A morphological classification of retinal ganglion cells in the Japanese catshark Scyliorhinus torazame.

Retinal ganglion cells (GCs) in the Japanese catshark Scyliorhinus torazame were labeled retrogradely with biotinylated dextran amine (BDA3000). First the labeled cells were classified into 5 morphological types (types I-III: small GCs; types IV and V: large GCs) according to the size of the soma and the dendritic arborization pattern as seen in retinal wholemounts. Type I cells were stellate, with dendrites radiating in different directions. Type II cells had bipolar dendritic trees, with 2 primary dendrites extending in opposite directions. Type III cells had a single thick primary dendrite. Type IV cells were stellate, with dendrites covering a large area centered on the cell body. Type V cells were asymmetric, with most dendrites extending opposite to the axon as a large, fan-shaped dendritic field. Subsequently a wholemount was cross-sectioned, and we classified cells further into multiple subtypes according to the level of dendritic arborization within the inner plexiform layer. The present results suggest the existence of many types of GCs in elasmobranchs in addition to the 3 types of large GCs that have been characterized previously. Some of the newly described GC subtypes in the catshark retina appear to be similar to some of those reported in actinopterygians.

Retinal ganglion cell distribution and spatial resolving power in the Japanese catshark Scyliorhinus torazame.

Topographic distribution of retinal ganglion cells (GCs) is linked with the visual capabilities and behavioral ecology of vertebrates. Studies on the distribution of different types of GCs, however, have been conducted in only a few species of elasmobranchs. In the present study, the distribution and peak cell density of GCs, and spatial resolving power (SRP) were examined in the Japanese catshark, Scyliorhinus torazame. Distinct populations of GCs were identified in the ganglion cell layer of S. torazame based on soma size: small and large GCs, which showed different spatial distribution patterns. A horizontal streak of high cell density was recognized in the dorsal retina for small GCs. The highest cell density occurred within the streak, and the peak SRPs of the three fish investigated in the present study were 2.32, 2.64, and 3.01 cycles/deg. In contrast, two spots of high cell density, or areae gigantocellulares, were identified for large GCs, one in the temporal and the other in the nasal retina. The highest cell density occurred in the temporal or nasal area gigantocellularis (SRP: 1.36, 1.55 and 1.83 cycles/deg). This is the first study reporting an elasmobranch species with a horizontal visual streak of small GCs and two areae gigantocellulares. The horizontal streak of small GCs in the dorsal retina, which serves for the inferior visual field, is likely important for food search on the bottom, and the areae gigantocellulares may be important to the detection of prey and/or predators approaching from the front or behind the catshark.

Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades.

The Meg1/Grb10 protein has been implicated as an adapter protein in the signaling pathways from insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in vitro. To elucidate its in vivo function, four independent Meg1/Grb10 transgenic mouse lines were established, and the effects of excess Meg1/Grb10 on both postnatal growth and glucose metabolism were examined. All of the Meg1/Grb10 transgenic mice showed growth retardation after weaning (3-4 weeks), which indicates that ectopic overexpression of Meg1/Grb10 inhibits postnatal growth that is mediated by IGF1 via IGF1R. In addition, the mice became hyperinsulinemic owing to high levels of insulin resistance, which demonstrates that Meg1/Grb10 also modulates the insulin receptor cascade negatively in vivo. Type II diabetes arose frequently in the two transgenic lines, which also showed impaired glucose tolerance. In these mice, severe atrophy of the pancreatic acinus cells was associated with high-level production of Meg1/Grb10 in the pancreas. These results suggest that Meg1/Grb10 inhibits the function of both insulin and IGF1 receptors in these cells, since a similar phenotype has been reported for Ir and Igf1r double knockout mice. Taken together, these results indicate that Meg1/Grb10 interacts with both insulin and IGF1 receptors in vivo, and negatively regulates the IGF growth pathways via these receptors.

The novel dominant mutation Dspd leads to a severe spermiogenesis defect in mice.

Spermiogenesis is a complex process that is regulated by a plethora of genes and interactions between germ and somatic cells. Here we report a novel mutant mouse strain that carries a transgene insertional/translocational mutation and exhibits dominant male sterility. We named the mutation dominant spermiogenesis defect (Dspd). In the testes of Dspd mutant mice, spermatids detached from the seminiferous epithelium at different steps of the differentiation process before the completion of spermiogenesis. Microinsemination using spermatids collected from the mutant testes resulted in the birth of normal offspring. These observations indicate that the major cause of Dspd infertility is (are) a defect(s) in the Sertoli cell-spermatid interaction or communication in the seminiferous tubules. Fluorescent in situ hybridization (FISH) analysis revealed a translocation between chromosomes 7F and 14C at the transgene insertion site. The deletion of a genomic region of chromosome 7F greater than 1 megabase and containing at least six genes (Cttn, Fadd, Fgf3, Fgf4, Fgf15, and Ccnd1) was associated with the translocation. Cttn encodes the actin-binding protein cortactin. Immunohistochemical analysis revealed localization of cortactin beside elongated spermatids in wild-type testes; abnormality of cortactin localization was found in mutant testes. These data suggest an important role of cortactin in Sertoli cell-spermatid interactions and in the Dspd phenotype.

A genetic quality testing system for early stage embryos in the mouse.

We have established a genetic quality testing system for early stage embryos of the mouse. A method of preparation of template DNA for PCR was established using the lysis buffer (1 x PCR reaction buffer supplemented with proteinase K at a concentration of 40 microg/ml) developed by the authors. We demonstrated that two 8-cell embryos of an inbred strain provide sufficient volumes of template DNA for PCR to identify the strain of embryos using four microsatellite markers (D3Mit54, D5Mit18, D6Mit15 and D8Mit50) differentiating 13 inbred strains of mice. This system will be useful in embryo banks that have recently been established worldwide for demonstrating the genetic accuracy of a given strain prior to recovery of live animals.

Chromosomal mapping of the peroneal muscular atrophy (pma) gene in the mouse.

We conducted chromosomal mapping of the pma gene that is a causative gene in the peroneal muscular atrophy mouse, which shows a club foot at birth and unusual gait due to a dropped foot in the adult. Linkage analyses using backcross progeny revealed a significant linkage between the pma gene and three microsatellite markers, D5Mit263 at 73 cM, D5Mit141 and D5Mit97 at 74 cM on Chr 5. The gene order was determined as follows: centromere-D5Mit263-[2.65 cM]-D5Mit141-[2.56 cM]-pma-[5.13 cM]-D5Mit97-telomere.

Direct production of gene-targeted mice from ES cells by nuclear transfer and gene transmission to their progeny.

In order to evaluate the usefulness of a cloning technique to produce gene-manipulated mice for the field of laboratory animal science, we produced mice cloned from gene-targeted embryonic stem (ES) cells and examined the vertical transmission of a targeted gene to their progeny. Of 1257 eggs constructed by nuclear transfer using M-phase ES donor cells targeted with an oviduct-specific glycoprotein (OGP) gene, 990 formed a pseudo-pronucleus and a polar body after activation. Of 504 cloned embryos transferred into recipients, 20 live cloned pups (2%) were recovered by Caesarean section at 19.5 days of gestation. Fourteen of these cloned mice were studied. Genotyping of the OGP locus and 20 microsatellite loci showed that they were genetically identical to the OGP gene-targeted TT2 cells. Eight cloned pups grew into adults, of which 7 were male and 1 was female (missing the Y chromosome). Mating experiments using the cloned mice were carried out. Of 89 F1 mice produced from the mating of cloned and C57BL/6J mice, 50 had the targeted OGP gene heterozygously. Thirty-seven F2 mice from 4 pairs of the OGP-/+ mice were composed of 9 OGP-/-, 18 OGP-/+, and 10 OGP+/+. Moreover, 26 offspring of one pair of the cloned mice were composed of 10 OGP-/-, 12 OGP-/+, and 4 OGP+/+. These offspring were fertile and transmitted the mutant OGP gene to the next generation. Comparison of these results with those of germline chimeric mice indicates that gene-targeted mice can be produced at least one generation earlier by nuclear transfer than by the conventional methods. In addition, the targeted OGP gene was constantly transmitted to the progeny of the gene-targeted mice. Cloning techniques are potentially a more efficient way to generate gene-manipulated mice for laboratory animal science, although such techniques include many unresolved problems, such as low production efficiency, and selection of a cell source for gene manipulation among others.

Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies in colonies.

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.

Transgene stability and features of rasH2 mice as an animal model for short-term carcinogenicity testing.

The transgenic mouse rasH2 line, in which the mouse carries the human c-Ha-ras gene under the control of its own enhancer and promoter, has been proposed as one of the alternative short-term models for carcinogenicity testing. To apply this purpose, we have produced a genetically homogeneous population as C57BL/6JJic-TgN(RASH2) (Tg-rasH2) by continuous backcrossing. In this study, we examined the transgene stability between different generations and the detailed transgene architecture of the integrated human c-Ha-ras gene. Fluorescence in situ hybridization analysis showed that the integrated human c-Ha-ras gene was stably located on chromosome 15E3 in Tg-rasH2 mice at generation number (N) 15 and 20. Southern and Northern blot analysis did not show any differences in the hybridized band pattern in each generation. Southern blot analyses showed that the Tg-rasH2 mouse contained three copies of the human c-Ha-ras gene arrayed in a head-to-tail configuration. We also determined the nucleotide sequence of the transgene in the Tg-rasH2 mouse at N20 and confirmed that the sequence of the coding region was perfectly matched with human c-Ha-ras cDNA. Cloning and sequencing of genome/transgene junctions revealed that integration of the microinjected human c-Ha-ras gene into mouse host genome resulted in a 1820-bp deletion in the rasH2 line. The deleted sequence did not have any sequence homologies with known functional genes. We assumed that either the deletion or the transgene insertion, or both, would not cause insertional mutation. In short-term carcinogenicity testing with a genetically engineered mouse model, confirmation of the transgene or modified gene stability at each generation is one of the important factors that affect the sensitivity to carcinogenic compounds in the same way as the genetic background, age and route of administration.