Secondary Neural Tube Research Articles

Molecular and morphogenetic features of neurulation

Neurulation occurs by two different mechanisms, called primary and secondary neurulation. In humans, primary neurulation occurs along most of the rostrocaudal axis of the embryo, while secondary neurulation occurs caudally, only in the lower sacral and coccygeal regions. Primary neurulation is responsible for a change in the neural plate shape, the lateral edges of which rise and then converge at the dorsal midline to merge into a tube. Initially, the neural tube, formed as a result of primary neurulation, is open at both ends through the so-called rostral and caudal neuropores. These neuropores connect the inner part of the neural tube with the environment (amniotic cavity) and later (by the end of primary neurulation) are closed. During primary neurulation, the brain and spinal cord are formed up to the upper sacral region (up to the level of junction between S1 and S2 vertebral bodies), however, the most caudal part of this anatomical region (sacral-coccygeal division of the spinal cord, conus medullaris and filum terminale) is formed at secondary neurulation. In humans, secondary neurulation occurs due to elongation and cavitation of the caudal cell mass into the medulla, which then transforms into a secondary neural tube. Thus, the main differences between primary and secondary neurulation are that the neural plate folds and invaginates into the body of the embryo and separates from the surface ectoderm, forming an underlying hollow tube in primary neurulation. Mesenchymal cell сlusters form a dense cord that undergoes mesenchymal-epithelial transition and forms cavities and an empty tube during secondary neurulation to form the terminal part of the spinal cord. Conclusions. Understanding the detailed molecular and genetic mechanisms of each stage of neurulation is relevant due to widespread congenital neural tube defects, and only perfect knowledge on each aspect of neurulation and all possible factors of potential influence on it will help to develop modern options for influencing some of them, and probably, cause a decrease in neural tube congenital defects.

The cellular dynamics of neural tube formation.

The vertebrate brain and spinal cord arise from a common precursor, the neural tube, which forms very early during embryonic development. To shape the forming neural tube, changes in cellular architecture must be tightly co-ordinated in space and time. Live imaging of different animal models has provided valuable insights into the cellular dynamics driving neural tube formation. The most well-characterised morphogenetic processes underlying this transformation are convergent extension and apical constriction, which elongate and bend the neural plate. Recent work has focused on understanding how these two processes are spatiotemporally integrated from the tissue- to the subcellular scale. Various mechanisms of neural tube closure have also been visualised, yielding a growing understanding of how cellular movements, junctional remodelling and interactions with the extracellular matrix promote fusion and zippering of the neural tube. Additionally, live imaging has also now revealed a mechanical role for apoptosis in neural plate bending, and how cell intercalation forms the lumen of the secondary neural tube. Here, we highlight the latest research on the cellular dynamics underlying neural tube formation and provide some perspectives for the future.

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.

The transcriptomic landscape of caudal cell mass in different developmental stages of the chick embryo.

The caudal cell mass (CCM) is an aggregate of undifferentiated pluripotent cells and the main player in secondary neurulation. Previous studies have elucidated the dynamic fate of the multipotent cell lineages, with arecent interest in the neuromesodermal progenitors. However, a transcriptomic analysis of the CCM during secondary neurulation has not been performed yet. We analyzed RNA sequencing data of CCM samples at three different developmental stages of chicken embryos; HH16 (largest CCM phase), HH20 (secondary neural tube formation phase), and HH28 (degeneration phase). The transcriptomic profiles were clearly distinguishable according to developmental stage, and HH20 was shown to have not only intermediate, but also unique properties in secondary neurulation. A total of 10,666 differentially expressed genes, including FGF18 and GDF11, were identified and enriched in several gene ontologies related to embryogenesis or organogenesis. We also found that genes encoding transcription factors, such as TWIST2, IRX4, HOXB4, HOXD13, LIN28A, CDX4, and Brachyury, were among the top-ranked differentially expressed genes. Through transcriptomic profiling, we provided a picture of the developmental process of the CCM. We identified several key molecules or pathways involved in secondary neurulation and the pathogenesis of related diseases.

Myelomeningocele as an anomaly of secondary neurulation.

Myelomeningocele (MMC) is the representative entity of open neural tube defects resulting from an error during primary neurulation. However, cases of MMC in the region of the secondary neural tube (below the junction of S1 and S2 vertebrae) are sometimes encountered. We aimed to analyze the clinical features of atypical "low-lying" MMC in comparison to the typical MMC and suggest possible pathoembryogenesis. From 1986 to 2020, 95 MMC patients were treated in our institute. A retrospective review of the radiological and clinical information was performed. We defined "low-lying" MMCs as those with fascia or lamina defects below the S1-2 interspinous ligament. Thirty-one out of the 95 MMC patients were identified as having low-lying MMC. The percentage of low-lying MMC within the entire MMC group increased dramatically (19% from 1990 to 1999 and 48% from 2000 to 2020). Thirty-nine percent of the low-lying MMCs were associated with hydrocephalus, and 36% showed the Chiari malformation. Clean intermittent catheterization was being performed by 52% of the patients and 46% had a motor weakness. The proportions of hydrocephalus, neurological symptoms, and the number of related procedures in the low-lying MMC were substantially lower than the typical MMC in our cohort and the literature. We present cases of atypical MMC occurring in the region of secondary neurulation. These cases provide clues that secondary neurulation may lead to open neural defects. Future experiments with animal models supporting what we have seen in the clinics will greatly enhance the understanding of the developmental process of neurulation and the corresponding anomalies.

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

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. 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. 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.

Junctional Neurulation : A Junction between Primary and Secondary Neural Tubes.

Recent case reports of junctional neural tube defect (JNTD) which is a peculiar type of spinal anomaly showing the functional disconnection of the primary and secondary neural tubes has risen interest in the process of junctional neurulation (the connection between the two neural tubes) during development. This article summarizes the clinical features of the JNTD and reviews the literature on the basic research on junctional neurulation.

Overview of Secondary Neurulation.

Secondary neurulation is a morphological process described since the second half of the 19th century; it accounts for the formation of the caudal spinal cord in mammals including humans. A similar process takes place in birds. This form of neurulation is caused by the growth of the tail bud region, the most caudal axial region of the embryo. Experimental work in different animal species leads to questioning dogmas widely disseminated in the medical literature. Thus, it is clearly established that the tail bud is not a mass of undifferentiated pluripotent cells but is made up of a juxtaposition of territories whose fate is different. The lumens of the two tubes generated by the two modes of neurulation are continuous. There seem to be multiple cavities in the human embryo, but discrepancies exist according to the authors. Finally, the tissues that generate the secondary neural tube are initially located in the most superficial layer of the embryo. These cells must undergo internalization to generate the secondary neurectoderm. A defect in internalization could lead to an open neural tube defect that contradicts the dogma that a secondary neurulation defect is closed by definition.

Cell intercalation driven by SMAD3 underlies secondary neural tube formation.

Body axis elongation is a hallmark of the vertebrate embryo, involving the architectural remodeling of the tail bud. Although it is clear how neuromesodermal progenitors (NMPs) contribute to embryo elongation, the dynamic events that lead to de novo lumen formation and that culminate in the formation of a 3-dimensional, neural tube from NMPs, are poorly understood. Here, we used invivo imaging of the chicken embryo to show that cell intercalation downstream of TGF-β/SMAD3 signaling is required for secondary neural tube formation. Our analysis describes the events in embryo elongation including lineage restriction, the epithelial-to-mesenchymal transition of NMPs, and the initiation of lumen formation. We show that the resolution of a single, centrally positioned lumen, which occurs through the intercalation of central cells, requires SMAD3/Yes-associated protein (YAP) activity. We anticipate that these findings will be relevant to understand caudal, skin-covered neural tube defects, among the most frequent birth defects detected in humans.

Junctional Neural Tube Defect.

Junctional neurulation represents the most recent adjunct to the well-known sequential embryological processes of primary and secondary neurulation. While its exact molecular processes, occurring at the end of primary and the beginning of secondary neurulation, are still being actively investigated, its pathological counterpart –junctional neural tube defect (JNTD)– had been described in 2017 based on three patients whose well-formed secondary neural tube, the conus, is widely separated from its corresponding primary neural tube and functionally disconnected from corticospinal control from above. Several other cases conforming to this bizarre neural tube arrangement have since appeared in the literature, reinforcing the validity of this entity. The cardinal clinical, neuroimaging, and electrophysiological features of JNTD, and the hypothesis of its embryogenetic mechanism, form part of this review.

Terminal Myelocystocele : Pathoembryogenesis and Clinical Features.

There has been confusion in the classification of terminal myelocystocele (TMCC) due to its diverse morphology and vague pathoembryogenesis. TMCC could be summarized as having the essential features of an elongated caudal spinal cord extruding out of the dorsal extraspinal space that fuses with the subcutaneous fat, which is in the shape of a trumpet-shaped cerebrospinal fluid-filled cyst. The extraspinal portion of the extruded spinal cord is nonfunctional. The morphological features suggest that TMCC is formed during secondary neurulation, specifically the failure of the degeneration of the secondary neural tube near the time of the terminal balloon. This review discusses the definition, as well as the clinical and surgical features, of TMCC with special emphasis on its pathoembryogenesis.

Do junctional neural tube defect and segmental spinal dysgenesis have the same pathoembryological background?

Junctional neural tube defect (JNTD) is a recently introduced form of congenital spinal dysraphism that is characterized by functional disconnection between the primary and secondary neural tubes. The upper and lower cords appeared to be connected by a non-functioning band-like structure. JNTD is suspected to arise from a developmental error not corresponding to either primary or secondary neurulation, but rather between the two neurulation processes. On the other hand, segmental spinal dysgenesis (SSD) is an older entity of spinal anomalies in which a segment of the spine and spinal cord does not develop properly. The anomaly had been noted for the bony abnormality, as it is the most prominent feature. Based on the recent encounter of two cases resembling both entities, we sought the possibility that the two diseases may have the same pathoembryogenesis. Based on the impression that the two entities share important features, we compared the details of the two anomalies. First, our two recently encountered cases of JNTD were described. Second, previous reports of SSD were comprehensively reviewed. The two cases had the essential anomaly of the neural structures satisfying the definition of JNTD, as well as the elaborate spinal deformity as seen in SSD. In the previous literature on SSD, it was recognized that in addition to the bone anomaly, disconnected spinal cord was present. Hence, the two entities seem to have many similar clinical and neuroimaging features. The dysgenic spinal level is similar, and the disconnection between the primary and secondary neural tubes is found in the two diseases. The two neural tubes are connected by a band-like structure, with severe stenosis of the spinal canal at the level of the band. Both entities show segmental anomalies of the vertebrae in the thoracolumbar region, especially in the posterior element. Although the extent of shared features seems high, the previously suggested hypothetical pathoembryogenesis of SSD did not involve the process of junctional neurulation. We suggest that SSD shares the same origin as JNTD, and the bony abnormality may be a secondary phenomenon to the core error during neural tube development. We propose that JNTD and SSD may be the same entity, originating from an error during junctional neurulation. As there is controversy regarding the treatment strategy for both entities, unified accumulation of clinical experience and analysis may help improve the management of patients.

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using EGFP transfection.

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.

Unjoined primary and secondary neural tubes: junctional neural tube defect, a new form of spinal dysraphism caused by disturbance of junctional neurulation

Unjoined primary and secondary neural tubes: junctional neural tube defect, a new form of spinal dysraphism caused by disturbance of junctional neurulation

Neurosphere formation potential resides not in the caudal cell mass, but in the secondary neural tube.

The caudal cell mass (CCM) is known as the main player in secondary neurulation, forming the secondary neural tube (2NT). This suggests that the CCM may have the character of neural progenitor cells. The neural potential of the CCM and the 2NT (CCM + 2NT) was assessed by in vitro culture of neurospheres during Hamburger and Hamilton stages (HH) of secondary neurulation (HH16 to HH32). We also analyzed the neural potential of the developing central nervous system (CNS) by comparing the neurosphere culture from the brain, upper / lower spinal cord, and CCM + 2NT from various HH stages. The CCM + 2NT was capable of forming neurospheres. Of the various HH stages, the greatest number of neurospheres from CCM + 2NT were cultured at HH28. Because the 2NT is most prominent at HH28, we hypothesized that the 2NT, rather than the CCM, had the main potential to produce neurospheres. When the neurospheres were cultured separately from the CCM and the 2NT, 2NT made significantly more neurospheres. When comparing different parts of the CNS, at HH16, the greatest number of neurospheres was formed from the brain. At HH32, it was the CCM + 2NT. The region with the greatest number of neurospheres progressed in a cephalo-caudal direction during development. This study showed that neurospheres can be cultured from CCM + 2NT, and the main player in neurosphere formation was the 2NT. The present study has also revealed cephalo-caudal trend in the neural potential of developing CNS.

Unjoined primary and secondary neural tubes: junctional neural tube defect, a new form of spinal dysraphism caused by disturbance of junctional neurulation

Primary and secondary neurulation are the two known processes that form the central neuraxis of vertebrates. Human phenotypes of neural tube defects (NTDs) mostly fall into two corresponding categories consistent with the two types of developmental sequence: primary NTD features an open skin defect, an exposed, unclosed neural plate (hence an open neural tube defect, or ONTD), and an unformed or poorly formed secondary neural tube, and secondary NTD with no skin abnormality (hence a closed NTD) and a malformed conus caudal to a well-developed primary neural tube. We encountered three cases of a previously unrecorded form of spinal dysraphism in which the primary and secondary neural tubes are individually formed but are physically separated far apart and functionally disconnected from each other. One patient was operated on, in whom both the lumbosacral spinal cord from primary neurulation and the conus from secondary neurulation are each anatomically complete and endowed with functioning segmental motor roots tested by intraoperative triggered electromyography and direct spinal cord stimulation. The remarkable feature is that the two neural tubes are unjoined except by a functionally inert, probably non-neural band. The developmental error of this peculiar malformation probably occurs during the critical transition between the end of primary and the beginning of secondary neurulation, in a stage aptly called junctional neurulation. We describe the current knowledge concerning junctional neurulation and speculate on the embryogenesis of this new class of spinal dysraphism, which we call junctional neural tube defect.

Median Sacral Artery, Sympathetic Nerves, and the Coccygeal Body: A Study Using Serial Sections of Human Embryos and Fetuses.

To examine how the median sacral artery (MSA) is involved with the coccygeal body or glomus coccygeum, we studied serial frontal or sagittal sections of 14 embryos (approximately 5-6 weeks of gestation) and 12 fetuses (10-18 weeks). At five weeks, the caudal end of the dorsal aorta (i.e., MSA) accompanied putative sympathetic ganglion cells in front of the upper coccygeal and lower sacral vertebrae. At six weeks, a candidate for the initial coccygeal body was identified as a longitudinal arterial plexus involving nerve fibers and sympathetic ganglion cells between arteries. At 10-18 weeks, the MSA exhibited a highly tortuous course at the lower sacral and coccygeal levels, and was attached to and surrounded by veins, nerve fibers, and sympathetic ganglion cells near and between the bilateral origins of the levator ani muscle. Immunohistochemistry demonstrated expression of tyrosine hydroxylase and chromogranin A in the nerves. However, throughout the stages examined, we found no evidence suggestive of an arteriovenous anastomosis, such as well-developed smooth muscle. An acute anterior flexure of the vertebrae at the lower sacrum, as well as regression of the secondary neural tube, seemed to induce arterial plexus formation from an initial straight MSA. Nerves and ganglion cells were likely to be secondarily involved with the plexus because of the close topographical relationship. However, these nerves might play a major role in the extreme change into adult morphology. An arteriovenous anastomosis along the MSA might be an overinterpretation, at least in the prenatal human. Anat Rec, 299:819-827, 2016. © 2016 Wiley Periodicals, Inc.

The Filum Terminale Revisited: A Histological Study in Human Fetuses

Previous studies have suggested that secondary neurulation provides no functional neurons but only the filum terminale. However, no studies have confirmed that the coccygeal and lower sacral nerves do not originate from the secondary neural tube but only from the primary tube. To obtain a better understanding of these relationships, we examined sagittal or frontal sections from 20 embryonic and fetal specimens ranging from 6 to 14 weeks of gestation. During the growth of the vertebral column as well as the subsequent upward migration of the caudal end of the dural sac, the secondary neural tube was stretched to maintain the original attachment to the coccyx or the lower sacral vertebra. The filum-like structure showed much individual variability but in all cases appeared to be derived from the stretched neural tube. Intermediate age morphology revealed that the secondary neural tube itself provided an initial filum terminale before the ascent of the dural sac. Given that the coccygeal and lower sacral nerves are likely to originate from the secondary neural tube, these parts of the tube persisted and differentiated into spinal neurons to form the anococcygeal nerves. Likewise, the filum terminale was also most likely to contain some neurons that persisted postnatally. Depending on the timing and site of degeneration of the secondary neural tube, individual variations could occur in proportion to the amount of sensory and motor elements in the anococcygeal nerve supply.

Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

Junctional neurulation: a unique developmental program shaping a discrete region of the spinal cord highly susceptible to neural tube defects.

In higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation.