Ryuju Hashimoto

Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness

Signaling through the Ror2 receptor tyrosine kinase promotes invadopodia formation for tumor invasion. Here, we identify intraflagellar transport 20 (IFT20) as a new target of this signaling in tumors that lack primary cilia, and find that IFT20 mediates the ability of Ror2 signaling to induce the invasiveness of these tumors. We also find that IFT20 regulates the nucleation of Golgi-derived microtubules by affecting the GM130-AKAP450 complex, which promotes Golgi ribbon formation in achieving polarized secretion for cell migration and invasion. Furthermore, IFT20 promotes the efficiency of transport through the Golgi complex. These findings shed new insights into how Ror2 signaling promotes tumor invasiveness, and also advance the understanding of how Golgi structure and transport can be regulated.

Rac1 is required for the formation of three germ layers during gastrulation

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.

Spatio‐temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system

Drosophila neurospecific receptor tyrosine kinases (RTKs), Dror and Dnrk, as well as Ror1 and Ror2 RTKs, isolated from human neuroblastoma, have been identified as a structurally related novel family of RTKs (Ror‐family RTKs). Thus far, little is known about the expression and function of mammalian Ror‐family RTKs.

Sulfated glycosaminoglycans are necessary for Nodal signal transmission from the node to the left lateral plate in the mouse embryo

Situs-specific organogenesis in the mouse results from leftward fluid flow in the node cavity and subsequent left-sided expression of Nodal in the lateral plate mesoderm (LPM). Nodal expression at the node is essential for the subsequent asymmetric Nodal expression in the left LPM, but the precise role of Nodal produced at the node has remained unknown. We have now investigated how the Nodal signal is transferred from the node to the LPM. Externally supplied Nodal protein failed to signal to the LPM, suggesting that the Nodal signal is transferred to the LPM via an internal route rather than an external one. Transgenic rescue experiments showed that the Nodal co-receptor Cryptic (Cfc1) is required only in the LPM, not at the node, for asymmetric Nodal expression in the LPM, indicating that the Nodal signal is not relayed indirectly between the node and LPM. Nodal interacts in vitro with sulfated glycosaminoglycans (GAGs), which are specifically localized to the basement membrane-like structure between the node and LPM in the mouse embryo. Inhibition of sulfated GAG biosynthesis prevents Nodal expression in the LPM. These data suggest that Nodal produced at the node might travel directly to the LPM via interaction with sulfated GAGs.

Roles of leptin in prenatal and perinatal brain development

ABSTRACT  Leptin is a hormone that reduces food intake and increases energy expenditure by acting on the arcuate nucleus in the hypothalamus. Recent studies indicated that the neuronal circuit related to food intake in the hypothalamus is formed in the neonatal period and that leptin is necessary for the formation of this circuit. Our studies have further suggested that leptin may act on the fetal cerebral cortex, including the cingulate cortex, which is involved in motor and cognitive processes, and that leptin may affect maintenance and differentiation of neural stem cells, glial‐restricted progenitor cells and/or neuronal lineage cells. These recent studies showed that leptin not only has homeostatic functions in adults, but also regulates brain development in the prenatal and neonatal periods. These findings suggest that leptin is related to formation of the normal brain structure and regenerative potency of neural cells as well as the predisposition to homeostatic dysfunction, low locomotor activity or impairment of cognitive function.

Ror2 is Required for Midgut Elongation During Mouse Development

The receptor tyrosine kinase Ror2 acts as a receptor for Wnt5a to mediate noncanonical Wnt signaling, and it plays essential roles in morphogenesis. Ror2−/− embryos exhibit phenotypes similar to, albeit generally milder than, those of Wnt5a−/− embryos. During mouse embryogenesis, Ror2 is expressed in various organs and regions, although little is known about its expression pattern and roles in the developing gut, while Wnt5a is expressed in the developing gut, where its absence causes abnormal phenotypes. Here, we demonstrated that Ror2 was strongly and differentially expressed in the rostral and middle midgut endoderm from embryonic day (E) 10.5 through embryonic day (E) 12.5. At E11.5, Ror2−/− embryos exhibited a shorter middle midgut with a larger diameter and more accumulation of epithelial cells in the middle midgut than control embryos, while the total cell numbers remained unaltered. These findings suggest that Ror2 plays important roles in midgut elongation by means of an epithelial convergent extension mechanism. Developmental Dynamics 239:941–953, 2010.

Role of Wnt5a-Ror2 signaling in morphogenesis of the metanephric mesenchyme during ureteric budding

ABSTRACT Development of the metanephric kidney begins with the induction of a single ureteric bud (UB) on the caudal Wolffian duct (WD) in response to GDNF (glial cell line-derived neurotrophic factor) produced by the adjacent metanephric mesenchyme (MM). Mutual interaction between the UB and MM maintains expression of GDNF in the MM, thereby supporting further outgrowth and branching morphogenesis of the UB, while the MM also grows and aggregates around the branched tips of the UB. Ror2, a member of the Ror family of receptor tyrosine kinases, has been shown to act as a receptor for Wnt5a to mediate noncanonical Wnt signaling. We show that Ror2 is predominantly expressed in the MM during UB induction and that Ror2- and Wnt5a-deficient mice exhibit duplicated ureters and kidneys due to ectopic UB induction. During initial UB formation, these mutant embryos show dysregulated positioning of the MM, resulting in spatiotemporally aberrant interaction between the MM and WD, which provides the WD with inappropriate GDNF signaling. Furthermore, the numbers of proliferating cells in the mutant MM are markedly reduced compared to the wild-type MM. These results indicate an important role of Wnt5a-Ror2 signaling in morphogenesis of the MM to ensure proper epithelial tubular formation of the UB required for kidney development.

The role of leptin in the development of the cortical neuron in mouse embryos

Leptin is an obese gene product that decreases appetite and raises energy expenditure in adults. We previously reported that leptin was detected in the sera of mouse embryos and leptin receptors were expressed in the mouse embryonic cerebrum, suggesting that leptin plays a role in cerebral development. In this study, we injected leptin into the lateral ventricle of the cerebrum in leptin-deficient ob/ob mouse embryos to investigate the function of leptin in cerebral development. When leptin was injected on embryonic day (E) 14, the ratio of the number of cells in the cortical plate (CP) to that in the intermediate zone (IZ) was higher in leptin-injected than in vehicle-injected ob/ob embryos on E16, although there was no significant difference in the number of cells in the ventricular zone (VZ), IZ, or CP between these groups. The number of postmitotic BrdU-positive CP cells was larger in leptin-injected than in vehicle-injected ob/ob embryos on E16 and E17 when BrdU labeling and leptin injection were performed on E14. By in situ hybridization, NPY mRNA expression in CP neurons on E18 was weaker in leptin-injected than in vehicle-injected ob/ob embryos when leptin was injected on E16. These results suggest that leptin promotes the migration of neuronal lineage cells to CP and that the leptin-NPY axis in neurons works in the cerebral cortex on E16.

Spatial and temporal patterns of expression of melanocortin type 2 and 5 receptors in the fetal mouse tissues and organs

In the present study to analyze the role of ACTH in fetal tissues and organs, we observed the expression of melanocortin type 2 (MC2) and 5 (MC5) receptors in ICR mouse embryos from E11.5 to E18.5 by immunohistochemistry. In the adrenal gland and testis, both receptors were expressed from E13.5 to E18.5. In the genital ridge and the ovary, melanocortin type 2 receptors (MC2R) was detected from E11.5 to E12.5 and from E13.5 to E18.5, respectively, while melanocortin type 5 receptors (MC5R) was not detected. In the mesonephros, MC2R and MC5R were expressed from E11.5 to E12.5, and in the metanephros, MC2R and MC5R were expressed from E12.5 to E18.5 and from E14.5 to E18.5, respectively. In the lung, MC2R was expressed from E11.5 to E14.5, but MC5R was not expressed at all. In blood cells, MC5R was detected at all stages examined, while MC2R was detected at none. MC2R was observed in the brain and spinal cord from E11.5 to E13.5, while MC5R was detected only in the telencephalon and only from E16.5 to E18.5. At different temporal patterns, MC2R, but not MC5R, was detected in the choroid plexus, while MC5R, but not MC2R, was expressed in the liver and in the nasal epithelium, and both MC2R and MC5R were expressed in the dorsal root ganglion and the trigeminal ganglion. These findings show the spatio-temporal specific expression patterns of MC2R and MC5R in the mouse embryo and suggest that ACTH may be related to histogenesis and/or prenatal function of various tissues and organs via MC2R and/or MC5R.

Development of the Innervation Pattern in the Upper Limb of Staged Human Embryos

The upper limb nerves of 8 human embryos (Carnegie stages 13-21) were studied by reconstruction. In stage 13, upper limb nerves (C5-T1) extended from the spinal cord. In stage 14, these nerves united to form the nascent brachial plexus. In stages 16 and 17, the median nerve, the radial nerve and the ulnar nerve entered into the hand plate. In stages 20 and 21, the upper limb nerves were observed in an orientation and arrangement similar to those in the adult.