(1) Molecular mechanisms of murine development
1) Lgr4 in development of eipididymis
The roles of the leucine-rich repeat domain containing GPCR 4 (
Lgr4 ), which is one of the orphan GPCRs, were analyzed using the
Lgr4 hypomorphic mutant mouse line (
Lgr4 Gt ). This homozygous mutant had only one-tenth of the normal transcription level; furthermore, 60% of them survived to adulthood. The homozygous male was infertile showing morphological abnormalities in both the testes and the epididymides (Fig. 2). In the testes, luminal swelling, loss of germinal epithelium in the seminiferous tubules, and rete testis dilation were observed. Cauda epididymidis sperm were immotile. Rete testis dilation was due to a water reabsorption failure caused by a decreased expression of ESR1 and SLC9A3 in the efferent ducts. Although we found differential regulation of ESR1 expression in the efferent ducts and the epididymis, the role of ESR1 in the epididymis remains unclear. The epididymis contained short and dilated tubules and completely lacked its initial segment. In the caput region, we observed multi-lamination and distortion of the basement membranes (BM) with an accumulation of laminin. Rupture of swollen epididymal ducts was observed, leading to an invasion of macrophages into the lumen. Male infertility was probably due to the combination of a developmental defect of the epididymis and the rupture of the epithelium resulting in the immotile spermatozoa. These results indicate that
Lgr4 has pivotal roles to play in the regulation of ESR1 expression, the control of duct elongation through BM remodeling, and the regional differentiation of the caput epididymidis.
2) Skt and intervertebral disc formation
We established the mutant mouse line, B6;CB-
Skt GtAyu8021IMEG (
Skt Gt ), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called
Sickle tail (
Skt ), is composed of 19 exons and encodes a protein of 1352 amino acids with a proline-rich region and a coiled-coil domain. Expression of a reporter gene (
b -geo ) was detected in the mesonephros and notochord during embryogenesis (Fig. 3), and in its derivative, the nucleus pulposus, of adult mice. Compression of some of the muclei pulposi in the center of intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and the annulus fibrosus of the IVDs in
Skt Gt/Gt adult mice . These phenotype and histological pictures were different from those in
Danforth's short tail ( Sd ) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to the annulus fibrosus in all IVDs . The
Skt gene maps to the proximal part of mouse chromosome 2, near the
Sd locus. The genetic distance between them was 0.95 cM. Adult Skt mutant mice showed kinky tail in the caudal region, but Sd mutant showed truncated caudal vertebrate. These suggest that Skt and Sd genes are not identical. The number of vertebrae in both [
Sd +/+
Skt Gt ] and [
Sd Skt Gt /+ +] compound heterozygotes was less than that of
Sd heterozygotes . Furthermore, the enhancer trap locus
Etl4 lacZ , which was previously reported to be an allele of
Sd, was located in the third intron of the
Skt gene. We are now trying to find the Sd gene.
3) C ranio-facial development
Little is known about the molecular mechanism which regulates how the whole cranium is shaped. Mouse models currently available for genetic research include several hundreds of unique inbred strains and genetically engineered mutants.
By cross comparing their genomic structures, we can elucidate the cause of any differences in the phenotype between two strains. The craniometry of subspecies, or closely related species, of mice provide a good systemic model to study the relationship between genetic variance and cranial shapeevolution. The lack of a quantified framework for comparing and analyzing mouse cranial shape has been a problem. For this reason, we performed quantitative analysis of cranial shape morphology among several mouse strains. We carried out a craniometric assay of seven mouse strains: four inbred strains (C57BL/6J, BALB/cA, C3H/HeJ, and CBA/JNCr) from
Mus musculus domesticus (
M. m. domesticus ); one closed colony strain (ICR) from
M. m. domesticus ; one inbred strain (MSM/Ms) from
Mus musculus molossinus ; and,
Mus spretus as a strain from a species other than
M. m. domesticus (Fig. 4) . We performed linear measurements and geometric morphometrics. Geometric morphometrics revealed that the cranial characteristics of each strains were clearly distinguishable, and that
M. spretus has a slender cranium when compared with our other strains. The mean cranial shape of C3H or CBA was more similar to MSM/Ms, which is derived from
M. m. molossinus , than to either C57BL/6J, BALB, or ICR which are derived from
M. m. domesticus . Future work in this field will aid in elucidating the mechanism of whole cranial shape regulation.
(2) Molecular pathogenesis for human genetic diseases.
1) Transthyretin gene and Familial amyloidotic polyneuropathy (FAP)
We established mouse models for human dominantly inherited disease, FAP. Using this mouse model, we are analysing the gene-environmental interactions in this disease development. So far, we showed that a mouse model did not develop amyloidosis under the specific pathogen free (SPF) conditions (Fig. 5), and that the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder. In addition, we established the new mouse model by replacing the mouse transthyretin gene with human transthyretin gene to analyze the role of each mutation in disease process. In any case, environmental factors can be divided into intrinsic and extrinsic factors, and these can affect the development of FAP in terms of onset, tissue distribution of amyloid deposition, and prognosis.
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2) Spink3 and Pancreatitis
Serine protease inhibitor Kazal Type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the
SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of
SPINK1 mutations remains controversial. To elucidate the role of SPINK1 , we generated
Spink3 -deficient (
Spink3 -/- ) mice by gene targeting in mouse embryonic stem cells. Surprisingly, in
Spink3 -/- mice, extensive autophagic degeneration of acinar cells occurred, resulting in death by 14.5 days postpartum ( dpp). Thus, Spink3 has essential roles for maintenance of integrity and regeneration of acinar cells. Autophagy is mostly a nonselective, bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and a killer of the cell, depending on the stage of the disease or the surrounding cellular environment. To analyze the role of macroautophagy in acute pancreatitis, we produced a conditional knockout mice, lacking the
Atg5 (autophagy-related 5) gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice (Fig. 6) . Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.
3)
Abhd2 and atherosclerosis
We previously reported that the mouse alpha/beta hydrolase domain containing 2 (Abhd2) was expressed in smooth muscle cells (SMCs) which suppressed their migration and inhibited the development of intimal hyperplasia by cuff placement; however, the role of ABHD2 in human remains to be elucidated. Thus, we examined ABHD2 expression in the human coronary atherosclerotic lesions of the patients with unstable angina (UA) and stable angina (SA). Our results showed that the ABHD2 was expressed in atherosclerotic lesions, and that the ABHD2 expression was significantly higher in the patients with UA than with SA. Immunohistochemistry analysis revealed abundant expression of ABHD2 in macrophages, but low expression in SMCs of atherosclerotic lesions. Using human vascular primary culture cell lines, we also demonstrated that the expression of ABHD2 was significantly higher in macrophages than in SMCs, and that the expression of ABHD2 significantly increased proportionally with differentiation from monocyte into macrophage.
