Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.

Membrane molecule bouncer regulates sperm binding activity in immature oocytes in the viviparous teleost species Poecilia reticulata (guppy).

Generally, in vertebrates, the first step toward fertilization is the ovulation of mature oocytes, followed by their binding to sperm cells outside of the ovary. Exceptionally, the oocytes of poeciliid fish are fertilized by sperm cells within the follicle, and the developmental embryo is subsequently released into the ovarian lumen before delivery. In the present study, we aimed to identify the factor(s) responsible for intrafollicular fertilization in a viviparous teleost species, Poecilia reticulata (guppy). Sperm tracking analysis in this regard indicated that in this species, sperm cells reached immature oocytes including the germinal vesicle, and the insemination assay indicated that the immature oocytes robustly adhered to the sperm cells; similar binding was not observed in Danio rerio (zebrafish) and Oryzias latipes (medaka). We also identified the Ly6/uPAR protein bouncer as the factor responsible for the observed sperm binding activity of the immature oocytes in this species. The recombinant bouncer peptide acted as an inhibitory decoy for the sperm-oocyte binding in guppy. On the other hand, ectopic expression of guppy bouncer in zebrafish oocytes resulted in interspecific sperm-oocyte binding. These results argue that bouncer is responsible for sperm-immature oocyte binding. Our findings highlight the unique reproductive strategies of guppy fish and enhance our understanding of the diverse reproductive mechanisms in vertebrates.

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of ß-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization.

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the "serial section method" has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.

Upregulated miR-224-5p suppresses osteoblast differentiation by increasing the expression of Pai-1 in the lumbar spine of a rat model of congenital kyphoscoliosis.

Congenital scoliosis is defined by the presence of structural anatomical malformations that arise from failures of vertebral formation or segmentation before and after birth. The understanding of genetic background and key genes for congenital scoliosis is still poor. We herein report that the excess expression of plasminogen activator inhibitor-1 (Pai-1) induced by the upregulation of miR-224-5p is involved in the pathogenesis of congenital kyphoscoliosis through impaired osteoblast differentiation. We first investigated the variety and progression of abnormalities of the lumbar spines in Ishibashi (IS) rats, a rat model of congenital kyphoscoliosis. The rats had already shown fusion and division of the primary ossification center at postnatal day 4. Over time, the rats showed various abnormalities of the lumbar spine, including the fusion of the annular epiphyseal nucleus. At postnatal day 42, spinal curvature was clearly observed due to the fusion of the vertebral bodies. Using a microRNA array, we found that the expression of miR-224-5p was increased in the lumbar spine of the rats at postnatal day 4. The expression of Pai-1, which is involved in osteoblast differentiation regulated by miR-224-5p, was also increased, while the levels of type I collagen, a marker of osteoblast differentiation, were decreased in the lumbar spine. These results indicate that the aberrant expression of miRNA-224-5p and its target genes is involved in the impaired osteoblast differentiation and may provide a partial molecular explanation for the pathogenesis of congenital scoliosis.

Loss of VAMP5 in mice results in duplication of the ureter and insufficient expansion of the lung.

BACKGROUND: Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which regulates the docking and fusion of membrane vesicles within cells. Previously, we reported ubiquitous expression of VAMP5 proteins in various organs except the brain and small intestine. However, the precise roles of VAMP5 in each organ remain unclear. To explore the roles of VAMP5 in vivo, we generated VAMP5 knockout (KO) mice. RESULTS: VAMP5 KO mice showed low birth rate and low body weight. KO embryos grew normally in the uterus, and tended to die around birth. Anatomical analysis revealed that viable KO mice often exhibited duplication of the ureter, and dead KO mice showed insufficient expansion of the lung. VAMP5 was localized in the epithelial cells of the ureter and terminal bronchiole. CONCLUSIONS: VAMP5 KO mice showed a low birth rate and abnormalities of the urinary and respiratory systems. VAMP5 KO mice died around birth, possibly due to defects in vesicoureteral flow and breathing. The results presented could provide a basis for future studies to understand the roles of VAMP5 protein. Developmental Dynamics 247:754-762, 2018. © 2018 Wiley Periodicals, Inc.

Filamentous structures in skeletal muscle: anchors for the subsarcolemmal space.

In skeletal muscle fibers, intermediate filaments and actin filaments provide structural support to the myofibrils and the sarcolemma. For many years, it was poorly understood from ultrastructural observations that how these filamentous structures were kept anchored. The present study was conducted to determine the architecture of filamentous anchoring structures in the subsarcolemmal space and the intermyofibrils. The diaphragms (Dp) of adult wild type and mdx mice (mdx is a model for Duchenne muscular dystrophy) were subjected to tension applied perpendicular to the long axis of the muscle fibers, with or without treatment with 1% Triton X-100 or 0.03% saponin. These experiments were conducted to confirm the presence and integrity of the filamentous anchoring structures. Transmission electron microscopy revealed that these structures provide firm transverse connections between the sarcolemma and peripheral myofibrils. Most of the filamentous structures appeared to be inserted into subsarcolemmal densities, forming anchoring connections between the sarcolemma and peripheral myofibrils. In some cases, actin filaments were found to run longitudinally in the subsarcolemmal space to connect to the sarcolemma or in some cases to connect to the intermyofibrils as elongated thin filaments. These filamentous anchoring structures were less common in the mdx Dp. Our data suggest that the transverse and longitudinal filamentous structures form an anchoring system in the subsarcolemmal space and the intermyofibrils.

Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins.

Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes.

Evaluation of ACL mid-substance cross-sectional area for reconstructed autograft selection.

PURPOSE: The purpose of this study was to compare the size of the native ACL mid-substance cross-sectional area and the size of commonly used autografts. Hypothesis of this study was that the reconstructed graft size with autografts would be smaller than the native ACL size. METHODS: Twelve non-paired human cadaver knees were used. The ACL was carefully dissected, and the mid-substance of the ACL was cross-sectioned parallel to the articular surface of the femoral posterior condyles at 90 degrees of knee flexion. The size of the cross-sectional area of the ACL, and the femoral and tibial footprints were measured using Image J software (National Institute of Health). The semitendinosus tendon (ST) and the gracilis (G) tendon were harvested and prepared for ACL grafts. Simulating an ST graft, the ST was cut in half. The bigger half was regarded as the antero-medial (AM) bundle, and the remaining half was regarded as the postero-lateral (PL) bundle. Simulating an ST-G graft, the bigger half of the ST and G were regarded as the AM bundle, and the smaller half of the ST was regarded as the PL bundle. Each graft diameter was measured, and the graft area was calculated. Simulating a rectangular bone-patella tendon-bone (BPTB) graft, a 10-mm-wide BPTB graft was harvested and the area calculated. RESULTS: The sizes of the ACL mid-substance cross-sectional area, femoral and tibial ACL footprint were 46.9 ± 18.3, 60.1 ± 16.9 and 123.5 ± 12.5 mm(2), respectively. The average areas of the ST, ST-G, and BPTB grafts were 52.0 ± 3.8, 64.4 ± 6.2, and 40.8 ± 6.7 mm(2), respectively. The ST and BPTB grafts showed no significant difference in graft size when compared with the ACL cross-sectional area. CONCLUSION: ST and BPTB autografts were able to reproduce the native size of the ACL mid-substance cross-sectional area. The ST-G graft was significantly larger than the ACL cross-sectional area. For clinical relevance, ST and BPTB grafts are recommended in order to reproduce the native size of the ACL in anatomical ACL reconstruction with autograft.

The localization of VAMP5 in skeletal and cardiac muscle.

Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which is generally thought to regulate the docking and fusion of vesicles with their target membranes. This study investigated the expression and localization of the VAMP5 protein. Immunoblotting analyses detected the VAMP5 protein in skeletal muscle, heart, spleen, lung, liver, and kidney tissue, but not in brain or small intestine tissue. Through the immunofluorescence microscopy of skeletal muscle, we found that the expression level of VAMP5 varies among fibers. Most of the fibers with high expression levels of VAMP5 were categorized as type IIa fibers on the basis of their myosin heavy chain subtypes. In addition, the expression patterns of VAMP5 and glucose transporter 4 (GLUT4) were similar. In cardiac muscle, we determined that VAMP5 was localized to the vicinity of intercalated discs. These results suggest that VAMP5 plays local roles in membrane trafficking in skeletal and cardiac muscle.

Alterations of biochemical marker levels and myonuclear numbers in rat skeletal muscle after ischemia-reperfusion.

Prolonged ischemia-reperfusion results in various damages in skeletal muscle. Following reperfusion, although the damaged muscles undergo regeneration, the precise process and mechanism of regeneration have not yet been fully understood. Here, we show the altered levels of plasma biochemical markers of muscle damage, and the change in myonuclear numbers in adult rat skeletal muscle by ischemia-reperfusion. Male Wistar rats were subjected to unilateral hindlimb ischemia by clamping the anterior tibial artery for 2 h before reperfusion. Both plasma creatine kinase activity and C-reactive protein levels in plasma were increased significantly at 0.5 h of reperfusion and returned to the control level at 24 h. The transverse sectional area of muscle belly of the anterior tibial muscles in ischemic side was significantly decreased by 20 % compared with those in sham-ischemic (control) side at 2 days, and returned to the control level at 5 days of reperfusion. Moreover, the number of interstitial nuclei in the ischemic side were significantly increased at 5-14 days and returned to the control level at 21 days of reperfusion. Central nuclei that are specifically observed in regenerating muscle, appeared at 5 days, reached a peak at 14 days, and disappeared at 28 days of reperfusion. Furthermore, MyoD, a regulatory factor for myogenesis, showed a transient expression at 5 days of reperfusion. These results indicate that, although the size of muscle seems to be recovered by 5 days of reperfusion, the most active muscle regeneration occurs much later, as shown by the increase in central nuclei.

Micro-abnormalities in the retina and choroid induced by anti-CTLA4 treatment.

Anti-Cytotoxic T-Lymphocyte Associated protein 4 agents, such as ipilimumab, are widely applied to various cancers. However, they cause immune-related adverse effects throughout the body, including the eye. This study examined whether ipilimumab induces retinal and choroidal abnormalities in rodents, and investigated potential underlying mechanisms. Female wild-type mice were injected with ipilimumab three times/week for 5 weeks. The mice underwent optical coherence tomography (OCT) on the first day of the 6th week. Retinal function and morphology were evaluated by light microscopy, immunohistochemistry and electroretinography (ERG). On OCT, the lines indicating the ellipsoid and interdigitation were obscure in treated mice, suggesting outer retina destruction. Haematoxylin-eosin staining revealed destruction, shortening, and outer segment vacuolization. Treated mice exhibited weaker, fragmented rhodamine peanut agglutinin staining in outer photoreceptor structures. The choroid of treated mice showed severe infiltration of CD45-positive cells. In addition, CD8-positive cells invaded into the outer retina. On ERG, rod, maximum responses of combined rods and cones, and cone response wave amplitudes were significantly reduced in treated mice. Ipilimumab may induce impairments in outer photoreceptor architecture accompanied with CD8- positive infiltration in the retina and CD45-positive cell infiltration in the choroid, which may contribute to retinal function deterioration.

Correlative microscopy and block-face imaging (CoMBI): a 3D imaging method with wide applicability in the field of biological science.

Correlative microscopy and block-face imaging (CoMBI) is an imaging method, which is characterized by the ability to obtain both serial block-face images as a 3-dimentional (3D) dataset and sections for 2-dimentional (2D) light microscopic analysis. These 3D and 2D morphological data can be correlated with each other to facilitate data interpretation. CoMBI is an easy-to-install and low-cost 3D imaging method since its system can be assembled by the researcher using a regular microtome, consumer digital camera, and some self-made devices, and its installation and instruction manuals are open-source. After the first release of CoMBI method from our laboratory, CoMBI systems have been installed in more than a dozen laboratories and are used for 3D analysis of various biological specimens. Typical application of CoMBI is 3D anatomical analysis using the natural color and contrast of the specimen. We have been using CoMBI for analyzing human brain to obtain the fine 3D anatomy as a reference to determine the causes of neurological diseases and to improve the effectiveness of surgery. Recently, we have been using CoMBI for detecting the colors of chromogens, which are used for labeling specific molecules. Mouse embryos colored with X-gal, a conventional chromogen for detecting LacZ products, were imaged using CoMBI, and the 3D distribution of X-gal was successfully visualized. Thus, CoMBI can now be used for many purposes, including 3D anatomical analysis, 2D microscopy using sections, and 3D distribution of specific molecules. These suggest that CoMBI should be more widely used in the field of biological research.

Recent technological advances in correlative light and electron microscopy for the comprehensive analysis of neural circuits.

Light microscopy (LM) covers a relatively wide area and is suitable for observing the entire neuronal network. However, resolution of LM is insufficient to identify synapses and determine whether neighboring neurons are connected via synapses. In contrast, the resolution of electron microscopy (EM) is sufficiently high to detect synapses and is useful for identifying neuronal connectivity; however, serial images cannot easily show the entire morphology of neurons, as EM covers a relatively narrow region. Thus, covering a large area requires a large dataset. Furthermore, the three-dimensional (3D) reconstruction of neurons by EM requires considerable time and effort, and the segmentation of neurons is laborious. Correlative light and electron microscopy (CLEM) is an approach for correlating images obtained via LM and EM. Because LM and EM are complementary in terms of compensating for their shortcomings, CLEM is a powerful technique for the comprehensive analysis of neural circuits. This review provides an overview of recent advances in CLEM tools and methods, particularly the fluorescent probes available for CLEM and near-infrared branding technique to match LM and EM images. We also discuss the challenges and limitations associated with contemporary CLEM technologies.

Maternal hypothyroidism is associated with M-opsin developmental delay.

Thyroid hormones are critical for the development of opsins involved in color vision. Hypothyroid mice show delayed M-opsin development and expanded distribution of S-opsin on the retina. However, the effects of maternal hypothyroidism on opsin development remain unknown. This study investigates the effects of congenital central hypothyroidism and maternal hypothyroidism on opsin development in thyrotropin-releasing hormone knockout (TRH-/-) mice. We examined the mRNA expression and protein distribution of S/M-opsin on postnatal days (P)12 and 17, as well as mRNA expression of type 2 and 3 iodothyronine deiodinase (DIO2 and DIO3, respectively) in the retina and type 1 iodothyronine deiodinase (DIO1) in the liver at P12 in TRH+/- mice born to TRH+/- or TRH-/- dams, and conducted S/M-opsin analysis in TRH+/+ or TRH-/- mice born to TRH+/- dams at P12, P17, and P30. M-opsin expression was lower in TRH+/- mice born to TRH-/- dams than in those born to TRH+/- dams, whereas S-opsin expression did not significantly differ between them. DIO1, DIO2, and DIO3 mRNA expression levels were not significantly different between the two groups; therefore, thyroid function in peripheral tissues in the pups was similar. S/M-opsin expression did not significantly differ between the TRH+/+ and TRH-/- mice born to TRH+/- dams on any postnatal day. These results demonstrate that maternal hypothyroidism causes M-opsin developmental delay during the early developmental stages of neonatal mice, and TRH-/- mice, a model of congenital central hypothyroidism, born to a euthyroid dam do not have delayed opsin development.

Correlative microscopy and block-face imaging (CoMBI) method for both paraffin-embedded and frozen specimens.

Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.

Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells.

Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (i.e. phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells.

VAMP2 marks quiescent satellite cells and myotubes, but not activated myoblasts.

We examined the expression and intracellular localization of vesicle-associated membrane protein 2 (VAMP2) during the differentiation of skeletal muscle cells by immunofluorescence microscopy. In isolated single myofibers, VAMP2 was expressed in quiescent satellite cells, downregulated in proliferating myoblastic cells, and re-expressed with differentiation. In the myoblastic cell line C2C12, VAMP2 was expressed at a low level in the proliferating stage, and then increased after differentiation into myotubes. Based on these results, we propose that VAMP2 can be used as a molecular marker for both quiescent satellite cells and myotubes, but not for proliferating myoblasts. We also found the partial colocalization of VAMP2 with transferrin- or Rab11-labeled vesicles in myotubes, suggesting a role of VAMP2 in the trafficking of recycling endosomes.

Neural regulation in tooth regeneration of Ambystoma mexicanum.

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.

Insufficient membrane fusion in dysferlin-deficient muscle fibers after heavy-ion irradiation.

Recently, SJL/J mice have been used as an animal model in studies of dysferlinopathy, a spectrum of muscle diseases caused by defects in dysferlin protein. In this study we irradiated muscle fibers isolated from skeletal muscle of SJL/J mice with heavy-ion microbeam, and the ultrastructural changes were observed by electron microscopy. The plasma membrane of heavy-ion beam irradiated areas showed irregular protrusions and invaginations. Disruption of sarcomeric structures and the enhancement of autophagy were also observed. In addition, many vesicles of varying size and shape were seen to be accumulated just beneath the plasma membrane. This finding further supports the recent hypothesis that dysferlin functions as a membrane fusion protein in the wound healing system of plasma membrane, and that the defect in dysferlin causes insufficient membrane fusion resulting in accumulation of vesicles.