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Biomedical subjects

Kohei Shiota

Publications and source records attributed to Kohei Shiota.

At least 19 recordsLinked to original sources
PubMed2007

Amniotic fluid induces rapid epithelialization in the experimentally ruptured fetal mouse palate--implications for fetal wound healing.

Cleft of the secondary palate is one of the most common congenital birth defects in humans. The primary cause of cleft palate formation is a failure of fusion of bilateral palatal shelves, but rupture of the once fused palate has also been suggested to take place in utero. The possibility of post-fusion rupture of the palate in humans has hardly been accepted, mainly because in all the cleft palate cases, the cleft palatal edge is always covered with intact epithelium. To verify whether the intrauterine environment of the fetus plays roles in wound healing when the once fused palate is torn apart, we artificially tore apart fetal mouse palates after fusion and cultivated them in culture medium with or without mouse or human amniotic fluid. We thereby found that the wounded palatal edge became completely covered with flattened epithelium after 36 hours in culture with amniotic fluid, but not in culture without amniotic fluid. Using histological and scanning electron microscopic analyses of the healing process, it was revealed that the epithelium covering the wound was almost exclusively derived from the adjacent nasal epithelium, but not from the oral epithelium. Such actions of amniotic fluid on the fetal wound were never simulated by exogenous epidermal growth factor (EGF), albumin, or both. In addition, the rapid epithelialization induced by amniotic fluid was not prevented by either PD168393 (an inhibitor of the EGF receptor-specific tyrosine kinase) or SB431542 (a specific inhibitor of TGFbeta receptor type I/ALK5). The present study provides new insights into the unique biological actions of amniotic fluid in the repair of injured fetal palate.

Activin Receptors, Type I↗
PubMed2006

Analysis of Fibroblast growth factor 15 cis-elements reveals two conserved enhancers which are closely related to cardiac outflow tract development.

Fibroblast growth factor 15 (Fgf15) is expressed in the developing mouse central nervous system and pharyngeal arches. Fgf15 mutant mice showed defects of the cardiac outflow tract probably because of aberrant behavior of the cardiac neural crest cells. In this study, we examined cis-elements of the Fgf15 gene by transient transgenic analysis using lacZ as a reporter. We identified two enhancers: one directed lacZ expression in the hindbrain/spinal cord and the other in the posterior midbrain (pmb), rhombomere1 (r1) and pharyngeal epithelia. Interestingly, human genomic regions which are highly homologous to these two mouse enhancers showed almost the same enhancer activities as those of mice in transgenic mouse embryos, indicating that the two enhancers are conserved between humans and mice. We also showed that the mouse and human pmb/r1 enhancer can regulate lacZ expression in chick embryos in almost the same way as in mouse embryos. We found that the lacZ expression domain with this enhancer was expanded by ectopic Fgf8b expression, suggesting that this enhancer is regulated by Fgf8 signaling. Moreover, over-expression of Fgf15 resulted in up-regulation of Fgf8 expression in the isthmus/r1. These findings suggest that a reciprocal positive regulation exists between Fgf15 and Fgf8 in the isthmus/r1. Together with cardiac outflow tract defects in Fgf15 mutants, the conservation of enhancers in the hindbrain/spinal cord and pharyngeal epithelia suggests that human FGF19 (ortholog of Fgf15) is involved in early development and the distribution of cardiac neural crest cells and is one of the candidate genes for congenital heart defects.

Animals↗
PubMed2006

Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection.

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time-consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer-based self-learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos.

Anatomy, Artistic↗
PubMed2006

Regional heterogeneity in the developing palate: morphological and molecular evidence for normal and abnormal palatogenesis.

Development of the mammalian secondary palate involves the growth, elevation, medial elongation and midline fusion of palatal shelves. Recent morphological and molecular studies on palatogenesis suggest that the developing palate is not a homogeneous organ but each part may behave differently during organogenesis. Especially, some key molecules involved in palate development have been shown to exhibit heterogeneous patterns of expression in the palatal tissue. Therefore it seems necessary to recognize the regional heterogeneity of the developing palate along the dorsoventral and anteroposterior axes when analyzing the mechanisms of normal and abnormal morphogenesis. Based on recent studies, we discuss the issue of the regional heterogeneity in the fetal palate and propose a principle that divides the fetal palate into several regions from the morphological and molecular standpoint.

Animals↗
PubMed2005

Mixed-mode pattern in Doublefoot mutant mouse limb--Turing reaction-diffusion model on a growing domain during limb development.

It has been suggested that the Turing reaction-diffusion model on a growing domain is applicable during limb development, but experimental evidence for this hypothesis has been lacking. In the present study, we found that in Doublefoot mutant mice, which have supernumerary digits due to overexpansion of the limb bud, thin digits exist in the proximal part of the hand or foot, which sometimes become normal abruptly at the distal part. We found that exactly the same behaviour can be reproduced by numerical simulation of the simplest possible Turing reaction-diffusion model on a growing domain. We analytically showed that this pattern is related to the saturation of activator kinetics in the model. Furthermore, we showed that a number of experimentally observed phenomena in this system can be explained within the context of a Turing reaction-diffusion model. Finally, we make some experimentally testable predictions.

Animals↗
PubMed2005

Disruption of actin cytoskeleton and anchorage-dependent cell spreading induces apoptotic death of mouse neural crest cells cultured in vitro.

In vertebrate embryos, neural crest cells emigrate out of the neural tube and contribute to the formation of a variety of neural and nonneural tissues. Some neural crest cells undergo apoptotic death during migration, but its biological significance and the underlying mechanism are not well understood. We carried out an in vitro study to examine how the morphology and survival of cranial neural crest (CNC) cells of the mouse embryo are affected when their actin cytoskeleton or anchorage-dependent cell spreading is perturbed. Disruption of actin fiber organization by cytochalasin D (1 microg/ml) and inhibition of cell attachment by matrix metalloproteinase-2 (MMP-2; 2.0 units/ml) were followed by morphologic changes and apoptotic death of cultured CNC cells. When the actin cytoskeleton was disrupted by cytochalasin D, the morphologic changes of cultured CNC cells preceded DNA fragmentation. These results indicate that the maintenance of cytoskeleton and anchorage-dependent cell spreading are required for survival of CNC cells. The spatially and temporally regulated expression of proteinases may be essential for the differentiation and migration of neural crest cells.

Actins↗
PubMed2005

Ectopic dermal ridge configurations on the interdigital webbings of Hammertoe mutant mice (Hm): another possible role of programmed cell death in limb development.

BACKGROUND: The mechanism underlying the development of aberrant phalangeal pads and dermal ridge configurations in malformed limbs is not well understood. The forelimbs of Hammertoe (Hm) mutant mouse fetuses were examined sequentially to clarify the relationship between the occurrence of abnormal programmed cell death (PCD) and the formation of phalangeal pads and dermal ridge patterns. METHODS: Relevant morphological features, with special emphasis on pads and dermal ridge configurations, were inspected on the exposed dermal surface of the forelimbs of adult Hm mutant mice. The forelimbs of Hm mutant mouse fetuses (GD13-18) and newborns were examined histologically. The forelimbs of GD13 fetuses were subjected to Nile blue (NB) vital staining for in situ labeling of PCD. RESULTS: In the forelimbs of +/+ mice, the formation of dermal ridges was confined to pads, while in Hm/+ and Hm/Hm animals, which have interdigital webbing involving digits II-V, dermal ridges were formed also on the ventral side of the webbing, specifically on its lateral margins between the neighboring digits and on the medial margin of the webbing extending toward the palmar pad. PCD was decreased in the interdigital zones II-IV in GD13 Hm/+ and Hm/Hm fetuses. CONCLUSIONS: Reduced PCD interdigital tissue of Hm/+ and Hm/Hm fetuses may result in the failure of physiological elimination of interdigital cells and in the persistence of soft tissue webbing between digits. The failure of PCD to occur may also interrupt the interdigital surviving cells to reach the neighboring digits and the distal area of the palm, thereby producing ectopic dermal ridges. It seems that interdigital PCD contributes not only to digit separation but also to the development of digital and palmar pads.

Animals↗
PubMed2005

Expression of the mouse Fgf15 gene is directly initiated by Sonic hedgehog signaling in the diencephalon and midbrain.

Sonic hedgehog (Shh) is a secreted molecule that is thought to regulate tissue growth and patterning in vertebrate embryos. Although it has been reported that Gli transcription factors mediate Shh signaling to the nucleus, little is known about developmental target genes of Gli. In the previous genetic study, we showed that Shh is required for Fgf15 expression in the diencephalon and midbrain. Here, we examined whether Fgf15 is a direct target of Shh signaling through Gli. Shh was expressed in the midline cells and Fgf15 in the medial region of the diencephalon/midbrain by the seven-somite stage. The Fgf15 expression domain coincided with that of Gli1 and overlapped with that of Gli2 at this stage. Fgf15 expression in the diencephalon/midbrain was greatly reduced in the seven-somite Shh mutant embryos. Transgenic analysis showed that the 3.6-kb 5'-flanking region of the Fgf15 gene is sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain. Luciferase assay showed that the 3.6-kb Fgf15 enhancer/promoter was activated by Gli2. A Gli-binding site was located 1 kb upstream of the transcription start site and was required for expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. These findings indicate that Fgf15 is directly regulated by Shh signaling through Gli proteins.

Amino Acid Motifs↗
PubMed2005

Embryogenesis of fused umbilical arteries in human embryos.

OBJECTIVE: The objective of this study was to elucidate the embryologic basis of fused umbilical arteries in the human. STUDY DESIGN: Twenty-nine human embryo specimens at Carnegie stages 11 through 15 (4-5 weeks after fertilization) were examined histologically, with special reference to the development of umbilical arteries. RESULTS: All embryos at Carnegie stage 11 and 12 had fused umbilical arteries, and 66% of Carnegie stage 13 embryos and 29% of Carnegie stage 14 embryos still had the condition. None of the embryos at Carnegie stage 15 or older had fused umbilical arteries, but there were always 2 arteries present in their umbilical cords. CONCLUSION: Our data suggest that (1) a single umbilical artery splits into 2 as the developmental stage of the embryo advances, (2) that fused umbilical arteries represent a remnant of the embryonic phenotype, and (3) that fused umbilical arteries are embryologically distinct from true single umbilical artery.

Cadaver↗
PubMed2005

Signaling cascade coordinating growth of dorsal and ventral tissues of the vertebrate brain, with special reference to the involvement of Sonic Hedgehog signaling.

The vertebrate brain is a complex and highly organized structure with numerous neurons and glial cells. During development, undifferentiated progenitor cells proliferate from neural stem/precursor cells and gradually restrict their fates according to their environment. Differentiated cells are arranged precisely to accomplish their function and to maintain integrity as a whole brain. In this respect, cells must receive signals to know where and when they determine their fates. Secreted and membrane molecules convey the information between cells. The secreted glycoprotein Sonic hedgehog (Shh) is one of such signaling molecules. Sonic hedgehog is widely known to specify ventral neuronal types according to the concentration of Shh, whereas differentiation of dorsal neurons is largely independent of Shh. However, in the diencephalon and midbrain, dorsal parts are also affected in Shh-mutant embryos. Detailed analysis demonstrated that Shh signaling indirectly regulates the growth of the dorsal tissue in these regions. One of the fibroblast growth factor (FGF) members, namely FGF15, has been reported to be downstream to Shh signaling in the mouse embryonic brain. Luciferase assays and transgenic analysis revealed that the Fgf15 gene is a direct target of Shh. Downregulation of Tcf4 and upregulation of Bmp4 in Shh mutants suggest that Wnt and BMP signals from the dorsal midline are also involved in the dorsal brain phenotype. These data suggest the coordinating role of the Shh-FGF15-Wnt/BMP signaling cascade between the ventral and dorsal parts of the brain.

Animals↗
PubMed2005

Assisted reproductive technologies and birth defects.

In vitro fertilization (IVF) and other assisted reproductive technologies (ART) are effective treatments for infertility and are widely provided at infertility clinics. Although IVF and related ART procedures are generally considered safe, some studies have suggested an excess occurrence of major malformations, low birth-weight and other perinatal complications in babies conceived by ART. Further, it was recently reported that IVF and intracytoplasmic sperm injection (ICSI) are associated with imprinting disorders in the offspring such as Beckwith-Wiedemann syndrome and Angelman syndrome. Here we review the human and animal studies investigating the potential risks of ART, and discuss the need for further investigation.

Animals↗
PubMed2005

Transforming growth factor beta 2 promotes the formation of the mouse cochleovestibular ganglion in organ culture.

The inner ear structures are derived from the otic vesicle (OV) which is formed by thickening and invagination of the otic placode of the surface ectoderm. A number of neuroblasts, which arise from epithelial cells of the otic vesicle, delaminate and differentiate into neurons of the cochleovestibular ganglion (CVG). We have found that transforming growth factor-BEta2 (Tgfbeta2 ) was expressed in the otic epithelium at the OV stages between Embryonic days (E) 9.5 and 11.5 and that anteroventrolateral localization of its expression in the OV overlapped with that of NeuroD, which is a marker of delaminating CVG precursors. The expression of TGFbeta type I and type II receptors in the otic epithelium and the nuclear localization of phosphorylated-Smad2 in both the otic epithelium and CVG suggested that TGFbeta2 signaling plays some roles in CVG formation. In order to examine the roles of TGFbeta2 in differentiation of the inner ear, otic vesicle explants of E10.5 mouse embryos were treated in vitro with TGFbeta2 or the TGFbeta type I receptor kinase inhibitor, SB431542. Addition of TGFbeta2 peptide to the culture led to Enlargement of the CVG, while the inhibitor reduced its size. These findings strongly imply that TGFbeta2 contributes to the development of the CVG in mouse embryos.

Animals↗
PubMed2004

Development of the posterior neural tube in human embryos.

Development of the posterior neural tube (PNT) in human embryos is a complicated process that involves both primary and secondary neurulation. Because normal development of the PNT is not fully understood, pathogenesis of spinal neural tube defects remains elusive. To clarify the mechanism of PNT development, we histologically examined 20 human embryos around the stage of posterior neuropore closure and found that the developing PNT can be divided into three parts: 1) the most rostral region, which corresponds to the posterior part of the primary neural tube, 2) the junctional region of the primary and secondary neural tubes, and 3) the caudal region, which emerges from the neural cord. In the junctional region, the axially-condensed mesenchyme (AM) intervened between the neural plate/tube and the notochord at the stage of posterior neuropore closure, while the notochord was directly attached to the neural plate/tube in the most rostral region. A single cavity was found to be formed in the AM as the presumptive luminal surface cells were radially aligned in the junctional region prior to the formation of the neural cord. The single cavity was continuous with the central cavity of the primary neural tube. In contrast, multiple or isolated cavities were frequently observed in the caudal region of the PNT. Our observation suggests that the junctional region of the PNT is distinct from other regions in terms of the relationship with the notochord and the mode of cavitation during secondary neurulation.

Cauda Equina↗
PubMed2004

Palatal shelf movement during palatogenesis: a fate map of the fetal mouse palate cultured in vitro.

Day-13 fetal mouse palates (plug day=day 0) were labeled with carbon particles at various sites of palatal shelves and cultivated in a chemically defined medium for up to 48 h. During the culture period, the bilateral palatal shelves came in contact and fused with each other, which simulated in vivo palatogenesis. The carbon study revealed that at the midpalatal region, the medial edge of the palatal shelf elevated to the horizontal plane, elongated toward the midline, and made contact with the medial edge of the opposing shelf. On the other hand, near the anterior and posterior ends of the shelf, some new tissue was formed at the medial edge of the shelf by remodeling and this newly formed tissue took part in palatal fusion. The results of the present study indicate that during mouse palatogenesis, the anterior and posterior regions of the palatal shelf behave differently from the midpalatal region. It seems that in the fetal mouse palate, the midpalate closes mainly by means of rotation and medial elongation of the shelf, whereas the anterior and posterior parts of the palate close mainly by tissue remodeling of the medial edge and partly by medial elongation of the shelf.

Animals↗
PubMed2004

Phenotypic variability in human embryonic holoprosencephaly in the Kyoto Collection.

BACKGROUND: Holoprosencephaly (HPE) is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Although numerous postnatal cases have been reported, early phases of its pathogenesis are not well understood. We examined over 200 cases of HPE human embryos both grossly and histologically, and studied their phenotypic variability and stage-specific characteristics. METHODS: Among over 44,000 human embryos in the Kyoto Collection of Human Embryos, 221 embryos have been diagnosed as HPE. Their developmental stages ranged from Carnegie stage (CS) 13 to CS 23. They were examined grossly and were also serially sectioned for detailed histological analysis. RESULTS: HPE embryos after CS 18 were classified into complete (true) cyclopia, synophthalmia (partially fused eyes in a single eye fissure), closely apposed separate eyes (possible forerunners of ethmocephaly and cebocephaly), and milder HPE with median cleft lip (premaxillary agenesis). At CS 13-17, when facial morphogenesis is not completed, HPE embryos had some facial characteristics that are specific to these stages and different from those in older HPE embryos. The midline structures of the brain, including the pituitary gland, were lacking or seriously hypoplastic in HPE embryos. Complete cyclopia was found in two cases after CS 18 but none at earlier stages. CONCLUSIONS: The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.

Embryonic Development↗
PubMed2004

A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis.

The vertebrate inner ear has been extensively studied as a model system of morphogenesis and differentiation. The interactions between epithelium and surrounding mesenchyme have not previously been studied directly, because an appropriate experimental system had not been established. Here we describe a mesenchyme-free culture system of E11.5 mouse otic vesicle which retains the ability for (1) formation of the cochlear loop, (2) emigration of ganglion cells from the epithelium and (3) invagination of semicircular canal epithelium. E10.5 otic vesicle was maintained using the same method, but morphogenesis was less successful. Culture of the E11.5 cochlear region alone resulted in regeneration of a structure with semicircular canal character from the cut end, indicating that region-specific cell fate within the otic vesicle is not irreversibly determined at this stage. Co-culturing otic vesicle with cochleovestibular ganglion (CVG) resulted in enhanced looping or ectopic diverticulum formation of the cochlear region, suggesting that the CVG provides a morphogenetic signal for cochlear looping. Cochlear looping was specifically blocked by inhibiting actin polymerization by cytochalasin D, while morphogenesis of the semicircular canal region remained intact. Hyaluronidase treatment inhibited semicircular canal morphogenesis, resulting in a cystic form of the otic vesicle. These data validate this culture system as a tool for elucidating the mechanism of morphogenesis of the otic vesicle.

Actins↗