Naoki Tsunekawa

FGF signaling directs a center-to-pole expansion of tubulogenesis in mouse testis differentiation

In mouse embryogenesis, Sry is transiently activated in a center-to-pole wavelike manner along the anteroposterior (AP) axis of developing XY gonads. However, the mechanism and significance of the center-to-pole expansion of testis initiation pathways downstream of Sry expression remain unclear. Here we demonstrate that FGF9 can act as a diffusible conductor for a poleward expansion of tubulogenic programs at early phases of testis differentiation. In XY genital ridge cultures of anterior, middle and posterior segments at 11.0-11.25 days post-coitum, male-specific activation of Sry and its target gene, Sox9, was still observed in both anterior and posterior pole segments despite their isolation from the central domain. However, high-level Sox9 expression was not maintained, resulting in the failure of testis cord organization in most pole segments. A reconstruction experiment using ROSA:lacZ middle segments showed rescue of the tubulogenic defect in the poles without any appreciable contribution of lacZ-positive gonadal parenchyma cells. A partition culture assay also showed a possible contribution of soluble/diffusible factors secreted from the gonadal center domain to proper tubulogenesis in the poles. Among various signaling factors, Fgf9 expression was significantly lower in both anterior and posterior pole segments than in the central domain. The supportive role of the central domain could be substituted by exogenous FGF9 supply, whereas reduction of Wnt4 activity did not rescue the tubulogenesis defect in the pole segments. These observations imply that center-to-pole FGF9 diffusion directs a poleward expansion of testiculogenic programs along the AP axis of developing XY gonads.

Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes.

Background and Aims In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. Methodology and Principal Findings Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. Conclusion/Significance Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

Sox17 haploinsufficiency results in perinatal biliary atresia and hepatitis in C57BL/6 background mice

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute ‘embryonic hepatitis’, leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17+/− B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17+/− gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

Expression and function of mouse Sox17 gene in the specification of gallbladder/bile-duct progenitors during early foregut morphogenesis.

In early-organogenesis-stage mouse embryos, the posteroventral foregut endoderm adjacent to the heart tube gives rise to liver, ventral pancreas and gallbladder. Hepatic and pancreatic primordia become specified in the posterior segment of the ventral foregut endoderm at early somite stages. The mechanisms for demarcating gallbladder and bile duct primordium, however, are poorly understood. Here, we demonstrate that the gallbladder and bile duct progenitors are specified in the paired lateral endoderm domains outside the heart field at almost the same timing as hepatic and pancreatic induction. In the anterior definitive endoderm, Sox17 reactivation occurs in a certain population within the most lateral domains posterolateral to the anterior intestinal portal (AIP) lip on both the left and right sides. During foregut formation, the paired Sox17-positive domains expand ventromedially to merge in the midline of the AIP lip and become localized between the liver and pancreatic primordia. In Sox17-null embryos, these lateral domains are missing, resulting in a complete loss of the gallbladder/bile-duct structure. Chimera analyses revealed that Sox17-null endoderm cells in the posteroventral foregut do not display any gallbladder/bile-duct molecular characters. Our findings show that Sox17 functions cell-autonomously to specify gallbladder/bile-duct in the mouse embryo.

Disappearance of Vimentin in Sertoli Cells: A Mono(2-ethylhexyl) Phthalate Effect

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V–FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V–FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries

Summary In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry-inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

A Niche for GFRα1‐Positive Spermatogonia in the Terminal Segments of the Seminiferous Tubules in Hamster Testes

In invertebrate species such as flies and nematodes, germline stem cells are maintained in a niche environment, which is restricted to the terminal end of the tubular structure in the gonads. In mice, spermatogonial stem cells (SSCs), a subpopulation of Asingle GFRα1 (glial cell line‐derived neurotrophic factor [GDNF] family receptor‐α1)‐positive spermatogonia, are widely distributed along the longitudinal axis in the convoluted seminiferous tubules, preferentially juxtaposed to the interstitial vasculature. However, whether this area is the only SSC niche is not known. In this study, we identified a valve‐like terminal segment of the seminiferous tubules, the Sertoli valve (SV), adjacent to the rete testis as another niche for GFRα1‐positive spermatogonia in hamsters. Here, we show that the SV epithelium is composed of the modified Sertoli cells that are still capable of proliferation and missing most spermatogenic activities in the adult stage. The SV epithelium constitutively expresses GDNF, a major niche factor for SSCs, and supports the stable proliferation and selective maintenance of an Asingle subpopulation of GFRα1‐positive spermatogonia in hamsters. The SV region of hamster seminiferous tubules has features that are similar to the stem cell niche in invertebrate gonads. Therefore, we propose that the SV may be a novel niche for Asingle GFRá1‐positive spermatogonia potentially including a SSC population, at the terminal segments of the seminiferous tubules in hamsters. Stem Cells 2015;33:2811–2824

Dynamics of GFRα1-positive spermatogonia at the early stages of colonization in the recipient testes of W/Wν male mice

Background: The spermatogonial transplantation experiment can be used as an unequivocal detection assay of spermatogenic stem cells (SSCs) in both a qualitative and quantitative manner, based on their regenerative capacity. In this study, the proliferative patterns and kinetics of donor‐derived GFRα1‐positive spermatogonia containing potential SSCs were examined during early colonization following spermatogonial transplantation.Results: Donor‐derived GFRα1‐positive cells frequently formed several aggregates of Aal(aligned)/morula‐like structures in a single spermatogenic cell patch before and on day 14 post‐transplant, indicating a possible involvement in the formation of a stable spermatogenic colony at 21 days post‐transplant. The appearance of these Aal/morula‐like aggregates is positively correlated with regional, high‐level expression of immunoreactive GDNF signals, a ligand for GFRα1, associated with colony expansion. Conclusions: These data raise the hypothesis that regional GDNF signals regulate the balance between donor‐derived Aal‐like cell aggregates and their differentiation in each small patch, which subsequently leads to further selection of survival colonies at later stages. Developmental Dynamics 241:1374–1384, 2012.

Ontogenetic Shape Change in the Chicken Brain: Implications for Paleontology.

Paleontologists have investigated brain morphology of extinct birds with little information on post-hatching changes in avian brain morphology. Without the knowledge of ontogenesis, assessing brain morphology in fossil taxa could lead to misinterpretation of the phylogeny or neurosensory development of extinct species. Hence, it is imperative to determine how avian brain morphology changes during post-hatching growth. In this study, chicken brain shape was compared at various developmental stages using three-dimensional (3D) geometric morphometric analysis and the growth rate of brain regions was evaluated to explore post-hatching morphological changes. Microscopic MRI (μMRI) was used to acquire in vivo data from living and post-mortem chicken brains. The telencephalon rotates caudoventrally during growth. This change in shape leads to a relative caudodorsal rotation of the cerebellum and myelencephalon. In addition, all brain regions elongate rostrocaudally and this leads to a more slender brain shape. The growth rates of each brain region were constant and the slopes from the growth formula were parallel. The dominant pattern of ontogenetic shape change corresponded with interspecific shape changes due to increasing brain size. That is, the interspecific and ontogenetic changes in brain shape due to increased size have similar patterns. Although the shape of the brain and each brain region changed considerably, the volume ratio of each brain region did not change. This suggests that the brain can change its shape after completing functional differentiation of the brain regions. Moreover, these results show that consideration of ontogenetic changes in brain shape is necessary for an accurate assessment of brain morphology in paleontological studies.

IL‐17A‐producing CD30+ Vδ1 T cells drive inflammation‐induced cancer progression

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL‐17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL‐17A was critical for amplifying such local inflammation, as observed in the production of IL‐1β and neutrophil infiltration following the cross‐talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi‐invariant TCR initiate cancer‐promoting inflammation by producing IL‐17A in an MyD88/IL‐23‐dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune‐escalation process. Collectively, these results reveal the importance of IL‐17A‐producing CD30+ Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.