Relationship between the left-right asymmetric motor-related conformation and the orientation of facial hair whorls in Japanese Kiso horses.

We examined the relationship between inherited motor-related conformation and orientation of facial hair whorls in Japanese Kiso horses. Eleven horses were divided into clockwise, counterclockwise, and radial groups according to facial hair whorls. We placed six markers on anatomical landmarks of each lateral side in a horse and measured the height of the landmarks, the distance between adjacent landmarks, and the angle of the adjacent landmarks. In the counterclockwise group, the horses tended to exhibit higher values on the left side than on the right side, and the comparison of the height of landmarks revealed a significant difference between both sides. Therefore, the orientation of facial hair whorls may suggest the tendency of motor-related conformation, at least in counterclockwise group.

Selective prosaposin expression in Langerhans islets of the mouse pancreas.

The islets of Langerhans are clusters of endocrine cells surrounded by exocrine acinar cells in the pancreas. Prosaposin is a housekeeping protein required for normal lysosomal function, but its expression level is significantly different among tissues. Prosaposin also exists in various body fluids including serum. Intracellularly, prosaposin activates lysosomes and may support autophagy, and extracellularly, prosaposin promotes survival of neurons via G protein-coupled receptors. In this study, prosaposin and its mRNA expression were examined in endocrine cells of the islets as well as in exocrine acinar cells in the pancreas of mice by in situ hybridization and immunostaining. High expression levels of prosaposin were found in Alpha, Beta and Delta cells in the islets, whereas prosaposin mRNA expression was faint or negative and prosaposin immunoreactivity was negative in exocrine acinar cells. The high expression levels of prosaposin in endocrine cells may indicate that prosaposin plays a crucial role in crinophagy, which is a characteristic autophagy in peptide-secreting endocrine cells, and/or that prosaposin is secreted from pancreatic islets. Since prosaposin has been reported in serum, this study suggests a new possible function of the Langerhans islets.

Expression patterns of prosaposin and neurotransmitter-related molecules in the chick paratympanic organ.

The paratympanic organ (PTO) is a small sense organ in the middle ear of birds that contains hair cells similar to those found in vestibuloauditory organs and receives afferent fibers from the geniculate ganglion. To consider the histochemical similarities between the PTO and vestibular hair cells, we examined the expression patterns of representative molecules in vestibular hair cells, including prosaposin, G protein-coupled receptor (GPR) 37 and GPR37L1 as prosaposin receptors, vesicular glutamate transporter (vGluT) 2 and vGluT3, nicotinic acetylcholine receptor subunit α9 (nAChRα9), and glutamic acid decarboxylase (GAD) 65 and GAD67, in the postnatal day 0 chick PTO and geniculate ganglion by in situ hybridization. Prosaposin mRNA was observed in PTO hair cells, supporting cells, and geniculate ganglion cells. vGluT3 mRNA was observed in PTO hair cells, whereas vGluT2 was observed in a small number of ganglion cells. nAChRα9 mRNA was observed in a small number of PTO hair cells. The results suggest that the histochemical character of PTO hair cells is more similar to that of vestibular hair cells than that of auditory hair cells in chicks.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in the mouse olfactory organ.

Prosaposin is a glycoprotein conserved widely in vertebrates, because it is a precursor for saposins that are required for normal lysosomal function and thus for autophagy, and acts as a neurotrophic factor. Most tetrapods possess two kinds of olfactory neuroepithelia, namely, the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). This study examined the expression patterns of prosaposin and its candidate receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in mouse OE and VNE by immunofluorescence and in situ hybridization. Prosaposin immunoreactivity was observed in the olfactory receptor neurons, vomeronasal receptor neurons, Bowman's gland (BG), and Jacobson's gland (JG). Prosaposin expression was mainly observed in mature neurons. Prosaposin mRNA expression was observed not only in these cells but also in the apical region of the VNE. GPR37 and GPR37L1 immunoreactivities were found only in the BG and/or the JG. Prosaposin was suggested to secrete and facilitate the autophagic activities of the neurons and modulate the mucus secretion in mouse olfactory organ.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1 mRNAs, in the chick inner ear.

Prosaposin is a glycoprotein that is widely conserved in vertebrates. It serves as a precursor for saposins A, B, C, and D, which are necessary activators of lysosomal sphingolipid hydrolases. It can also act as a neurotrophic factor. Prosaposin plays a crucial role in the mammalian vestibuloauditory system because it prevents progressive deafness and severe vestibular dysfunction. Prosaposin can exhibit a neurotrophic effect through the G protein-coupled receptor (GPR), and GPR37 and GPR37L1 are its candidate receptors. In this study, we examined the expression patterns of prosaposin, GPR37, and GPR37L1 mRNAs in postnatal day 0 chick vestibuloauditory organs by in situ hybridization. Prosaposin mRNA expression was observed in all vestibular end organs, the vestibular and spiral ganglions, whereas no hybridization signal was observed in the auditory organ, namely basilar papilla. While GPR37L1 mRNA expression was observed in the oligodendrocytes/Schwann cells in the vestibular ganglion, GPR37 mRNA expression was observed in the crista ampullaris base region. These findings suggest that prosaposin expression in the auditory hair cells is acquired uniquely in mammals partly due to the loss of regeneration upon maturation and improved autophagic activity in mammalian auditory hair cells. In addition, as GPR37L1 expression in the chick glial cells differed from GPR37 expression in mammalian glial cells, the roles of GPR37 and GPR37L1 for prosaposin may differ between birds and mammals.

Expression of prosaposin and its G protein-coupled receptor (GPR) 37 in mouse cochlear and vestibular nuclei.

Prosaposin is a precursor of lysosomal hydrolases activator proteins, saposins, and also acts as a secretory protein that is not processed into saposins. Prosaposin elicits neurotrophic function via G protein-coupled receptor (GPR) 37, and prosaposin deficiency causes abnormal vestibuloauditory end-organ development. In this study, immunohistochemistry was used to examine prosaposin and GPR37 expression patterns in the mouse cochlear and vestibular nuclei. Prosaposin immunoreactivity was observed in neurons and glial cells in both nuclei. GPR37 immunoreactivity was observed in only some neurons, and its immunoreactivity in the vestibular nucleus was weaker than that in the cochlear nucleus. This study suggests a possibility that prosaposin deficiency affects not only the end-organs but also the first center of the vestibuloauditory system.

Morphological variations in the transverse foramen of the axis in Japanese serows (Capricornis crispus).

The presence of transverse foramina in the axes of Japanese serows, a special national natural treasure in Japan, has been reported to be unstable, but other variations are unknown. In this study, we analysed the shape, cross-sectional area, length, and volume of the transverse foramen in the axes of 19 specimens using gross anatomy and computed tomography (CT) scan. There were four types in the transverse foramen: type 1, having the transverse foramina; type 2, having two cranial openings; type 3, sifting a caudal opening to the ventral side of the transverse process; and type 4, having no transverse foramina. Although the transverse foramina showed different types on the left and right sides in several specimens, there were no statistically significant differences in the length and volume. This variation may be related to running patterns of the vertebral artery penetrating the transverse foramina. Two goats without the transverse foramina were examined to infer a running pattern of the vertebral artery instead of Japanese serows. The vertebral artery in the goats branched in two directions (spinal and muscle), between the axis and the third cervical vertebra. This passage of the goat vertebral artery might be presumed in type 4 of Japanese serows. This study reveals the instability of the transverse foramina in the axes of Japanese serows and provides new data to compare the axes of other ruminants.

Morphometric study of the vestibuloauditory organ of the African clawed frog, Xenopus laevis.

Independent auditory end-organs appear first in amphibians in vertebrate phylogeny. In amphibians, sound detection is carried out by the amphibian papilla, basilar papilla and macula saccule. Amphibians inhabit distinct habitats and exhibit specific behaviours and sound frequency responses, so the amphibian vestibuloauditory system is an excellent model for considering the relationships between behaviour and physiological/anatomical vestibuloauditory properties. The African clawed frog, Xenopus laevis, lives in shallow water throughout its life and is thought to use sound in a higher frequency range compared with terrestrial anurans. In this study, the size of each vestibuloauditory end-organ and the distribution of ganglion cells in the vestibuloauditory ganglion were examined using haematoxylin and eosin staining and lectin histochemistry in Xenopus laevis. This study revealed that the size ratios among end-organs in Xenopus are similar to those in terrestrial anurans. Large and small cells were observed in the ganglion, but their distribution patterns are different from those in general terrestrial anurans. Lycopersicon esculentum lectin stained a large number of ganglion cells. Lectin-stained cells were found throughout the whole ganglion, but were especially abundant in the caudal part. These results suggested a unique distribution pattern of the vestibuloauditory ganglion cells in Xenopus.

Morphological features of the mouse duodenocolic fold in foetus and adult.

For the mechanism of duodenojejunal flexure (DJF) morphogenesis in mice, we consider the gut tube itself and the gut mesentery as important players. In this study, we focussed on the morphological features of the gut mesentery around the mouse duodenum, especially the duodenocolic fold at embryonic day (E) 18.5 and the adult phase. The duodenocolic fold, a sheet of the mesentery, was located between the entire ascending duodenum and the descending colon. At E18.5, in the cranial area near the DJF, the duodenocolic fold joined both the mesocolon and the mesojejunal part of the root of the mesentery. In the middle and caudal areas, the duodenocolic fold joined the mesocolon. Interestingly, along with the ascending duodenum, the duodenocolic fold contained a smooth muscle bundle. The smooth muscle bundle continued from the outer muscular layer of the middle to the caudal part of the ascending duodenum. The three-dimensional imaging of the foetal duodenocolic fold revealed that the smooth muscle bundle had short and long apexes towards the proximal and distal parts of the root of the mesentery, respectively. At the adult phase, the duodenocolic fold had a much thinner connective tissue with a larger surface area in comparison with the duodenocolic fold at E18.5. The adult duodenocolic fold also contained the smooth muscle bundle which was similar to the foetal duodenocolic fold. A part of the duodenocolic fold connecting to the mesojejunal part of the root of the mesentery seemed to be homologous to the superior duodenal fold in humans, known as the duodenojejunal fold; by contrast, most of the duodenocolic fold seemed to be homologous to the inferior duodenal fold in humans, known as the duodenomesocolic fold. The smooth muscle bundle in the mouse duodenocolic fold seemed to play a role in keeping the ascending duodenum in the abdominal cavity because the duodenum in animals did not belong to a retroperitoneal organ in contrast to humans owing to the difference in the direction of gravity on the abdominal organs between mice and humans. Moreover, the smooth muscle bundle shared common and uncommon points in its location and nerve supply to the suspensory muscle of the duodenum in humans, known as the ligament of Treitz. This study had insufficient evidence that the smooth muscle bundle of the mouse duodenocolic fold was homologous to the suspensory muscle of the duodenum in humans. In conclusion, this study revealed the detailed structure of the mouse duodenocolic fold, including the relationship between the fold and other mesenteries. Particularly, the smooth muscle bundle is a specific feature of the mouse duodenocolic fold and might play several roles in DJF morphogenesis, especially the ascending duodenum and the caudal duodenal flexure during development.

Expression of the G protein-coupled receptor (GPR) 37 and GPR37L1 in the mouse digestive system.

G protein-coupled receptor (GPR) 37 and GPR37L1 are known to modulate the dopaminergic neuron activity, and recently, they are identified as candidate prosaposin receptors. Intercellular prosaposin is proteolytically processed into four saposins, each of which acts as a sphingolipid hydrolase activator in the lysosome. In contrast, extracellular prosaposin exerts a trophic effect on neurons via GPR37 and GPR37L1. In this study, the expression patterns of GPR37 and GPR37L1 in the mouse digestive system were examined immunohistochemically. The islets of Langerhans of the pancreas showed intense immunoreactivity for GPR37 and GPR37L1. Weak immunoreactivity for GPR37 and GPR37L1 was found in the nerve plexuses of the esophagus and small and large intestines. Colocalization of GPR37 and tyrosine hydroxylase immunoreactivity was observed in the neuron of the nerve plexus of the large intestine. This study suggests the possibility that prosaposin affects the function of islet-secreting cells. Also, the expression of GPR37 and GPR37L1 in the nerve plexus suggests that prosaposin exerts a trophic effect not only in the central nervous system, but also in the enteric nervous system.

ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats.

The aim of the present study was to clarify roles of ATP-dependent potassium channels (KATP channels) in motility of the striated muscle portion in the esophagus. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response of striated muscle in the esophageal segment. Application of glibenclamide, an antagonist of KATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of KATP channels, decreased amplitude of twitch contractions. RT-PCR revealed the expression of subunits of KATP channels in esophageal tissue. In addition, immunopositivity for subunits of KATP channels was observed in the striated muscle cells of the esophageal muscle layer. These findings indicate that KATP channels contribute to motor regulation of striated muscle in the rat esophagus.

Spatiotemporal distribution of extracellular matrix changes during mouse duodenojejunal flexure formation.

Although gut flexures characterize gut morphology, the mechanisms underlying flexure formation remain obscure. Previously, we analyzed the mouse duodenojejunal flexure (DJF) as a model for its formation and reported asymmetric morphologies between the inner and outer bending sides of the fetal mouse DJF, implying their contribution to DJF formation. We now present the extracellular matrix (ECM) as an important factor for gut morphogenesis. We investigate ECM distribution during mouse DJF formation by histological techniques. In the intercellular space of the gut wall, high Alcian-Blue positivity for proteoglycans shifted from the outer to the inner side of the gut wall during DJF formation. Immunopositivity for fibronectin, collagen I, or pan-tenascin was higher at the inner than at the outer side. Collagen IV and laminins localized to the epithelial basement membrane. Beneath the mesothelium at the pre-formation stage, collagen IV and laminin immunopositivity showed inverse results, corresponding to the different cellular characteristics at this site. At the post-formation stage, however, laminin positivity beneath the mesothelium was the reverse of that observed during the pre-formation stage. High immunopositivity for collagen IV and laminins at the inner gut wall mesenchyme of the post-formation DJF implied a different blood vessel distribution. We conclude that ECM distribution changes spatiotemporally during mouse DJF formation, indicating ECM association with the establishment of asymmetric morphologies during this process.

Asymmetric morphology of the cells comprising the inner and outer bending sides of the murine duodenojejunal flexure.

The asymmetric shape of component cells determines the asymmetric features of developing organs. Here, we focused on the murine duodenojejunal flexure (DJF), which bends without affecting the mesentery, and analyzed the morphological asymmetries of the mucosal epithelium and gut wall cells between the inner and outer bending sides at embryonic days 10.75-11.75. In the mucosal epithelium, the cell shape and the expression of epithelial markers (Cdx2, E-cadherin) showed no differences between the two DJF sides. In contrast, the gut wall cells comprising the inner and outer sides of the DJF were elongated along the inner-outer axis and perpendicular to this axis, respectively. Furthermore, the gut wall cells in the outer side possessed cytoplasmic processes connecting cells via adherens junctions, but those in the inner side were attached via adherens junctions of juxtaposed cell bodies and were relatively more crowded. In immunohistochemistry experiments, there was no remarkable difference in the positive reactions of markers for mesenchyme (vimentin), smooth muscle cells (αSMA), endothelial cells (LYVE-1, CD34), and undifferentiated neurons (Sox10) between the DJF sides. Interestingly, Tuj1-positive cells, indicating differentiated neurons, were observed in the middle layer of the gut wall, and these cells were significantly more abundant and tended to be larger in the inner side than in the outer side of the DJF. In conclusion, we clarified the asymmetries of gut wall cell morphology and neural differentiation between the inner and outer sides of the DJF. These characteristics of the developing murine DJF indicate its asymmetric formation.

Analysis of duodenojejunal flexure formation in mice: implications for understanding the genetic basis for gastrointestinal morphology in mammals.

The mammalian gut undergoes morphological changes during development. We studied the developing mouse duodenojejunal flexure (DJF) to elucidate the mechanism of formation. During embryonic days 10.75-13.75, DJF formation was morphologically classified into three stages: the expansion stage, flexure formation stage, and flexure elongation stage. From the expansion to the flexure formation stages, the DJF wall showed asymmetric morphology and proliferation along the left-right intestinal axis. From the flexure formation to the flexure elongation stage, the DJF started to bend dorsally with counterclockwise rotation along the antero-caudal intestinal axis, indicating that the original right side of the duodenum was rotated towards the dorsal body wall during development of the DJF. The direction of attachment of the dorsal mesentery to the DJF did not correspond to the bending direction of the DJF during flexure formation, and this finding indicated that the dorsal mesentery contributed very little to DJF formation. During DJF formation, Aldh1a2 and hedgehog mRNAs were detected at the DJF, and their expression levels differed along the bending axis. In conclusion, DJF formation might be triggered by asymmetric morphology and proliferation along the left-right intestinal axis under the control of retinoic acid and hedgehog signaling.