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

INTRODUCTION: 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 a recent interest in the neuromesodermal progenitors. However, a transcriptomic analysis of the CCM during secondary neurulation has not been performed yet. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

STAT3 is a key molecule in the oncogenic behavior of diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is one of the most lethal childhood brain tumors. This tumor is unique because it is detected exclusively in the ventral pons of patients aged between 6 and 7 years, which suggests a developmental nature of its formation. Signal transducer and activator of transcription 3 (STAT3) is a critical molecule for the differentiation of neural stem cells into astrocytes during neurodevelopment. Additionally, STAT3 is associated with oncogenesis and the epithelial-mesenchymal transition (EMT) in various types of tumor. In recent years, several studies have demonstrated the oncogenic role of STAT3 in high-grade gliomas. However, the role of STAT3 in DIPG at the cellular level remains unknown. To assess the possible association between gliogenesis and DIPG, the expression levels of various molecules participating in the differentiation of neural stem cells were compared between normal brain control tissues and DIPG tissues using open public data. All of the screened genes exhibited significantly increased expression in DIPG tissues compared with normal tissues. As STAT3 expression was the most increased, the effect of STAT3 inhibition in a DIPG cell line was assessed via STAT3 short hairpin (sh)RNA transfection and treatment with AG490, a STAT3 inhibitor. Changes in viability, apoptosis, EMT and radiation therapy efficiency were also evaluated. Downregulation of STAT3 resulted in decreased cyclin D1 expression and cell viability, migration and invasion. Additionally, treatment with STAT3 shRNA or AG490 suppressed the EMT phenotype. Finally, when radiation was administered in combination with STAT3 inhibition, the therapeutic efficiency, assessed by cell viability and DNA damage repair, was increased. The present results suggest that STAT3 is a potential therapeutic target in DIPG, especially when combined with radiation therapy.

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.

A rat model of chronic syringomyelia induced by epidural compression of the lumbar spinal cord.

OBJECTIVE There has been no established animal model of syringomyelia associated with lumbosacral spinal lipoma. The research on the pathophysiology of syringomyelia has been focused on Chiari malformation, trauma, and inflammation. To understand the pathophysiology of syringomyelia associated with occult spinal dysraphism, a novel animal model of syringomyelia induced by chronic mechanical compression of the lumbar spinal cord was created. METHODS The model was made by epidural injection of highly concentrated paste-like kaolin solution through windows created by partial laminectomy of L-1 and L-5 vertebrae. Behavioral outcome in terms of motor (Basso-Beattie-Bresnahan score) and urinary function was assessed serially for 12 weeks. Magnetic resonance images were obtained in some animals to confirm the formation of a syrinx and to monitor changes in its size. Immunohistochemical studies, including analysis for glial fibrillary acidic protein, NeuN, CC1, ED-1, and caspase-3, were done. RESULTS By 12 weeks after the epidural compression procedure, syringomyelia formation was confirmed in 85% of the rats (34 of 40) on histology and/or MRI. The syrinx cavities were found rostral to the epidural compression. Motor deficit of varying degrees was seen immediately after the procedure in 28% of the rats (11 of 40). In 13 rats (33%), lower urinary tract dysfunction was seen. Motor deficit improved by 5 weeks after the procedure, whereas urinary dysfunction mostly improved by 2 weeks. Five rats (13%, 5 of 40) died 1 month postoperatively or later, and 3 of the 5 had developed urinary tract infection. At 12 weeks after the operation, IHC showed no inflammatory process, demyelination, or accelerated apoptosis in the spinal cords surrounding the syrinx cavities, similar to sham-operated animals. CONCLUSIONS A novel experimental model for syringomyelia by epidural compression of the lumbar spinal cord has been created. The authors hope that it will serve as an important research tool to elucidate the pathogenesis of this type of syringomyelia, as well as the CSF hydrodynamics of the lumbar spinal cord.

Pathoembryogenesis of terminal myelocystocele: terminal balloon in secondary neurulation of the chick embryo.

PURPOSE: Terminal myelocystocele (TMC) is thought to be caused by a misstep during secondary neurulation. However, due to the paucity of data on secondary neurulation and the rarity of TMC, proofs of this pathogenetic mechanism are unavailable. Based on a previous observation that TMC resembles a step of secondary neurulation in chick, a closer look was taken at secondary neurulation of chick embryos focusing on the cerebrospinal fluid-filled distal neural tube (terminal balloon). METHODS: Chick embryos at Hamburger and Hamilton (H-H) stages of 28, 30, 33, 35, 37, and 40 were harvested. Hematoxying-eosin staining, additional immunohistochemistry (laminin, cytokeratin, nestin), and scanning electron microscopy were performed. RESULTS: In H-H stages 28 to 30, after merging of the lumina of the primary and secondary neural tubes, the caudal end of the confluent tube dilates into a balloon-like structure (terminal balloon). As the proximal tube progressively becomes narrower, the terminal balloon dilates even further, and its wall fuses with the surface ectoderm (H-H stage 33). Later in H-H stages 35 to 40, the terminal balloon shrinks and becomes detached from the surface ectoderm and ultimately disappears, as the proximal lumen of the secondary neural tube continues to collapse. CONCLUSION: A dilated balloon doubtlessly exists in the terminal secondary neural tube in chick embryos, and its subsequent disappearance occurs in a variable time course and sequence. Arrest of apoptosis resulting in failure of detachment of the terminal balloon from the surface ectoderm may well be the basis for human TMC.