Yoshinobu Asada

The Zinc Transporter SLC39A13/ZIP13 Is Required for Connective Tissue Development; Its Involvement in BMP/TGF-β Signaling Pathways

Background Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. Methodology/Principal Findings Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. Conclusions/Significance Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction.

Quantitative Trait Loci on Chromosomes 10 and 11 Influencing Mandible Size of SMXA RI Mouse Strains

Predicting the mandible size before the termination of growth of the maxillofacial bones is essential in pedodontics as well as for the predictions needed for genetic analysis. Here, Quantitative Trait Locus (QTL) analysis was used to detect the chromosomal regions responsible for the mandible length between the menton and gonion in an SMXA recombinant inbred strain of mice. Around the region 60 cM from the centromere in chromosome 10, the logarithm of the odds score showed a higher than suggestive level. Around the regions 13 cM and 16 cM in chromosome 11, two significant QTLs were detected. Analysis of genotypes from loci corresponding to those QTLs revealed a large mandible when the region between the markers Hba and D11Mit163 and D10Mit70 and D10Mit136 indicated the genotype from the A/J and SM/J alleles, respectively. These results suggest that the major gene(s) responsible for mandible length are located in these regions.

Craniofacial and dental characteristics of Kabuki syndrome

We describe oral manifestations in six patients (three females and three males aged 6 to 24 years) with Kabuki syndrome (KS), based on their physical, orthopantomographic, and cephalometric findings. All six patients had a high-arched palate, malocclusion, most commonly unilateral posterior cross-bite (5/6), severe maxillary recession and mid-facial hypoplasia. Other frequently observed oral manifestations included small dental arch and hypodontia. Three patients lacked permanent teeth, mostly the central/lateral incisors. Both tooth size (in primary and permanent teeth) and dental arch (in length and width) tended to be small. We would like to stress that oral care and management is a must for the well-being of KS patients.

DNA pooling as a quick method for finding candidate linkages in multigenic trait analysis: an example involving susceptibility to germ cell tumors

A logistical challenge m multigenic trait analysis is typing large numbers of segregants for large numbers of loci with the goal of mapping alleles contributing to the phenotype of interest. We and others showed that DNA pooling, which is also called bulk segregant analysis, is an efficient way to find candidate linkages for monogenic traits such as endogenous provirnses, RAPDs, RFLVs, SSLPs, and mutant genes (Taylor and Rowe 1989; Michelmore et al. 1991; Williams et al. 1993; Asada et al. 1994; Taylor et al. 1994; Carmi et al. 1995). The advantage of pooling is that the number of individuals needing to be typed can be reduced dramatically. Here we show that DNA pooling is also effective for finding candidate linkages in multigenic traits such as susceptibility to germ cell tumors in male 129/Sv mice. Given the number of independently segregating loci, mapping germ cell tumor susceptibility alleles is a formidable challenge. Segregation analysis shows that many independently segregating loci with recessive effects control susceptibility to germ cell tumors in 129/Sv mice (Stevens and Mackensen 1961; Stevens 1981; J.H. Nadeau and D.S. Varnum, unpublished). Stevens (1981) found only one tumor among more than 11,000 males that were surveyed after crossing 129/Sv mice to each of seven other inbred strains and then backcrossing the F 1 hybrids to 129/Sv. These results suggest that as many as 13 genes control susceptibility. Neither the map location nor the identity of any of these 129derived tumor susceptibility alleles has been determined. Presumably, comparable genetic complexity accounts for largely sporadic occurrence of male germ cell tumors in humans (Dieckmann et al. 1987; Douketis and Burkes 1993). We are taking advantage of a spontaneous mutation called Ter to simplify the genetic analysis. Ter causes severe germ cell deficiency on many if not all inbred strain backgrounds and increases susceptibility to germ cell tumors on the 129/Sv inbred background. Ter is not required for teratocarcinogenesis because -1% of 129/Sv males without Ter have tumors (Stevens and Little 1954; Stevens and Hummel 1957). Similarly, Ter is not sufficient for teratocarcinogenesis because on other inbred backgrounds Ter causes germ cell deficiency but not germ cell tumors (Stevens 1973). Instead, Ter acts as a modifier, increasing tumor incidence from 1% in 129 +/+ mice to 94% in 129-Ter/Ter mice (Stevens 1973; Noguchi and Noguchi 1985; Asada et al. 1994; Sakurai et al. 1994). Because of its dramatic effect on tumor incidence, mice with tumors are more readily obtained in crosses where Ter is segregating. DNA pooling suggested a candidate linkage for Ter on Chromosome (Chr) 18 (Asada et al. 1994). QTL-analysis of

Requirement of Zinc Transporter SLC39A7/ZIP7 for Dermal Development to Fine-Tune Endoplasmic Reticulum Function by Regulating Protein Disulfide Isomerase

Skin is the first area that manifests zinc deficiency. However, the molecular mechanisms by which zinc homeostasis affects skin development remain largely unknown. Here, we show that zinc-regulation transporter-/iron-regulation transporter-like protein 7 (ZIP7) localized to the endoplasmic reticulum plays critical roles in connective tissue development. Mice lacking the Slc39a7/Zip7 gene in collagen 1-expressing tissue exhibited dermal dysplasia. Ablation of ZIP7 in mesenchymal stem cells inhibited cell proliferation thereby preventing proper dermis formation, indicating that ZIP7 is required for dermal development. We also found that mesenchymal stem cells lacking ZIP7 accumulated zinc in the endoplasmic reticulum, which triggered zinc-dependent aggregation and inhibition of protein disulfide isomerase, leading to endoplasmic reticulum dysfunction. These results suggest that ZIP7 is necessary for endoplasmic reticulum function in mesenchymal stem cells and, as such, is essential for dermal development.

Enhanced induction of a histamine-forming enzyme, histidine decarboxylase, in mice primed with NOD1 or NOD2 ligand in response to various Toll-like receptor agonists

We investigated the immunopharmacological aspects of innate immune responses via Toll-like receptors (TLRs), NOD1 and NOD2, in terms of induction of the histamine-forming enzyme, histidine decarboxylase (HDC), activity in mice. Intravenous injection of TLR4-agonistic synthetic lipid A definitely induced HDC activity in the liver, spleen, and lungs, especially the lungs, in mice, where maximum activity was induced about 3 h after the injection of lipid A. The TLR2/6 agonistic synthetic diacyl-type lipopeptide FSL-1 and TLR3-agonistic poly I:C were also effective in inducing HDC, while the NOD2-agonistic synthetic muramyldipeptide (MDP) and NOD1-agonistic synthetic FK156 and FK565 exhibited only weak activities in this respect. Mice primed with intravenous injection of NOD1 or NOD2 agonists produced higher HDC activity following the 4–6 h later intravenous challenge with the above TLR agonists. Among the priming agents, FK565 exhibited the strongest activity, and it was effective via various administration routes – intraperitoneal, subcutaneous, intramuscular, as well as intravenous injection; furthermore, oral (gastric) administration was effective, although it needed a dose 10 times higher than that required for other administration routes. These findings suggest that HDC is induced in association with TLRs and NOD1/2, and that the newly formed histamine by the induced HDC might play important roles in the regulation of inflammatory and immune responses in various organs.We investigated the immunopharmacological aspects of innate immune responses via Toll-like receptors (TLRs), NOD1 and NOD2, in terms of induction of the histamine-forming enzyme, histidine decarboxylase (HDC), activity in mice. Intravenous injection of TLR4-agonistic synthetic lipid A definitely induced HDC activity in the liver, spleen, and lungs, especially the lungs, in mice, where maximum activity was induced about 3 h after the injection of lipid A. The TLR2/6 agonistic synthetic diacyl-type lipopeptide FSL-1 and TLR3-agonistic poly I:C were also effective in inducing HDC, while the NOD2-agonistic synthetic muramyldipeptide (MDP) and NOD1-agonistic synthetic FK156 and FK565 exhibited only weak activities in this respect. Mice primed with intravenous injection of NOD1 or NOD2 agonists produced higher HDC activity following the 4-6 h later intravenous challenge with the above TLR agonists. Among the priming agents, FK565 exhibited the strongest activity, and it was effective via various administration routes - intraperitoneal, subcutaneous, intramuscular, as well as intravenous injection; furthermore, oral (gastric) administration was effective, although it needed a dose 10 times higher than that required for other administration routes. These findings suggest that HDC is induced in association with TLRs and NOD1/2, and that the newly formed histamine by the induced HDC might play important roles in the regulation of inflammatory and immune responses in various organs.

Slc39a13/Zip13: A Crucial Zinc Transporter Involved in Tooth Development and Inherited Disorders

Zinc (Zn) is an essential trace element and it plays indispensable roles in cellular processes for embryonic and postnatal development in mammals. Zn deficiency causes growth retardation, reduced bone volume, dental decay, skin diseases, and other conditions. Zn homeostasis is tightly controlled by the action of Zn transporters and metallothioneins, which together intricately regulate the Zn concentration and distribution in cells. We recently investigated the role of the Zn transporter Slc39a13/Zip13 in mice and humans. Mice deficient in Zip13 show changes in the tooth, bone, and connective tissues, and impairments in BMP and TGF-β signaling. A unique variant of Ehlers-Danlos syndrome (EDS) with hypodontia was found, in which Zip13 possesses a loss-of-function mutation, indicating that Zip13-mediated Zn homeostasis is crucial for tooth, bone and connective tissue development in mice and humans. In this review, we describe how Zn affects biological events especially in tooth development, with recent progress uncovering the roles of the Zn transporter Zip13 in mammalian health and diseases.

Genetic Mapping of the Absence of Third Molars in EL Mice to Chromosome 3

We noted the absence of all 4 third molars (M3) in Epilepsy-Like disorder (EL) mice, an animal model for the study of epilepsy. This study was conducted to identify the major candidate chromosome and to detect the region that included the candidate gene causing the absence of M3 in EL mice. Linkage analysis was performed on genetic crosses of EL mice and MSM (Mus musculus molossinus) strain mice, which had a normal complement of teeth. Genome-wide screening by individual genotyping of F2 intercross mice identified mouse chromosome 3 as one of the candidate chromosomes. Based on high linkage scores in detailed genotyping of F2 intercross and N2 backcross mice, the candidate locus for the absence of M3 in EL mice was mapped on the middle of chromosome 3.

Simple sequence length polymorphisms (SSLPs) that distinguish MOLF/Ei and 129/Sv inbred strains of laboratory mice

SSRs (simple sequence repeats) are valuable for high-resolution mapping as a prelude to mutant gene identification by positional cloning and candidate gene evaluation (Dietrich et al. 1994, 1996), for surveying the genome for mapping quantitative trait loci (QTL) as part of complex trait analyses (Dietrich et al. 1994, 1996), and for making congenic and consomic strains of mice (Nadeau et al. 1995; Markel et al. 1997). To date 7,377 SSLPs have been mapped onto mouse chromosomes using the MIT (C3H ob × CAST)F1 intercross and C57BL/6J × M. spretus backcross (Dietrich et al. 1996). The sequence of the clone that defined the particular CA repeat, the size of the PCR product revealed by primers that flank the CA repeat, and the map locations of these SSRs are available electronically (www.genome.wi.mit.edu/cgi-bin/mouse/index; see also www.informatics.jax.org). All three applications of SSRs depend on the availability of polymorphisms in the mouse strains of interest. Linkage testing crosses are often made between conventional laboratory inbred strains of laboratory mice and inbred strains recently made from wild-derived mice, e.g., strains such as CAST/Ei, SPRET/Ei, and MOLF/Ei (Dietrich et al. 1994, 1996). The MOLF/Ei strain was inbred from wild-derived Mus musculus molossinus, a naturally occurring hybrid species found in the Japanese archipelago (Silver 1995; Moriwaki 1994; Bonhomme and Guénet 1996). This strain has been used for interspecific crosses in gene mapping (Asada et al. 1994) and genome-wide scans (Collin et al. 1996) and for making congenic and consomic strains (A. Matin, J. Nadeau, et al. in preparation). It is separated from principal progenitors of conventional inbred strains by an evolutionary history spanning ∼100,000 years (Moriwaki 1994; Bonhomme and Guénet 1996). They are good breeders under laboratory conditions and are easy to handle. MOLF/Ei has better intercross reproductive performance than strains derived from M. spretus (Silver 1995), and, unlike progeny of interspecific crosses with mice derived from M. spretus, both sexes of the F1 and F2 generations are usually fertile (Moriwaki 1994; Bonhomme and Guénet 1996). The purpose of this communication is to share information about SSLPs (simple sequence length polymorphisms) that distinguish MOLF/Ei, 129/Sv, and other inbred strains. We have used MOLF/Ei in several intercross and backcross linkage studies (Asada et al. 1994; Collin et al. 1996) and have found many polymorphic SSRs between MOLF/Ei and 129/Sv. SSLPs involving the 129 strains and other strain combinations have been tabulated previously (Matouk et al. 1996; Simpson et al. 1997; Threadgill et al. 1997). These serve as references through which MOLF/ Ei SSLPs can be compared with other inbred strains. Genomic DNA was prepared from spleen or blood from the MOLF/Ei and 129/Sv − + + Tyrc-ch Ter/+ or 129/Sv−+ + +/J strains. DNA (50–100 ng) was used in a 25-ml PCR reaction (Collin et al. 1996). Aliquots of the PCR reaction products were electrophoresed on 15 cm 7% non-denaturing polyacrylamide gels, and PCR products were detected by staining with ethidium bromide. A 100-bp ladder was used as a size standard to estimate the PCR product sizes in MOLF/Ei and 129. Of the 209 SSLPs loci examined, 153 (73.2%) were polymorphic between 129 and MOLF/Ei. This compares with the reported 94% polymorphism rate between laboratory strains and CAST/Ei and SPRET/Ei and the less than 48% rate among conventional inbred strains (Dietrich et al. 1996). An important caveat is that Dietrich and associates used a more sensitive method to detect PCR product size differences and, as a result, our polymorphism rate reported here is under-estimated. The SSLPs and their estimated PCR product sizes are listed in Table 1. These data facilitate use of the MOLF/Ei strain as an alternative to CAST/Ei and SPRET/Ei in intersubspecific crosses.

Localization of am3 using EL Congenic Mouse Strains

EL/Sea mice have 100% incidence of the absence of third molars (M3). Our previous linkage analysis using EL/Sea and MSM/Msf mouse strains provides statistical evidence of a major locus for the absence of M3, designated am3, of EL/Sea at the middle region of chromosome 3. To obtain independent evidence for linkage and more precisely determine the location of the am3 locus, we generated EL/Sea congenic strains for am3 in which the restricted interval of chromosome 3 of EL/Sea was replaced by an MSM/Msf-derived homologue. EL/Sea congenic mice that were either heterozygous or homozygous for the MSM/Msf-derived interval exhibited a significant decrease in the incidence of the absence of third molars, confirming previous genome scan results. These results confine the am3 locus to an approximately 4.4-cM region, and demonstrate that other unmapped genes are also involved in the absence of M3 in EL/Sea mice.