Yasuhide Furuta

Driven by the same Ig enhancer and SV40 T promoter ras induced lung adenomatous tumors, myc induced pre-B cell lymphomas and SV40 large T gene a variety of tumors in transgenic mice.

Different types of tumors developed in transgenic mice following the introduction of the entire coding region of ras, myc or SV40 large T gene (T) linked to the same regulatory unit, consisting of a human immunoglobulin gene enhancer (Ig) and SV40 early gene promoter (Tp) with a 21‐bp repeat. All the 12 transgenic mice harboring the intact T gene developed a variety of tumors including choroid plexus tumor, B cell lymphoma, histiocytic lymphoma, thymoma and others. This suggests that the Ig/Tp regulatory unit has transcriptional activity in these heterologous tissues. With this regulatory unit, myc gene induced solely pre‐B cell lymphomas (five out of nine mice). Contrary to our expectation, however, the mutated ras gene induced lung adenomatous tumors in six out of eight transgenic mice over the 10‐month observation period; the tumors are histologically comparable to adenocarcinomas in man. The tumors developed as early as 4 weeks after birth and the introduced ras gene was as efficiently expressed in both normal and neoplastic bronchioloalveolar epithelial cells as in normal lymphoid cells. An unidentified secondary event thus appears to be necessary for these ras‐expressing cells to become neoplastic, as observed for myc (Leder et al., 1986). In a variety of tumors induced by Ig/Tp‐T, on the other hand, T gene was expressed only in the tumor cells, but not in normal cells. Thus, derepression of T gene in normal cells appears to be closely related to their malignant change as observed in development of pancreatic acinar cell tumors by the T gene (Ornitz et al., 1985). These results suggest that ras and myc oncogenes penetrate differentially specific types of cells, while the SV40 T gene is tumorigenic in a variety of cell types.

Induction of a variety of tumors by c-erbB2 and clonal nature of lymphomas even with the mutated gene (Val659----Glu659).

The c‐erbB2 gene is expressed uniquely in fetal epithelium in vivo and has been suggested to contribute to the development and/or progression of adenocarcinomas in man. In order to assess the oncogenicity of the c‐erbB2 gene in vivo, normal c‐erbB2 and mutant c‐erbB2 encoding glutamic acid instead of valine at position 659 within the transmembrane domain were introduced into mice under the transcriptional regulatory unit of mouse mammary tumor virus long terminal repeat (MMTV‐LTR) or immunoglobulin enhancer‐‐SV40 early gene promoter (Ig/Tp). In transgenic mice with normal c‐erbB2 under MMTV‐LTR, not only adenocarcinomas but also a variety of tumors including B lymphomas were induced at relatively late onset. Induction of pre‐B cell lymphomas with normal c‐erbB2 was also observed using the Ig/Tp regulatory unit within 6‐10 months in some members of one transgenic family among seven lines established. In contrast, with the mutant c‐erbB2 under the Ig/Tp regulatory unit, the lymphoma was induced neonatally in all members of four transgenic families among ten lines obtained. However, the immunoglobulin heavy chain gene rearrangement pattern indicated that even with the mutant c‐erbB2 the induced lymphomas were clonal.

A new strategy of gene trapping in ES cells using 3'RACE

Abstract“Gene trapping” in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses theneor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of theneor gene as fusion transcripts with the 3′ end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3′ rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

Env-derived gp55 gene of Friend spleen focus-forming virus specifically induces neoplastic proliferation of erythroid progenitor cells.

A group of retroviruses carrying truncated viral genes has recently been suggested as the cause of new patterns of diseases. One such virus is the replication defective component of the Friend murine leukemia virus (F‐MuLV) complex, called Friend spleen focus forming virus (F‐SFFV). This virus induces erythroblastosis, and a virion envelope‐related glycoprotein, gp55, encoded by F‐SFFV has been suggested as the pathogenic gene. The role of the gp55 gene is, however, yet unclear in the apparently multistep erythroleukemogenesis. By separately producing transgenic mice harboring the whole F‐SFFV DNA, the gp55 gene alone under the control of the retroviral long terminal repeat (LTR) and the gp55 gene under the control of cytoplasmic beta actin transcriptional regulatory unit, we show here that the gp55 gene is capable of inducing neoplastic proliferation of erythroid progenitor cells specifically in the absence of helper virus and other F‐SFFV sequences. Under the control of the viral LTR the gp55 expression was detected only in leukemic tissues, but under the control of cytoplasmic beta‐actin regulatory sequences, the gp55 was also expressed in a variety of normal tissues including preleukemic normal spleens. The development of erythroleukemia was suppressed under the genetic background of C57B1/6 mouse (resistant to F‐MuLV; Fv‐2rr), and required additional events even under the background of DDD mouse (susceptible to F‐MuLV; Fv‐2ss). The p53 and Spi‐1 genes were frequently aberrant in transplanted tumors and cell lines derived from them, but were not in primary leukemic spleens.

Hepatocarcinogenesis in Transgenic Mice Carrying Albumin‐promoted SV40 T Antigen Gene

We have developed transgenic mice that inherit albumin promoter‐regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi‐regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.

Degeneration of skeletal and cardiac muscles in c-myb transgenic mice.

In order to reveal cellular processes sensitive to abnormal c-myb expressionin vivo, transgenic mice were produced by introducing the c-myb nuclear proto-oncogene under the ubiquitous transcriptional regulatory unit of the cytoplasmic β-actin gene. Expression of c-myb in thymus did not cause apparent abnormality, but the mice unexpectedly developed degenerative abnormalities in skeletal and cardiac muscles; this occurred predominantly in males. Expression of c-myb in skeletal muscle was correlated with an inflammation of muscle and was accompanied by vacuolar degeneration of muscle fibres, their regeneration, and lymphocyte infiltration. The identical pathological progression in cardiac muscle was associated with cardiomegaly.

Ovarian Teratomas in Mice Lacking the Protooncogene c‐mos

Parthenogenesis has been suggested to be tightly coupled with development of ovarian teratomas. Indeed, ovarian tumors developed in c‐mos‐delieicnt female mice, which are characterized by the parthenogenetic activation of oocytes. The tumors appeared at a frequency of 30% between 4 and 8 months of age, and did not develop in younger or older mice. Most of the tumors were benign and consisted of multi‐focal cysts most notably with mature ectodermal components, but also with mesodermal and endodermal components. One among 17 tumors observed consisted of extraembryonic tissues alone, and two bore malignant components with metastasis to peritoneal organs. The results strongly suggest the involvement of c‐mos mutations in human germ cell tumors.

Apical constriction in distal visceral endoderm cells initiates global, collective cell rearrangement in embryonic visceral endoderm to form anterior visceral endoderm

The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.

ROSA26 reporter mouse lines and image analyses reveal the distinct region-specific cell behaviors in the visceral endoderm

ABSTRACT The early post-implantation mouse embryo changes dramatically in both size and shape. These morphological changes are based on characteristic cellular behaviors, including cell growth and allocation. To perform clonal analysis, we established a Cre/loxP-based reporter mouse line, R26R-ManGeKyou, that enables clonal labeling with multiple colors. We also developed a novel ImageJ plugin, LP-Clonal, for quantitative measurement of the tilt angle of clonal cluster shape, enabling identification of the direction of cluster expansion. We carried out long-term and short-term lineage tracking. We also performed time-lapse imaging to characterize cellular behaviors using R26-PHA7-EGFP and R26R-EGFP. These images were subjected to quantitative image analyses. We found that the proximal visceral endoderm overlying the extra-embryonic ectoderm shows coherent cell growth in a proximal-anterior to distal-posterior direction. We also observed that directional cell migration is coupled with cell elongation in the anterior region. Our observations suggest that the behaviors of visceral endoderm cells vary between regions during peri-implantation stages. Summary: Cell lineage analysis and live imaging reveals distinct region-specific cell behaviors in the visceral endoderm of mouse embryos during peri-implantation stages.