|Budget Amount *help
¥7,100,000 (Direct Cost : ¥7,100,000)
Fiscal Year 1996 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1994 : ¥3,000,000 (Direct Cost : ¥3,000,000)
1 Analysis of Uchida Rat (rSay)
Pax-6 gene is a member of the Pax family of vertebrate developmental genes that hae the conserved "paired box motif", which is originally defined in Drosophila segmentation genes. Mutations that reduce Pax-6 dosage cause dominantly inherited eye malformations in the mouse, rat, and human. Uchida rat (rSey) is a mutant rat spontaneously found in the colony of Sprague-Dowley rats, and has a mutation in an exon of Pax-6 gene, resulting in generation of truncated transceipts.
(1) We found a new type of Uchida rat (rSay^2) that has a mutation in an exon which is down stream of paired box.
(2) From Dil labeling study, neural crest cells originated from the anterior midbrain, which migrate toward the frontonasal mass via the eye rudiment in normal embryos, do not reach the frontonasal region but stay at the area dorsal the optic vesicle in rSey/rSey embryos. Pax-6 gene is not expressed in the crest cells and orthotopic injection of normal midbrain crest cells into
homozygous host embryos have confirmed that migration defect in rSey homozygotes is caused by abnormal environment to which midbrain crest cells encounter.
(3) We revealed, from recombination experiments using the presumptive lens ectoderm and optic cup obtained from homozygous and/or wild type embryos, taht the filure of lens differentiation in the mutant is due to the ectoderm ; the ectoderm taken from the homozygous mutant cannot respond to the signal from the optic cup, while the homozygous optic cup can induce lens in the wild type ectoderm.
(4) In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pir of cartilaginous rods in place of the nasal capsule. This suggest that the primary cranial defect is restricted to the lateral nasal prominence (LNP) derivatives.
(5) Pax-6 gene expression patterns in the central nervous systems are spatially and temporally complex. In accordance, various structural devects were observed in the brain of the Uchida rat. These included lack of the olfactory bulb, lateral olfactory tract, and commissura rostralis, 'as well as deformity of the cerebral and cerebellar cortex, and of the thalamus.
(6) We found impaired development of the peripheral nervous system in the Uchida rat. First, the homozygous ophthalmic nerve (N.V1) shows abnormal feature of innervation ; it is not well fasciculated and strays into ectopic regions. It is suggested that this abnormality is indirectly caused by the failure in formation of LNP,the structure being innervated by N.V1 in normal development. The other branches of the trigeminal ganglion did not seem to be affected in the homozygote. More interestingly, two cranial motor nerves, N.abducens (N.VI) and N.hypogossal (N.XII) nerves, whose nuclei lie at the postotic levels of the hidbrain, are absent in homozygous embryos, though spinal nerves (C1 and C2) appear to innervate the tongue muscles. Histological observation of homozygotes shows that Islet-1+/MAP1+cells are observed in the otic hindbrain but that they do not sprout axons out of the neural tube. Thus, Pax-6 is most likely to be involved in development of some motor neurons.
2 alysis of HIV Transgenic Mouse.
In order to elucidate the genetic effects on the growth and development of craniofacial regios, HIV I transgneic mice (TG mice) which exhibited the phenotype of anterior crossbites were investigated by various methods. Northern hybridization showed that the introduced gene was expressed from 14.5 days post coitum to adult. In morphological measurements using digital converter "GRADICON,significant differences betwee TG mice and/wild type mice (WT mice) were found at 5 weeks of age (5W) for most measurement items. Especially at 7W,the results indicated remarkable reductions in maxilla and catch up growth in the mandlble. On the other hand, the serrum alkaline phosphatase activity of TG mice (3W,5W,and 7W) was significantly higher than that of WT mice, and also the low level of inorganic phosphate was detected at 3W for TG mice. These results suggested that TG mice had a disorder of bone metabolism. Other TG mouse lines carrying the same DNA construct showed normal figures in the craniofacial regions, suggesting the possibility that the phenotype of anterior crossbites and the disorder of bone metabolism were not caused by functions of HIV-1 genome but by insertional mutation in craniofacial development. Less