Biology subjects

Wakayama, T.

Publications and source records attributed to Wakayama, T..

4 recordsLinked to original sources

bioRxiv2026

A conserved lamination pattern in the paleocortex and the neocortex revealed by single-cell RNA analyses

The piriform cortex, the largest paleocortical domain and a central olfactory cortex, is classically described as a three-layered structure, with layer II subdivided into semilunar and superficial pyramidal neurons. However, its cellular composition, developmental logic, and evolutionary relationship with the six-layered neocortex remain incompletely defined. Here, we aimed to resolve piriform cortical cell types and laminar organization and compare their molecular programs across mammalian cortical regions and the reptilian cortex. We performed single-nucleus RNA sequencing of the microdissected piriform cortex and integrated neuronal lineage data with published datasets, with spatial validation via the Allen Mouse Brain Atlas. Interspecies analyses identified piriform-dominant glutamatergic populations marked by Unc13c/Lmo3/Rora and an Rorb/Reln/Ntng1-enriched sensory-recipient subtype localized to the superficial layer IIa, consistent with semilunar neurons. Spatial transcriptomic mapping revealed that piriform layer IIb contains neocortical upper-layer-like corticocortical neurons, while piriform layer III segregates into layer V-like (IIIa), layer VI-like (IIIb), and VIb/VII (subplate)-equivalent populations, indicating a neocortex-like laminar framework with an inverted positioning of sensory recipients and corticocortical compartments relative to the neocortex. GABAergic neurons largely conformed to canonical MGE- and CGE-derived lineages but included a piriform-specific Rgs9+Pde7b+ subtype consistent with an LGE-related origin, suggesting region-specific diversification of GABAergic neurons. Disease ontology enrichment linked hippocampus-dominant glutamatergic programs to Alzheimers-related genes and piriform-dominant (and pan-cortical) excitatory and inhibitory programs to autism spectrum disorder and intellectual disability, implicating coordinated E/I circuit specializations in area-selective vulnerability. These findings support a revised view of the paleocortex as a divergent specialization of a conserved, multilayered cortical program and provide molecular markers and a comparative framework for studying cortical evolution and region-specific disease susceptibility.

developmental biology↗

bioRxiv2024

Claudin-11 regulates immunological barrier formation and spermatogonial proliferation through stem cell factor

Tight junctions (TJs) between adjacent Sertoli cells are believed to form immunological barriers that protect spermatogenic cells expressing autoantigens from autoimmune responses. However, there is no direct evidence that Sertoli cell TJs (SCTJs) do indeed form immunological barriers. Here, we analyzed male mice lacking claudin-11 (Cldn11), which encodes a SCTJ component, and found autoantibodies against antigens of spermatocytes/spermatids in their sera. Defective spermatogenesis was not restored in Cldn11-deficient mice on a genetic background mimicking a severely impaired adaptive immune system. This suggests that defective spermatogenesis is not caused by autoimmune responses against spermatogenic cells. Further analyses showed that Cldn11 knockout impaired Sertoli cell polarization, localization of stem cell factor (SCF) (a key molecule for maintaining differentiating spermatogonia) to the basal compartment of seminiferous tubules, and also proliferation of differentiating spermatogonia. We propose that CLDN11 creates a microenvironment for SCF-mediated spermatogonial proliferation at the basal compartment via Sertoli cell polarization.

developmental biology↗

bioRxiv2023

Reactive blue dye detects male reproductive toxicity

BackgroundReactive blue 2 (RB2) dye specifically binds to the nuclei of human spermatozoa under weakly alkaline conditions, thus providing a new method to assess sperm quality. However, this technique has not yet been applied to other mammalian species, such as well-established rodent models, which could enable evaluation of the male reproductive toxicity of drug candidates in non-clinical studies. ObjectivesWe aimed to evaluate the usefulness of RB2 staining in assessing testicular and epididymal sperm toxicity in mice using a busulfan-induced infertility model. MethodsMale C57BL/6J mice were intraperitoneally administered 40 mg/kg of busulfan. After 28 days, the testes and epididymis were collected and stained with RB2 at pH 10. In vitro evaluations were conducted on uncoated glass slides with RB2 mixed with either protamines extracted from the spermatozoa or intracellular protein components from somatic cells without protamines. ResultsFollowing peanut agglutinin (PNA) lectin histochemistry, RB2-positive cells were observed in elongating and elongated spermatids at all stages except for stages IX-XI of the seminiferous epithelium. After busulfan administration, the proportion of RB2-positive germ cells in the seminiferous tubules decreased significantly, and no RB2-positive spermatozoa were found in the caput epididymis of treated mice. Aggregates were observed in the mixture of RB2 dye (pH 10) with protamines but not in the mixture of intracellular protein components without protamines, and this specificity was lost at neutral pH. Discussion and ConclusionOur study demonstrates that RB2 specifically stains steps 12-16 spermatids, indicating specific binding to protamine expressed in these spermatids. The RB2 staining technique has potential as a biomarker for male reproductive toxicity, allowing for the rapid visualization of protamination.

pharmacology and toxicology↗

bioRxiv2022

Development of a non-invasive method for testicular toxicity evaluation using a novel compact magnetic resonance imaging system

In non-clinical animal studies for drug discovery, histopathological evaluation is the most powerful tool to assess testicular toxicity. However, histological analysis is extremely invasive; many experimental animals are needed to evaluate changes in the pathology and anatomy of the testes over time. As an alternative, small animal magnetic resonance imaging (MRI) offers a non-invasive methodology to examine testicular toxicity without radiation. The present study demonstrated the suitability of a new, ready-to-use compact MRI platform using a high-field permanent magnet to assist with the evaluation of testicular toxicity. To validate the utility of the MRI platform, male mice were treated with busulfan (40 mg/kg, intraperitoneal injection). Tenty-eight days after treatment, both testes in busulfan-treated and control mice (n = 3/group) were non-invasively scanned in situ by MRI at 1 tesla. On a T1-weighted, 3D gradient-echo MRI sequences (voxel size: 0.23 x 0.23 x 0.50 mm), the total testicular volume in busulfan-treated mice was significantly smaller than in controls. On T1-weighted images, the signal intensity of the testes was significantly higher in busulfan-treated mice than in controls. The mice were sacrificed, and the testes were isolated for histopathological analysis. The weight of the testes in busulfan-treated mice significantly decreased, similar to the results of the non-invasive analysis. Additionally, periodic acid-Schiff stain-positive effusions were observed in the interstitium of the busulfan-treated mouse testes, potentially explaining T1 shortening due to a high concentration of glycoproteinaceous content. The present data demonstrated a rapid evaluation of testicular toxicity in vivo by compact MRI.

pharmacology and toxicology↗