The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene

Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein.     

Desmoglein genes are up-regulated in the pk mutant mouse

Plucked (pk) is an autosomal recessive mouse mutation with a hair phenotype that arose spontaneously in the DBA/2J strain. Histological studies indicate that adult pk mutant mice lose truncal hair because of the scarring of follicles due to an apparent obstruction of the outward movement of the hair shaft within the follicular canal. We mapped the pk mutant phenotype to a 1.1cM region of chromosome 18 (between 6.6 and 7.7 cM from the centromere) using 370 backcross progeny. Within this region, among others, are genes for desmosome cadherins. Desmosome cadherins are interesting candidates because of their critical roles for cell-cell adhesion in epidermal function. Northern Blot analysis of wild-type and pk mutant mice indicates that expression of both desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) is up-regulated in the skin of mutant pk mice.     

Intragenic deletion in the Desmoglein 4 gene underlies the skin phenotype in the Iffa Credo "hairless" rat

The Iffa Credo (IC) "hairless" rat is an autosomal recessive hypotrichotic animal model actively used in pharmacological and dermatological studies. Although the molecular basis of the IC rat phenotype was never defined, the designation "hr/hr" (hairless) has been used for this rat mutation. Despite the observation that IC rats share many phenotypic similarities with Charles River (CR) 'hairless rats', crossbreeding between CR and IC rats indicated that these mutations are not allelic, and moreover, genetic analysis of both CR and IC hairless mutant rats showed no mutations in the hr gene. Here, we present a detailed analysis of the skin phenotype in the IC rat. While the initial stages of hair follicle (HF) morphogenesis reveal no significant abnormalities, the subsequent processes of inner root sheath and hair shaft formation are severely disturbed due to impaired proliferation in the hair matrix and abnormal differentiation in the precortex zone. This results in significant reduction of hair bulb volume, and the formation of dysmorphic "blebbed" hair shafts lacking medullar structure and resembling "lanceolate" hairs. Based on the presence of lance-head hairs typical of rodent lanceolate mutants, we performed molecular analysis of the desmoglein 4 gene and found a large intragenic deletion encompassing nine exons of the gene. This finding, together with specific morphological features of skin and hairs, confirms that the IC rat is allelic with the lanceolate hair (lah) mutations in mice and rats. Our results elucidate the genetic and morphological basis of the IC rat mutation, thus providing a new model to study molecular mechanisms of hair growth control.     

Derivation of SHR-chromosome 4 congenic sublines for fine genetic mapping of quantitative trait loci with major effects on insulin resistance and blood pressure

Recently, we have found that transfer of a segment of chromosome 4 between I16 and Npy markers from the Brown Norway (BN) rat into the spontaneously hypertensive rat (SHR) significantly attenuated both hypertension (measured by telemetry) and insulin resistance (measured as plasma insulin/glucose ratios before and after a high fructose diet) in the SHR progenitor strain. To map the putative quantitative trait loci (QTL) more precisely, we derived an (SHR×SHR.BN-chr.4)F2 population to search for recombinants that will enable us to produce congenic sublines. The F2 animals were genotyped in markers equally distributed along the interval of the chromosome 4 differential segment. Altogether, five new congenic sublines with overlapping segments of the differential chromosome 4 are being produced. New congenic sublines will enable us to test the hypothesis that insulin resistance and hypertension can be influenced by closely linked genes or perhaps even the same gene(s) on chromosome 4.     

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.     

Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle

Desmosomes are critical for the tissue integrity of stratified epithelia and their appendages. Desmogleins (DSGs) and desmocollins (DSCs) are transmembrane desmosomal cadherins that interact extracellularly to link neighboring epithelial cells. We recently identified a new member of the DSG family, designated desmoglein 4, whose mutations cause hypotrichosis in human, mouse and rat. In this study, we analyzed in detail the expression domains of human desmoglein 4 protein (DSG4) in human skin relative to differentiation markers and other DSGs. Our results show that DSG4 protein is expressed in the more highly differentiated layers of the epidermis. This expression pattern in vivo is recapitulated in highly differentiated HaCaT human keratinocytes and normal human keratinocytes in vitro. In the human hair follicle, DSG4 is expressed specifically in the hair shaft cortex, the lower hair cuticle, and the upper inner root sheath (IRS) cuticle. Using a green fluorescent protein-tagged version of mouse or rat desmoglein 4 protein (Dsg4) and immuno-electron microscopy, we demonstrate that Dsg4 localizes to desmosomes both in vitro and in vivo. The highly specific expression pattern of DSG4 in the human hair follicle, combined with the phenotype of rodent models and human patients with desmoglein 4 mutations, underscores the importance of this adhesion molecule in the integrity of the hair shaft.     

Mast cell hyperplasia in the skin of Dsg4-deficient hypotrichosis mice, which are long-living mutants of lupus-prone mice

Desmosomal cadherins are essential cell adhesion molecules expressed in the epidermis. We identified a mutation of a cadherin superfamily member, namely, desmoglein 4 (Dsg4), in early onset of death (EOD)( hage ) mice with hypotrichosis. The mutation was induced by the insertion of an early transposon II-beta into intron 8 of Dsg4. Mast cell hyperplasia was observed in the skin of EOD( hage ) mice. The abnormally expanded population of lpr T cells, i.e., CD4(-)CD8(-)B220(+)Thy1.2(+) alphabetaT cells, in the splenocytes of EOD mice was reduced in EOD( hage ) mice. Therefore, it was suspected that the long-living mutant EOD( hage ) mice were selected from lupus-prone EOD mice because of their immunological immaturity. These findings clearly indicate that Dsg4 is an important molecule for the formation of hair follicles and hypothesize that unorganized hyperplastic hair follicles in anagen due to the Dsg4 mutation provide niches for mast cell precursors in the skin.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

Targeted Disruption of the Pemphigus Vulgaris Antigen (Desmoglein 3) Gene in Mice Causes Loss of Keratinocyte Cell Adhesion with a Phenotype Similar to Pemphigus Vulgaris

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell–cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 −/− mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8–10 d weighed less than DSG3 +/− or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and “tombstoning” of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 −/− mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.     

Sparse and wavy hair: a new model for hypoplasia of hair follicle and mammary glands on rat chromosome 17

Mutant animals in the skin and hair have been used to identify important genes in biomedical research. We describe a new mutant rat, sparse and wavy hair (swh), that spontaneously arose in a colony of inbred WTC rats. The mutant phenotype was characterized by sparse and wavy hair, which was most prominent at age 3-4 weeks, and was inherited in an autosomal recessive manner. The swh/swh rats showed impaired gain of body weight, and their hair follicles were reduced both in number and size, associated with hypoplasia of the sebaceous glands and the subcutaneous fat tissue. Female swh/swh rats were unable to suckle their offspring. Their mammary glands were hypoplastic, and differentiation of mammary epithelial and myoepithelial cells was impaired. Linkage analysis of 579 backcross rats localized the swh locus to a .35-cM region between D17Rat131 and D17Rat50 in the distal end of rat Chr 17. The swh locus spanned the 3.7-Mb genomic region where 24 genes have been mapped and corresponded to the centromere region of the mouse Chr 2 or the region of the human Chr 10p11.1-p14. None of the genes or loci described in mouse or human hair and skin diseases mapped to these regions. These findings suggest that the rat swh is a novel mutation associated with impaired development of the skin appendages, such as hair follicles, sebaceous glands, and mammary glands, and will provide an experimental model to clarify a gene and mechanisms for development of skin appendages.     

Phenotypic selection: a successful strategy to fix major genes of hypertension

Spontaneously diabetic BB/OK rats are not genetically susceptible to develop diabetic complications as hypertension or nephropathy. Recently, we generated 5 congenic BB. SHR rat strains by transferring different chromosomal regions of the spontaneously hypertensive rat (SHR) onto the genetic background of BB/OK rats. Four out of 5 strains showed a weak increase of blood pressure (8 mmHg). This weak blood pressure effect indicated that the transferred regions fo not contain major genes for hypertension. That prompted us to choose the classical procedure of phenotypic selection to fix major genes causing hypertension in a BB/OK rat subline generated by cross of BB/OK and SHR and repeated backcrossing of animals with highest blood pressure onto normotensive BB/OK rats. After 7 backcrosses (N8), all backcross parents were genetically analysed with the aid of 259 microsatellites to identify loci causing blood pressure of 177 ± 10 mmHg in this BB/OK rat subline. The data revealed, that loci on chromosome 1, 14 and 18 were heterozygous until BC5, BC6 and BC7, respectively. Considering the relative stable high blood pressure during the backcross procedure, these loci might be of essential importance for the development of hypertension in the SHR.     

Evidence for a female-specific effect of a chromosome 4 locus on anxiety-related behaviors and ethanol drinking in rats

Previous studies using the inbred rat strains Lewis (LEW) and spontaneously hypertensive rats (SHR) led to the mapping of two quantitative trait loci, named Ofil1 (on chromosome 4 of the rat) and Ofil2 (on chromosome 7), for open-field inner locomotion, a behavioral index of anxiety. Studies using other strains showed that the region next to Ofil1 influences measures of not only anxiety but also ethanol consumption. In view of the high prevalence of psychiatric disorders such as anxiety and alcoholism, as well as the comorbidity between them, the present study was designed to better characterize the contribution of these two loci to complex emotional and consummatory responses. Rats deriving from an F2 intercross between the LEW and the SHR strains were selected according to their genotype at markers flanking the loci Ofil1 and Ofil2 and bred to obtain lines of rats homozygous LEW/LEW or SHR/SHR for each of the two loci, thus generating four genotypic combinations. These selected animals as well as purebred LEW and SHR rats of both sexes were submitted to a battery of tests including measures of locomotor activity, anxiety, sweet and bitter taste reinforcement and ethanol intake. Lewis rats displayed more anxiety-like behavior and less ethanol intake than SHR rats. Ofil1 (on chromosome 4) affected both the activity in the center of the open field and ethanol drinking in females only. These results suggest that Ofil1 contains either linked genes with independent influences on anxiety-related responses and ethanol drinking or a pleiotropic gene with simultaneous effects on both traits.     

Hypotensive effect associated with a phospholipase C-delta 1 gene mutation in the spontaneously hypertensive rat.

To identify the genes responsible for blood pressure in the spontaneously hypertensive rat strain, we performed a cosegregation analysis between the genotype and blood pressure in a set of male F2 rats obtained by crossmating SHR with Wistar-Kyoto rats, a parental normotensive strain. Our investigation revealed that the phospholipase C-delta 1 polymorphism, which resulted in missense mutation, cosegregates with the lower blood pressure in SHR, and that PLC-delta 1 gene is located on chromosome 8. On the other hand, we found the lack of cosegregation between blood pressure and the nerve growth factor receptor gene, which is linked to a hypertensinogenic gene locus (denoted as BP/SP-1) on chromosome 10. We propose that PLC-delta 1 gene itself of closely linked gene on chromosome 8 is a new candidate with the hypotensive effect, and that BP-SP1 locus does not directly contribute to blood pressure elevation in original SHR.     

Does Cd36 gene play a key role in disturbed glucose and fatty acid metabolism in Prague hypertensive hypertriglyceridemic rats?

Close links between hypertension, hypertriglyceridemia, insulin resistance and other symptoms of metabolic syndrome was demonstrated in humans and experimental animals. Quantitative trait loci for defects in glucose and fatty acid metabolism, hypertriglyceridemia and hypertension were mapped in spontaneously hypertensive rats (SHR) on chromosome 4 and defective Cd36 gene was identified in this region. Here we investigated the polymorphism of Cd36 gene in Prague hereditary hypertriglyceridemic (HTG) rats, which represent another model of genetic hypertension and metabolic syndrome. These animals were compared with NIH-derived SHR and two different normotensive control strains (WKY, LEW). In spite of the fact that HTG and SHR rats had similar metabolic disturbances, genotype analysis of PCR products has shown that Cd36 mutation was not present in HTG rats. In conclusion, we have revealed that defective Cd36 is probably a candidate gene for disorded fatty-acid metabolism, glucose intolerance and insulin resistance in NIH-derived SHR, but other genes might play a role in pathogenesis of metabolic syndrome in Prague hereditary hypertriglyceridemic rats. This is in accordance with the absence of defective Cd36 gene in original SHR from Japan.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Evaluation of insulin resistance linkage to rat chromosome 4 in SHR of a Japanese colony

The spontaneously hypertensive rat (SHR) is a model of human insulin resistance syndrome. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism have been mapped to an overlapping region of rat chromosome (RNO) RNO4 in SHR of the National Institute of Health colony, where a deletion in the Cd36 gene has been implicated as the causative mutation of insulin resistance. The present study has examined the potential presence of RNO4 linkage to a series of metabolic phenotypes in F(2) progeny derived from SHR of a Japanese colony (SHR/Izm) without the Cd36 mutation. Our data demonstrate that 'major' insulin resistance gene(s) are unlikely to exist on RNO4 in SHR/Izm and in vitro phenotypes measured in isolated adipocytes do not cosegregate in the F(2) population studied. Thus, it seems to be difficult to explain the underlying genetic mechanisms of insulin resistance by a single major gene on RNO4.     

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

Oca2^p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2^p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2^p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.