Souichi Oe

Sulfatide (3‐O‐sulfogalactosylceramide) is a major glycolipid component of myelin synthesized by oligodendrocytes in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS). Although sulfatide diversity has been characterized in CNS myelin, its species‐level composition, spatial organization, and developmental dynamics in the PNS remain poorly defined. Here, we used imaging mass spectrometry (IMS) to characterize sulfatide molecular species in murine dorsal root ganglia (DRGs) during development. Thirteen sulfatide species were identified in adult DRGs and classified as long‐chain (LC) or very‐long‐chain (VLC), with hydroxylated or non‐hydroxylated forms. VLC non‐hydroxylated sulfatides were preferentially enriched in compact myelin‐rich regions, whereas LC and hydroxylated species exhibited broader distributions extending into neuronal soma‐associated regions. Multivariate analyses further demonstrated that regional sulfatide molecular species profiles were associated with fatty acyl chain length and hydroxylation status. Developmentally, structurally diverse sulfatide classes, including LC non‐hydroxylated, VLC non‐hydroxylated, and VLC hydroxylated species, were detectable by embryonic day 14.5, and all species were present by postnatal day 2, indicating that sulfatide diversification is established before mature myelin formation. Sulfatide species exhibited ventral enrichment within developing DRGs, a pattern partially conserved in chick embryos. Analysis of Cst ‐null mice confirmed previously reported abnormalities in myelinating SC–axon units and further revealed defective Remak bundle organization characterized by incomplete axonal ensheathment, together with altered dynamics of Sox10+/Sox2+ SCs. These findings reveal a previously unrecognized spatiotemporal heterogeneity of sulfatide species in the developing PNS and support roles for sulfatide diversity in SC maturation and peripheral nerve organization.

The lateral line is a mechanosensory organ found in cyclostomes, teleosts, and amphibians. Hair cells within its neuromasts have kinocilia and stereocilia that sense water flow, and the lateral line nerve transduces this sensory input. Hair cells in the lateral line are analogous to those in the mammalian inner ear. Lateral line development and regeneration have been studied extensively in zebrafish. However, lateral line formation in other species remains poorly understood. The Iberian ribbed newt ( Pleurodeles waltl ) is a versatile model for regeneration with a short period of sexual maturation and aseasonal/prolific ovulation. Here, we investigated the features, regeneration processes, and additional neuromast formation in the posterior lateral line of Iberian ribbed newts using Brainbow reporters. The lateral line in the Iberian ribbed newts shares common features with other species. Laser ablation of hair cells or transection of the posterior lateral line nerve induced additional neuromast formation, to which epidermal cells contributed at least in part. We refer to it as “replicative neuromast regeneration” to allow distinction from several modes of neuromast formation in normal development. Based on these results, we propose that in response to injury, the posterior lateral line nerve induces epidermal cells to contribute to newly formed neuromasts in a replicative manner. Our results provide insights into lateral line morphogenesis.

Many treatments for spinal cord injury (SCI) have been developed; however, none of these treatments enable complete functional repair. In this study, we utilized a model amphibian, Xenopus laevis , which has regeneration capacity of the spinal cord in the tadpole stages but loses it during metamorphosis, to identify differences in spinal cord properties between the regenerative and non‐regenerative stages. We compared the spatiotemporal expression patterns of transcription factors important for neural tube development as well as cell proliferation markers between tadpoles and metamorphosed young adults (froglets) in Xenopus as well as between Xenopus and mouse embryos. We found that Xenopus drastically changed properties in ependymal cells of the spinal cord during the transition from regenerative to non‐regenerative stages, and that similar change occurred in the mouse neural tube between 12.5 and 14.5 dpc, during which neurogenesis is known to cease. We then investigated neurogenic activity during Xenopus metamorphosis by labeling newly generated neurons and found that it was correlated with regeneration capacity. We also found that neurogenesis actively occurred after SCI at the regenerative stages but was scarcely reactivated at the non‐regenerative stages, suggesting that neurogenesis may play an important role in the process of spontaneous regeneration in Xenopus . We propose that supplying neurons as a treatment for SCI is a valid strategy based on principles of regeneration, which are naturally possessed by regenerative animals.

Living organisms exhibit varying regenerative abilities depending on the species. Among them, urodele amphibians have been widely used in regeneration biology due to their remarkable regenerative capacity. Iberian ribbed newts, in particular, have been established as a prominent model for regeneration research, offering advantages such as a large number of eggs spawned, a short period of sexual maturation, and the development of genetic manipulation techniques. Cell tracing is an essential method for deciphering cellular processes during organ regeneration. The multicolor reporter Brainbow, which stochastically manifests multiple fluorescent proteins based on the Cre/lox recombination system, has been utilized for clonal analysis in regenerative animal models. In this study, we aimed to utilize this valuable multicolor reporter in Iberian ribbed newts, which are gaining increasing importance as a regenerative animal model. We generated transgenic Iberian ribbed newts carrying the Brainbow3.0 reporter cassette under the control of the CAG (cytomegalovirus early enhancer/chicken beta‐actin promoter/rabbit beta‐globin splice acceptor) promoter. Cre recombinase induction via electroporation led to recombinant reporter expression in the brain, spinal cord, and muscle. Recombinant reporter‐expressing cells could be traced in regenerating tail muscle, midbrain, and spinal cord. Additionally, we applied laser ablation to reporter‐positive epithelial cells of Brainbow3.0 newts, enabling clonal analyses at the cellular level. We expect that this long‐lasting multicolor reporter will prove versatile for a broad range of research fields.

In the M‐phase, the nuclear membrane is broken down, nucleosomes are condensed as mitotic chromosomes, and transcription factors are generally known to be dislocated from their recognition sequences and dispersed to the cytoplasm. However, some transcription factors have recently been reported to remain on mitotic chromosomes and facilitate the rapid re‐activation of the target genes in early G1‐phase. Paired‐like homeobox 2B (PHOX2B) is a transcription factor exhibiting chromosomal localization during M‐phase. PHOX2B mutations are associated with congenital central hypoventilation syndrome, Hirschsprung disease, and neuroblastoma. In this study, we investigated PHOX2B chromosomal localization during M‐phase through immunostaining and fluorescence recovery after photobleaching analysis to determine whether the chromosomal localization of disease‐associated PHOX2B mutants is altered during M‐phase. Missense mutations in the homeodomain and the frameshift mutation in the C‐terminal domain disrupted the chromosomal localization of PHOX2B in M‐phase, leading to its dispersion in the cell. Furthermore, a PHOX2B mutant with polyalanine expansion showed a line‐shaped localization to the restricted region of mitotic chromosomes. Our findings suggest an association between the disease‐associated mutations and defective chromosomal localization of transcription factors during M‐phase. Further investigations of PHOX2B chromosomal localization during M‐phase could reveal pathogenic mechanisms of such diseases.

Mesenchymal glioma stem cells (MES‐GSCs) are a major subtype of GSCs that reside within glioma tissues and contribute to metastasis, therapy resistance, and glioma recurrence. However, the precise molecular mechanisms governing MES‐GSC functions remain elusive. Our findings revealed that expression levels of miR‐505‐5p/‐3p are elevated in MES‐GSCs compared with those in proneural (PN)‐GSCs, glioma cell lines, and normal brain tissue and that miR‐505‐5p/‐3p expression levels are decreased in differentiated MES‐GSCs. We assumed that miR‐505‐5p/‐3p would play distinctive roles in MES‐GSCs and performed loss‐of‐function experiments targeting miR‐505‐5p/‐3p. Knockdown of miR‐505‐5p/‐3p increased proliferation and promoted differentiation in MES‐GSCs while suppressing invasion, temozolomide resistance, and enhancing apoptosis in MES‐GSCs. Mechanistically, miR‐505‐5p/‐3p directly targets the 3′ UTR of AUF1, leading to its repression in MES‐GSCs. Notably, AUF1 expression levels were significantly lower in MES‐GSCs compared with those in PN‐GSCs, glioma cell lines, and normal brain tissues. Co‐inhibition of AUF1 expression with miR‐505‐5p/‐3p knockdown ameliorated the observed cellular phenotypes caused by miR‐505‐5p/‐3p knockdown in MES‐GSCs. These results suggest that miR‐505‐5p/‐3p exerts MES‐GSC‐specific roles in regulating proliferation, differentiation, invasion, apoptosis, and temozolomide resistance by repressing AUF1 expression.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain‐ and spinal cord‐derived cells of post‐metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord‐derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.