Tissue-Targeted Transcriptomics Reveals SEMA3D Control of Hypoglossal Nerve Projection to Mouse Tongue Primordia.

The mouse hypoglossal nerve originates in the occipital motor nuclei at embryonic day (E)10.5 and projects a long distance, reaching the vicinity of the tongue primordia, the lateral lingual swellings, at E11.5. However, the details of how the hypoglossal nerve correctly projects to the primordia are poorly understood. To investigate the molecular basis of hypoglossal nerve elongation, we used a novel transcriptomic approach using the ROKU method. The ROKU algorithm identified 3825 genes specific for lateral lingual swellings at E11.5, of which 34 genes were predicted to be involved in axon guidance. Ingenuity Pathway Analysis-assisted enrichment revealed activation of the semaphorin signaling pathway during tongue development, and quantitative PCR showed that the expressions of Sema3d and Nrp1 in this pathway peaked at E11.5. Immunohistochemistry detected NRP1 in the hypoglossal nerve and SEMA3D as tiny granules in the extracellular space beneath the epithelium of the tongue primordia and in lateral and anterior regions of the mandibular arch. Fewer SEMA3D granules were localized around hypoglossal nerve axons and in the space where they elongated. In developing tongue primordia, tissue-specific regulation of SEMA3D might control the route of hypoglossal nerve projection via its repulsive effect on NRP1.

The relationship between Meckel's cartilage resorption and incisor tooth germ in mice.

Our understanding of the initiation and cellular mechanisms underlying endochondral resorption of Meckel's cartilage (MC) remains limited. Several studies have shown that the resorption site of MC and the mandibular incisor tooth germ are located close to each other. However, whether incisor tooth germ development is involved in MC resorption remains unclear. In this study, we aimed to elucidate the spatio-temporal interaction between the initiation site of MC resorption and the development of incisor tooth germs in an embryonic mouse model. To this effect, we developed a histology-based three-dimensional (3D) reconstruction technique using paraffin-embedded serial sections of various tissues in the jaw. The serial sections were cut in the frontal section and the tissue constituents (e.g., MC, incisor, and mineralized mandible) were studied using conventional and enzyme-based histochemistry. The outline of each component was marked on the frontal sectional images and 3D structures were constructed. To assess the vascular architecture at the site of MC resorption, immunohistochemical staining using anti-laminin, anti-factor VIII, and anti-VEGF antibodies was performed. MC resorption was first observed on the lateral incisor-facing side of the cartilage rods at sites anterior to the mental foramen on E16.0. The 3D analysis suggested that: (a) the posterior region of the clastic cartilage resorption corresponds to the cervical loop of the incisor; (b) the cervical portion of the tooth germ inflates probably due to temporal cellular congestion prior to differentiation into matrix-producing cells; (c) the incisor tooth germ tissue is present in close proximity to MC even in mouse with continuously growing tooth and determines the disappearance of MC as the tooth development.

Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue.

OBJECTIVES: The tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. METHODS: Tongue primordia were dissected from mouse embryos at embryonic day (E)10.5-E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. RESULTS: Microarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5-E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. CONCLUSIONS: These results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.

Aicardi-Goutières syndrome-like encephalitis in mutant mice with constitutively active MDA5.

MDA5 is a cytoplasmic sensor of viral RNA, triggering type I interferon (IFN-I) production. Constitutively active MDA5 has been linked to autoimmune diseases such as systemic lupus erythematosus, Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome (AGS), a genetically determined inflammatory encephalopathy. However, AGS research is challenging due to the lack of animal models. We previously reported lupus-like nephritis and SMS-like bone abnormalities in adult mice with constitutively active MDA5 (Ifih1G821S/+), and herein demonstrate that these mice also exhibit high lethality and spontaneous encephalitis with high IFN-I production during the early postnatal period. Increases in the number of microglia were observed in MDA5/MAVS signaling- and IFN-I-dependent manners. Furthermore, microglia showed an activated state with an increased phagocytic capability and reduced expression of neurotrophic factors. Although multiple auto-antibodies including lupus-related ones were detected in the sera of the mice as well as AGS patients, Ifih1G821S/+Rag2-/- mice also exhibited up-regulation of IFN-I, astrogliosis and microgliosis, indicating that auto-antibodies or lymphocytes are not required for the development of the encephalitis. The IFN-I signature without lymphocytic infiltration observed in Ifih1G821S/+ mice is a typical feature of AGS. Collectively, our results suggest that the Ifih1G821S/+ mice are a model recapitulating AGS and that microglia are a potential target for AGS therapy.

Malignant Leydig cell tumor in dogs: two cases and a review of the literature.

Malignant Leydig cell tumor (MLCT) is a rare testicular tumor in dogs. We report herein 2 dogs with MLCT and cutaneous metastasis. Grossly, marked enlargement and distortion of the involved testes were noted; on cut surface, the parenchyma was completely replaced by neoplastic tissue. In addition, these tumors had extensive necrosis and hemorrhage. Case 1 had a rapidly growing cutaneous mass in the left angle of the mouth; the lesion was well-circumscribed and had an indistinct lobular pattern. Case 2 had multiple cutaneous masses in the dorsal neck region, the thoracic back region, and the right hindlimb. Microscopically, the tumor lobules were composed of oval-to-polyhedral cells with eosinophilic cytoplasm and resembled testicular tumors. By immunohistochemistry, the neoplastic cells in both the testicular and cutaneous tumors were positive for inhibin-alpha and melan A. The mitotic counts of the primary tumors from cases 1 and 2 were 21 and 11 per 10 high-power fields, respectively. Based on these findings, the cases were diagnosed as MLCT with cutaneous metastasis. Ki-67 expression in the neoplastic cells of the 2 cases was higher than in benign Leydig cell tumors. Our findings may be helpful for the diagnosis of canine MLCT.

Suspected panosteitis in a crossbred calf.

A male crossbred calf developed a limp and pain upon deep pressure on the right hind limb and the right forelimb. The radiographic findings of affected limbs and pathological findings of bone biopsy were similar to those observed in canine panosteitis. This is the first case of suspected panosteitis reported in cattle.

Panostéite suspectée chez un veau de race croisée. Un veau mâle de race croisée a développé une boiterie et de la douleur à l'application d'une pression profonde sur la jambe arrière droite et la jambe avant droite. Les résultats de la radiographie des membres touchés et les résultats pathologiques d'une biopsie osseuse étaient semblables à ceux observés dans la panostéite canine. Il s'agit du premier cas de panostéite suspectée chez le bétail.(Traduit par Isabelle Vallières).

First report of Veronaea botryosa as a causal agent of chromomycosis in frogs.

A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran.

Lymphatic Stomata in the Adult Human Pulmonary Ligament.

BACKGROUND: Lymphatic stomata are small lymphatic openings in the serosal membrane that communicate with the serosal cavity. Although these stomata have primarily been studied in experimental mammals, little is known concerning the presence and properties of lymphatic stomata in the adult human pleura. Thus, adult human pleurae were examined for the presence or absence of lymphatic stomata. METHODS AND RESULTS: A total of 26 pulmonary ligaments (13 left and 13 right) were obtained from 15 adult human autopsy cases and examined using electron and light microscopy. The microscopic studies revealed the presence of apertures fringed with D2-40-positive, CD31-positive, and cytokeratin-negative endothelial cells directly communicating with submesothelial lymphatics in all of the pulmonary ligaments. The apertures' sizes and densities varied from case to case according to the serial tissue section. The medians of these aperture sizes ranged from 2.25 to 8.75 µm in the left pulmonary ligaments and from 2.50 to 12.50 µm in the right pulmonary ligaments. The densities of the apertures ranged from 2 to 9 per mm(2) in the left pulmonary ligaments and from 2 to 18 per mm(2) in the right pulmonary ligaments. However, no significant differences were found regarding the aperture size (p=0.359) and density (p=0.438) between the left and the right pulmonary ligaments. CONCLUSIONS: Our study revealed that apertures exhibit structural adequacy as lymphatic stomata on the surface of the pulmonary ligament, thereby providing evidence that lymphatic stomata are present in the adult human pleura.

Exposure to High Doses of Lipopolysaccharide during Ovalbumin Sensitization Prevents the Development of Allergic Th2 Responses to a Dietary Antigen.

Food allergies are driven by aberrant T helper (Th) 2 cells. Lipopolysaccharide (LPS) influences the development of Th2-mediated diseases, but its role in food allergy and tolerance remains unclear. To address this issue, we established mouse models presenting allergic or tolerant responses to ovalbumin (OVA). Mice sensitized with crude OVA developed Th2 responses including acute diarrhea, increases in serum OVA-specific IgE, dominant production of serum OVA-specific IgG1, increases in Th2-type cytokines and proliferation of mast cells in duodenal and colonic tissues. Sensitization of mice with crude OVA and LPS abrogated Th2-type responses observed in allergic mice. The level of OVA-specific proliferation in mesenteric lymph node CD4(+) T cells was comparable in allergic and tolerant mice, indicating that the tolerance is not caused by anergy and apoptosis of antigen-primed T cells. Expression of Th1- and Th2-type cytokines was suppressed in whole spleen cells and/or purified spleen CD4(+) T cells of tolerant mice, indicating that the tolerance was not caused by the shift from Th2 to Th1. On the other hand, interleukin (IL)-10, a regulatory cytokine produced by regulatory T cells, was upregulated in whole spleen cells and purified spleen CD4(+) T cells of tolerant mice. Furthermore, spleen CD4(+) T cells from tolerant mice suppressed the growth of CD4(+) T cells from DO11.10 mice in co-culture. These results indicate that tolerance is induced in allergic mice by simultaneous exposure to LPS during sensitization with OVA and that a population of T cells producing IL-10 plays an important role in the tolerance induction.

Effects of chronic γ-irradiation on growth and survival of the Tohoku hynobiid salamander, Hynobius lichenatus.

The Tohoku hynobiid salamanders, Hynobius lichenatus, were chronically irradiated with γ-rays from embryonic to juvenile stages for 450 days. At 490 µGy h(-1) or lower dose rates, growth and survival were not significantly affected by irradiation, and any morphological aberrations and histological damages were not observed. At 4600 µGy h(-1), growth was severely inhibited, and all the individuals died mostly at the juvenile stage. Chronic LD50 was 42 Gy as a total dose. In the liver, the number of hematopoietic cells was significantly reduced in the living juveniles, and these cells disappeared in the dead juveniles. In the spleen, mature lymphocytes were depleted in the living larvae, and almost all the heamtopoietic cells disappeared in the dead juveniles. These results suggest that this salamander died due to acute radiation syndrome, i.e., hematopoietic damage and subsequent sepsis caused by immune depression. The death would be also attributed to skin damage inducing infection. At 18,000 µGy h(-1), morphological aberrations and severe growth inhibition were observed. All the individuals died at the larval stage due to a multiple organ failure. Chronic LD50 was 28 Gy as a total dose. Assuming that chronic LD50 was 42 Gy at lower dose rates than 4600 µGy h(-1), a chronic median lethal dose rate could be estimated to be <340 µGy h(-1) for the whole life (>14 years). These results suggest that, among guidance dose rates, i.e., 4-400 µGy h(-1), proposed by various organisations and research programmes for protection of amphibians and taxonomic groups or ecosystems including amphibians, most of them would protect this salamander but the highest value may not on the whole life scale.

Three-dimensional visualization of perlecan-rich neoplastic stroma induced concurrently with the invasion of oral squamous cell carcinoma.

BACKGROUND: We have demonstrated the induction of perlecan-rich stroma of oral squamous cell carcinoma (SCC) on and after its start of invasion. However, it remains unknown how such a neoplastic stroma is actually arranged in tumor tissues. METHODS: To this end, tissue microarray samples, in which keratin and perlecan were contrastively labeled by immunohistochemistry, were three-dimensionally analyzed using digital images and image analysis software to demonstrate the relationship between SCC foci and the perlecan-positive stromal space or that between carcinoma in situ (CIS) and invasive SCC foci. RESULTS: The three-dimensional (3D) reconstruction demonstrated three kinds of perlecan profiles for inside (I) and outside (O) areas of the carcinoma cell focus: mode 1, I(+)/O(-) ; mode 2, I(+)/O(+) ; and mode 3, I(-)/O(+). Mode 1 was seen in CIS as well as SCC tumor massifs in the surface part. Mode 2 was seen in small SCC foci, which seemed isolated in 2D sections but were mostly continuous with the tumor massif in 3D reconstructions. Mode 3 was limited to small SCC foci, which were truly segregated from the tumor massif. CONCLUSIONS: The results indicated that the 2D SCC focus isolation could not be regarded as invasion but that the SCC foci surrounded by perlecan-positive stroma (modes 2 and 3) could be regarded as a more objective measure for invasion of SCC. This is the first 3D tissue-level demonstration of the neoplastic stroma space induced with oral SCC invasion, the presence of which we have predicted based on our previous 2D and tissue culture evidence.

Three-dimensional reconstruction of oral tongue squamous cell carcinoma at invasion front.

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 µ m thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.

Generation of a mouse model with down-regulated U50 snoRNA (SNORD50) expression and its organ-specific phenotypic modulation.

Box C/D-type small nucleolar RNAs (snoRNAs) are functional RNAs responsible for mediating 2'-O-ribose methylation of ribosomal RNAs (rRNAs) within the nucleolus. In the past years, evidence for the involvement of human U50 snoRNA in tumorigenesis has been accumulating. We previously identified U50HG, a non-protein-coding gene that hosted a box C/D-type U50 snoRNA, in a chromosomal breakpoint in a human B-cell lymphoma. Mouse genome analysis revealed four mouse U50 (mU50) host-genes: three mU50HG-a gene variants that were clustered in the genome and an mU50HG-b gene that we supposed to be the U50HG ortholog. In this study, to investigate the physiological importance of mU50 snoRNA and its involvement in tumorigenesis, we eliminated mU50 snoRNA sequences from the mU50HG-b gene. The established mouse line (ΔmU50(HG-b)) showed a significant reduction of mU50 snoRNA expression without alteration of the host-gene length and exon-intron structure, and the corresponding target rRNA methylation in various organs was reduced. Lifelong phenotypic monitoring showed that the ΔmU50(HG-b) mice looked almost normal without accelerated tumorigenicity; however, a notable difference was the propensity for anomalies in the lymphoid organs. Transcriptome analysis showed that dozens of genes, including heat shock proteins, were differentially expressed in ΔmU50(HG-b) mouse lymphocytes. This unique model of a single snoRNA knockdown with intact host-gene expression revealed further new insights into the discrete transcriptional regulation of multiple mU50 host-genes and the complicated dynamics involved in organ-specific processing and maintenance of snoRNAs.

Carcinosarcoma in a white Rhinoceros (Ceratotherium simum simum).

In Rhinocerotidae, there are very few reports of tumors and no reports of a mixed tumor. This paper reports the case of a male 33-year-old southern white rhinoceros. Grossly, there were two masses in the coelomic cavity and solid nodules in the liver. Histologically, all tumors had a biphasic pattern that consisted of malignant epithelial cells (cytokeratin- and E-cadherin-positive) and non-epithelial cells (vimentin-positive) with cartilage. In this case, the prostate could not be identified, and instead, the largest tumor mass was present at that site. Furthermore, since structures regarded as the prostate duct remained in this tumor, we considered that this tumor was very likely to be of prostate gland origin. This case is the first report of carcinosarcoma in Rhinocerotidae.

Crescentic glomerulonephritis in a polar bear (Ursus maritimus).

Spontaneous crescentic glomerulonephritis (CrGN) in animals has only been reported in dog and sheep. We report the pathological features of CrGN in a 17-year-old male polar bear that died due to renal failure. Histologically, the lesions were characterized by fibrocellular crescents, adhesion between Bowman's capsule and the glomerular capillary tuft and an increase in the mesangial matrix in glomeruli. The proliferating cells in the crescent were partly immunopositive for cytokeratin and intensely positive for vimentin, WT-1 and α-smooth muscle actin, suggesting they originated from parietal epithelial cells. Ultrastructually, thickening of the glomerular basement membrane and loss of epithelial cell foot processes were observed with electron-dense deposits.

Spontaneous nephroblastoma in a Japanese giant salamander (Andrias japonicus).

An adult male Japanese giant salamander (Andrias japonicus) died accidentally, and necropsy showed a white mass (23 × 15 mm) in the left kidney and hepatorrhexis with hemoperitoneum. Histologically, the renal mass was mainly composed of immature nephroblastic tumor cells. In the tumor tissue, a trabecular pattern lined by oval to polygonal tumor cells with a rich interstitium, solid growth and a few tubular structures was observed. Nephroblastic tumor cells were strongly positive for vimentin and weakly positive, and epithelium-like tumor cells were strongly positive for cytokeratin. However, antibody for Wilms' tumor protein 1 did not react with the salamander's cells. On electron microscopy, a desmosome junction was observed between tumor cells. This is the first report of nephroblastoma in a Japanese giant salamander.