIONS: ASCO/BMO offset underlies neural canal direction, obliqueness, and NCMCA. RNFLT is more strongly correlated to NCMCA than to BMO or ASCO, and its peripapillary distribution is influenced by neural canal direction.

Comparison of the clinical disc margin seen in stereo disc photographs with neural canal opening seen in optical coherence tomography images.

PURPOSE: To compare neural canal opening (NCO) with the clinical optic disc margin (DM) seen and to investigate the planarity of the NCO in normal human optic nerve heads (ONH). METHODS: Sixteen eyes were imaged. Twelve healthy eyes were selected for planarity and 9 for NCO and DM correspondence. All subjects were subjected to a visual field examination, stereo disc photograph (SDP), scanning laser ophthalmoscopy, clinical examination with a fellowship trained glaucoma specialist, and optical coherence tomography imaging. Three reviewers delineated the NCO and inner limiting membrane on optical coherence tomography images. The clinical DM was delineated by a glaucoma specialist while viewing SDPs. Plane error was calculated for NCO and for Bruch membrane (BM) at distances 80 and 120 µm from NCO. RESULTS: The NCO segmentation interrater variability was low with an average coefficient of variation of 2.7%. A regional variation of the SDP and NCO correspondence was observed, wherein the temporal region had the largest coefficient of variation. The plane error of the NCO and BM were similar and was approximately 12 µm, which is small relative to an average DM diameter of 1.7 mm. CONCLUSIONS: The BM opening has a good correspondence with the clinical DM seen in SDPs. NCO delineation seemed to be reliable. The BM and NCO are relatively planar in normal humans and can be further evaluated for longitudinal studies to observe stability.

PCP effector proteins inturned and fuzzy play nonredundant roles in the patterning but not convergent extension of mammalian neural tube.

PCP effector proteins Inturned (Intu) and Fuzzy (Fuz) play important roles in mammalian neural development and ciliogenesis, but the developmental defects in Intu and Fuz mutants are not the same as those with the complete loss of cilia. Furthermore, it remains unclear whether mouse Intu and Fuz play a role in convergent extension, a process regulated by PCP signaling. In the current study, we show that the functions of both Intu and Fuz in neural tube patterning are dependent on the presence of cilia. We further show that neither gene exhibits obvious genetic interaction with the core PCP regulator Vangl2 in convergent extension or patterning of the neural tube. Finally, we show in Intu; Fuz double mutants that the lack of convergent extension and more severe patterning defects in Intu and Fuz mutants does not result from a functional redundancy between these two proteins.

[Microanatomic and three-dimensional reconstruction study of lateral wall and related structures of optic canal].

OBJECTIVE: To investigate the key microanatomic and radiological structures of optic canal comprehensively so as to provide anatomic parameters and procedural flows for the decompression of optic canal. METHODS: Gross observations and microscopic measurements were applied on 10 (20 sides) formalin-treated cadaveric specimens and 15 (30 sides) adult skulls. Using multislice helical CT (computed tomography)-aided three-dimensional reconstruction in combination with direct anatomic measurement, the investigators dissected, photographed, measured and analyzed the shape of optic canal and analyze its anatomic relationship with the adjoining structures. RESULTS: Optic canal was formed by the superior, inferior, medial and external walls and distal proximal opening. The lateral wall of optic canal was formed by anterior clinoid process with a length of (9.87 ± 1.34) mm, a width of (11.66 ± 2.35) mm, a base thickness of (5.35 ± 1.07) mm and a middle thickness of (4.50 ± 1.06) mm. Optic strut separating the optic canal from the superior orbital fissure was located inferiorly. And the distance between the apex of anterior clinoid process and the middle of ICA (internal carotid artery) groove was (4.25 ± 2.30) mm. The CSF (cerebrospinal fluid) leakage and secondary injury of optic nerve and injury of ICA, ophthalmic artery might occur during the surgical procedures due to the variation of anterior clinoid process. The microanatomic figures and radiological measurements had a mean difference very close to each other at (0.08 - 0.48) mm. No statistical difference was found (P > 0.05). CONCLUSION: Optic nerve, ophthalmic artery and ICA may be exposed by a high-speed drilling of the lateral wall of optic canal. The drilling dissection of lateral wall plays a vital role during a successful optic canal decompression. Radiological measurement and three-dimensional reconstruction of skull base may be of great clinical significance in lesion visualization. And it helps to make a better choice of surgical approaches. The measurements provide valuable references for surgeons and researchers.

Foxa2 regulates the expression of Nato3 in the floor plate by a novel evolutionarily conserved promoter.

The development of the neural tube into a complex central nervous system involves morphological, cellular and molecular changes, all of which are tightly regulated. The floor plate (FP) is a critical organizing center located at the ventral-most midline of the neural tube. FP cells regulate dorsoventral patterning, differentiation and axon guidance by secreting morphogens. Here we show that the bHLH transcription factor Nato3 (Ferd3l) is specifically expressed in the spinal FP of chick and mouse embryos. Using in ovo electroporation to understand the regulation of the FP-specific expression of Nato3, we have identified an evolutionarily conserved 204 bp genomic region, which is necessary and sufficient to drive expression to the chick FP. This promoter contains two Foxa2-binding sites, which are highly conserved among distant phyla. The two sites can bind Foxa2 in vitro, and are necessary for the expression in the FP in vivo. Gain and loss of Foxa2 function in vivo further emphasize its role in Nato3 promoter activity. Thus, our data suggest that Nato3 is a direct target of Foxa2, a transcription activator and effector of Sonic hedgehog, the hallmark regulator of FP induction and spinal cord development. The identification of the FP-specific promoter is an important step towards a better understanding of the molecular mechanisms through which Nato3 transcription is regulated and for uncovering its function during nervous system development. Moreover, the promoter provides us with a powerful tool for conditional genetic manipulations in the FP.

Automated segmentation of 3-D spectral OCT retinal blood vessels by neural canal opening false positive suppression.

We present a method for automatically segmenting the blood vessels in optic nerve head (ONH) centered spectral-domain optical coherence tomography (SD-OCT) volumes, with a focus on the ability to segment the vessels in the region near the neural canal opening (NCO). The algorithm first pre-segments the NCO using a graph-theoretic approach. Oriented Gabor wavelets rotated around the center of the NCO are applied to extract features in a 2-D vessel-aimed projection image. Corresponding oriented NCO-based templates are utilized to help suppress the false positive tendency near the NCO boundary. The vessels are identified in a vessel-aimed projection image using a pixel classification algorithm. Based on the 2-D vessel profiles, 3-D vessel segmentation is performed by a triangular-mesh-based graph search approach in the SD-OCT volume. The segmentation method is trained on 5 and is tested on 10 randomly chosen independent ONH-centered SD-OCT volumes from 15 subjects with glaucoma. Using ROC analysis, for the 2-D vessel segmentation, we demonstrate an improvement over the closest previous work with an area under the curve (AUC) of 0.81 (0.72 for previously reported approach) for the region around the NCO and 0.84 for the region outside the NCO (0.81 for previously reported approach).

Neurons derive from the more apical daughter in asymmetric divisions in the zebrafish neural tube.

In the developing CNS, asymmetric cell division is critical for maintaining the balanced production of differentiating neurons while renewing the population of neural progenitors. In invertebrates, this process depends on asymmetric inheritance of fate determinants during progenitor divisions. A similar mechanism is widely believed to underlie asymmetrically fated divisions in vertebrates, but compelling evidence for this is missing. We used live imaging of individual progenitors in the intact zebrafish embryo CNS to test this hypothesis. We found that asymmetric inheritance of a subcellular domain is strongly correlated with asymmetric daughter fates and our results reveal an unexpected feature of this process. The daughter cell destined to become a neuron was derived from the more apical of the two daughters, whereas the more basal daughter inherited the basal process and replenished the apical progenitor pool.

Nectin-2 and N-cadherin interact through extracellular domains and induce apical accumulation of F-actin in apical constriction of Xenopus neural tube morphogenesis.

Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules regulating its initiation are mostly unknown. Here, we demonstrated that nectin-2, an immunoglobulin-like cell adhesion molecule, is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, we found that, at the beginning of neurulation, nectin-2 was strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impaired neural fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated F-actin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2 and F-actin, was not required for the generation of the ectopic apical constriction. Furthermore, we found that nectin-2 physically interacts with N-cadherin through extracellular domains, and they cooperatively enhanced apical constriction by driving the accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of neuroepithelium was dependent on the presence of nectin-2, but that of nectin-2 was not affected by depletion of N-cadherin. We propose a novel mechanism of neural tube morphogenesis regulated by the two types of cell adhesion molecules.

Neural stem cell self-renewal requires the Mrj co-chaperone.

The Mrj co-chaperone is expressed throughout the mouse conceptus, yet its requirement for placental development has prohibited a full understanding of its embryonic function. Here, we show that Mrj(-/-) embryos exhibit neural tube defects independent of the placenta phenotype, including exencephaly and thin-walled neural tubes. Molecular analyses revealed fewer proliferating cells and a down-regulation of early neural progenitor (Pax6, Olig2, Hes5) and neuronal (Nscl2, SCG10) cell markers in Mrj(-/-) neuroepithelial cells. Furthermore, Mrj(-/-) neurospheres are significantly smaller and form fewer secondary neurospheres indicating that Mrj is necessary for self-renewal of neural stem cells. However, the molecular function of Mrj in this context remains elusive because Mrj does not colocalize with Bmi-1, a self-renewal protein. Furthermore, unlike in Mrj(-/-) placentas, intermediate filament-containing aggregates do not accumulate in Mrj(-/-) neuroepithelium, ruling out nestin as a substrate for Mrj. Regardless, Mrj plays an important role in neural stem cell self-renewal.

Morphogenetic movements driving neural tube closure in Xenopus require myosin IIB.

Vertebrate neural tube formation involves two distinct morphogenetic events--convergent extension (CE) driven by mediolateral cell intercalation, and bending of the neural plate driven largely by cellular apical constriction. However, the cellular and molecular biomechanics of these processes are not understood. Here, using tissue-targeting techniques, we show that the myosin IIB motor protein complex is essential for both these processes, as well as for conferring resistance to deformation to the neural plate tissue. We show that myosin IIB is required for actin-cytoskeletal organization in both superficial and deep layers of the Xenopus neural plate. In the superficial layer, myosin IIB is needed for apical actin accumulation, which underlies constriction of the neuroepithelial cells, and that ultimately drive neural plate bending, whereas in the deep neural cells myosin IIB organizes a cortical actin cytoskeleton, which we describe for the first time, and that is necessary for both normal neural cell cortical tension and shape and for autonomous CE of the neural tissue. We also show that myosin IIB is required for resistance to deformation ("stiffness") in the neural plate, indicating that the cytoskeleton-organizing roles of this protein translate in regulation of the biomechanical properties of the neural plate at the tissue-level.

Bmp2 is required for cephalic neural tube closure in the mouse.

BMPs have been shown to play a role in neural tube development particularly as dorsalizing factors. To explore the possibility that BMP2 could play a role in the developing neural tube (NT) beyond the lethality of Bmp2 null embryos, we created Bmp2 chimeras from Bmp2 null ES cells and WT blastocysts. Analysis of Bmp2 chimeras reveals NT defects at day 9.5 (E9.5). We found that exclusion of Bmp2 null ES cells from the dorsal NT did not always prevent defects. For further comparison, we used a Bmp2 mutant line in a mixed background. Phenotypes observed were similar to chimeras including open NT defects, postneurulation defects, and abnormal neural ectoderm in heterozygous and homozygous null embryos demonstrating a pattern of dose-dependent signaling. Our data exposes BMP2 as a unique player in the developing NT for dorsal patterning and identity, and normal cephalic neural tube closure in a dose-dependent manner.

Detection of optic nerve head neural canal opening within histomorphometric and spectral domain optical coherence tomography data sets.

PURPOSE: To assess the ability to detect the neural canal opening (NCO) and its characteristics within three-dimensional (3-D) histomorphometric and 3-D spectral domain optical coherence tomography (SD-OCT) reconstructions of the optic nerve head from nonhuman primate (NHP) eyes. METHODS: NCO was delineated within 40 radial, sagittal sections of 3-D histomorphometric reconstructions of 44 normal eyes of 38 NHPs, each perfusion fixed at IOP 10 mm Hg, and 3-D SD-OCT (Spectralis; Heidelberg Engineering, Heidelberg, Germany) volumes acquired in vivo from a separate group of 33 normal eyes of 24 NHPs. Within all reconstructions, a least-squares ellipse was fitted to the 80 NCO points. For each eye, the dimensions and plane error (a gauge of planarity) of the fitted ellipse were calculated. RESULTS: The NCO was successfully delineated within every section of each histomorphometric and SD-OCT reconstruction. Median plane error was similar within histomorphometric and SD-OCT volumes (8 microm, range 4-19, histomorphometry, and 10 microm, range 4-26, SD-OCT) and was small relative to the size of the ellipse. Median histomorphometric ellipse dimensions were 1453 mum (major axis, range 1218-1737) and 1066 microm (minor axis, range 808-1263). Median SD-OCT ellipse dimensions were 1512 microm (major axis, range 1191-1865) and 1060 microm (minor axis, range 772-1248). CONCLUSIONS: The NCO is biologically continuous and relatively planar within all 3-D histomorphometric and SD-OCT reconstructions. These characteristics support its further evaluation as a reference plane for cross-sectional and longitudinal measurement of optic nerve head structures using 3-D SD-OCT.

Central projections of anuran optic nerves penetrating hindbrain or spinal cord regions of the neural tube.

To identify parameters of optic tract growth that may be independent of the region of the CNS in which the tract develops, eye primordia of embryonic Rana pipiens were transplanted to a variety of ectopic locations adjacent to the hindbrain and spinal cord. Longitudinal growth direction, cross-sectional growth position and possible termination sites of ectopic optic tracts from transplanted eyes were analyzed in serial section autoradiographs following intraocular injection of [3H]proline. Sixteen animals ranging in age from Taylor and Kollros Stage I32 to one month postmetamorphosis were used in this study. The analysis revealed that retinal ganglion cell axons are capable of growing in both rostral and caudal directions within all regions of the hindbrain and spinal cord. Within comparable regions of the hindbrain and spinal cord, all ectopic optic tracts were located in identical cross-sectional positions. The positions were always sub-adjacent to the pial surface of the CNS and contiguous to regions of the neural axis in which sensory fiber tracts are normally found. Possible termination sites of ascending ectopic optic tracts were detected within sensory regions of the hindbrain and posterior midbrain tegmentum. However, there was no consistent selection of particular regions of termination. No ectopic optic nerve fibers extended significantly beyond the hindbrain-midbrain junction and no fibers could be traced to the optic tecta.