Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F.

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.

Expression of Pcdh15 in the inner ear, nervous system and various epithelia of the developing embryo.

We previously determined that Protocadherin 15 (Pcdh15) is associated with the Ames waltzer mutation in the mouse. Here we describe where the Pcdh15 gene is expressed at specific times during mouse development using RNA in situ hybridization. The expression of Pcdh15 is found in the sensory epithelium in the developing inner ear, in Rathke's pouch, and broadly throughout the brain with the highest level of expression being detected at embryonic day 16 (E16). Pcdh15 transcripts are also found in the developing eye, dorsal root ganglion, and the dorsal aspect of the neural tube, floor plate and ependymal cells adjacent to the neural canal. Additionally, expression is also detected in the developing glomeruli of the kidney, surface of the tongue, vibrissae, bronchi of the lung, and in the epithelium of the olfactory apparatus, gut and lung.

The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene.

The neuroepithelia of the inner ear contain hair cells that function as mechanoreceptors to transduce sound and motion signals. Mutations affecting these neuroepithelia cause deafness and vestibular dysfuction in humans. Ames waltzer (av) is a recessive mutation found in mice that causes deafness and a balance disorder associated with the degeneration of inner ear neuroepithelia. Here we report that the gene that harbours the av mutation encodes a novel protocadherin. Cochlear hair cells in the av mutants show abnormal stereocilia by 10 days after birth (P10). This is the first evidence for the requirement of a protocadherin for normal function of the mammalian inner ear.

Phenotypic variations of orpk mutation and chromosomal localization of modifiers influencing kidney phenotype.

The Oak Ridge polycystic kidney (orpk) mutant mouse model resulted from a transgene insertion into the Tg737 gene and exhibits a pleiotropic syndrome with lesions in the kidney, liver, and pancreas. We found marked differences in the phenotypic expression of the orpk mutation when bred on different genetic backgrounds. In the FVB/N background, the phenotype is very severe for kidney, pancreas, and liver lesions. To evaluate better how genetic background might influence the expressivity of the orpk phenotype, we bred the transgene into the C3HeB/FeJLe (C3H) genetic background. We performed a genome-wide scan using backcross and intercross populations with more than 150 markers to map the chromosomal location of the modifier genes that differ in the FVB/N and C3H genetic backgrounds that affect the severity of kidney disease in the orpk mouse. Low-resolution interval mapping was performed using the Map Manager QTb program, with the interval explaining a significant portion of the variance being the distal end of chromosome 4.

Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous in transgenic line TgN3261Rpw.

Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous are congenital ocular anomalies that can lead to cataract formation. A line of insertional mutant mice, TgN3261Rpw, generated at the Oak Ridge National Laboratory in a large-scale insertional mutagenesis program was found to have a low incidence (8/243; 3.29%) of multiple developmental ocular abnormalities. The ocular abnormalities include persistent hyperplastic primary vitreous, persistent hyperplastic tunica vasculosa lentis, failure of cleavage of the anterior segment, retrolental fibrovascular membrane, posterior polar cataract, and detached retina. This transgenic mouse line provides an ontogenetic model because of the high degree of similarity of this entity in humans, dogs, and mice.

Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer.

This report presents new findings regarding a recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice homozygous for the mutation. The mutant locus was mapped to a region on mouse chromosome 10 close to three spontaneous recessive mutations causing deafness: Ames waltzer (av), Waltzer (v), and Jackson circler (jc). Complementation testing revealed that the TgN2742Rpw mutation is allelic with av. Histological and auditory brainstem response (ABR) evaluation of animals that have the new allele balanced with the av(J) allele (called compound heterozygotes, TgN2742Rpw/av(J)) supports our genetic analysis. ABR evaluation shows complete absence of auditory response throughout the life span of TgN2742Rpw/av(J) compound heterozygotes. Scanning electron microscopy revealed abnormalities of inner and outer hair cell stereocilia in the cochleae of TgN2742Rpw mutants at 10 days after birth (DAB). The organ of Corti subsequently undergoes degeneration, leading to nearly complete loss of the cochlear neuroepithelium in older mutants by about 50 DAB. The vestibular neuroepithelia remain morphologically normal until at least 30 DAB. However, by 50 days, degenerative changes are evident in the saccular macula, which progresses to total loss of the saccular neuroepithelium in older animals. The new allele of av reported here will be designated av(TgN2742Rpw).

The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination.

Analysis of several mutations in the mouse is providing useful insights into the nature of the genes required for the establishment of the left-right axis during early development. Here we describe a new targeted allele of the mouse Tg737 gene, Tg737(Delta)2-3(beta)Gal), which causes defects in left-right asymmetry and other abnormalities during embryogenesis. The Tg737 gene was originally identified based on its association with the mouse Oak Ridge Polycystic Kidney (orpk) insertional mutation, which causes polycystic kidney disease and other defects. Complementation tests between the original orpk mutation and the new targeted knock-out mutation demonstrate that Tg737(Delta)2-3(beta)Gal) behaves as an allele of Tg737. The differences in the phenotype between the two mutations suggest that the orpk mutation is a hypomorphic allele of the Tg737 gene. Unlike the orpk allele, where all homozygotes survive to birth, embryos homozygous for the Tg737(Delta)2-3(beta)Gal) mutation arrest in development at mid-gestation and exhibit neural tube defects, enlargement of the pericardial sac and, most notably, left-right asymmetry defects. At mid-gestation the direction of heart looping is randomized, and at earlier stages in development lefty-2 and nodal, which are normally expressed asymmetrically, exhibit symmetrical expression in the mutant embryos. Additionally, we determined that the ventral node cells in mutant embryos fail to express the central cilium, which is a characteristic and potentially functional feature of these cells. The expression of both Shh and Hnf3(beta) is downregulated in the midline at E8.0, indicating that there are significant alterations in midline development in the Tg737(Delta)2-3(beta)Gal) homozygous embryos. We propose that the failure of ventral node cells to fully mature alters their ability to undergo differentiation as they migrate out of the node to contribute to the developing midline structures. Analysis of this new knockout allele allows us to define a critical role for the Tg737 gene during early embryogenesis. We have named the product of the Tg737 gene Polaris, which is based on the various polarity related defects associated with the different alleles of the Tg737 gene.

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Cellular pathophysiology of cystic kidney disease: insight into future therapies.

Polycystic kidney disease (PKD) is a developmental kidney disorder which can be inherited as either an autosomal dominant trait, with an incidence of 1:50 to 1:1000, or as an autosomal recessive trait with an incidence of 1:6,000 to 1:40,000. Three different genes have now been cloned that are associated with mutations that cause PKD. Two of these are linked to the most common forms of the dominant disease while the third is associated with the orpk mouse model of recessive polycystic kidney disease. Advances in understanding the molecular genetics of PKD have been paralleled by new insights into the cellular pathophysiology of cyst formation and progressive enlargement. Current data suggest that a number of PKD proteins may interact in a complex, which when disrupted by mutations in PKD genes may lead to altered epithelial proliferative activity, secretion, and cell matrix biology. The identification of a unique cystic epithelial phenotype presents new opportunities for targeted therapies. These include targeted gene therapy, gene complementation, and specific immunological or pharmacological interruption of growth factor pathways.

Alternative processing of the human and mouse raly genes(1).

A human homolog(RALY) of the mouse Raly gene was isolated and sequenced, and shown to encode a novel protein isoform containing a 16 amino acid in-frame insert in the variable region of the protein. Analysis of the corresponding region of the mouse Raly gene demonstrated that this novel protein isoform is also present in the mouse. Comparative analysis of RALY cDNA and EST sequences suggests the presence of additional alternatively processed RALY transcripts. As in the mouse, the human RALY gene is widely expressed as a 1.7-kb transcript.

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.

An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.

Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.

Insertional mutagenesis in transgenic mice generated by the pronuclear microinjection procedure.

Insertional mutagenesis in transgenic mice is a powerful method to study structure/function relationships between individual genes and complex developmental traits in the whole organism. Unlike spontaneous or chemical-induced mutations, insertional mutations have the advantage that the mutant locus is "tagged" with the transgene and, therefore, readily accessible at the molecular level. Starting with the work on the limb deformity locus, we describe here the characterization of several mouse mutants generated by insertional mutagenesis with the pronuclear microinjection procedure. These transgenic lines have proven to be ideal as models for human disease and for studying the function of novel genes during development. We also describe the unique features of insertional mutations that arise in transgenic mice produced with the pronuclear microinjection procedure and provide recommendations on how to clone and characterize these mutations at the molecular level. Finally, we discuss future prospects for the use of this unique form of germline mutagenesis in the mouse.

Functional genomics in the post-genome era.

As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease. New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously. Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease.

Utilization of microhomologous recombination in yeast to generate targeting constructs for mammalian genes.

We have developed a new procedure utilizing microhomologous recombination in yeast to generate targeting constructs for producing targeted mutations in mice. This procedure is rapid and efficient, and should be directly applicable to all mammalian genes. Moreover, only minimal information about the locus being targeted is required. The feasibility of this approach was demonstrated by producing another allele of the mouse Tg737 polycystic kidney gene.

Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease.

A consistent phenotype observed in both human patients and several different mouse models of autosomal recessive polycystic kidney disease (ARPKD) is an increased activity of the epidermal growth factor receptor (EGFR) in the affected kidneys. To determine whether this increased activity of the EGFR is a functional event that is directly part of the disease pathway of renal cyst formation, we used a genetic approach to introduce a mutant EGFR with decreased tyrosine kinase activity into a murine model of ARPKD. We found that the modified form of the EGFR could block the increase in EGFR-specific tyrosine kinase activity that normally accompanies the development of renal cysts, and this correlated with an improvement in kidney function and a substantial decrease in cyst formation in the collecting ducts. These results suggest that changes in the expression of the EGFR contribute to the formation of cysts in the collecting ducts, and that drugs that target the tyrosine kinase activity of the EGFR may potentially be therapeutic in ARPKD.

Characterization of growth factor responsiveness and alterations in growth factor homeostasis involved in the tumorigenic conversion of mouse oval cells.

Five mouse oval cell lines were investigated in regards to their growth and differentiation factor (GDF) responsiveness and to changes in their GDF responsiveness following tumorigenic conversion. In all 59 GDFs and 11 comitogens were evaluated with variable responsiveness, depending on the mouse oval cell line under study, observed. Analysis of oval cell GDF responsiveness during tumorigenic conversion revealed that tumorigenic variants displayed alterations in GDF responsiveness which correlated with tumorigenicity. In addition, analysis of autocrine/paracrine growth factor production demonstrates that most tumorigenic variants produce growth factors. These studies demonstrate for the first time that (1) mouse oval cells respond to a wide variety of GDFs including various members of the interleukin, chemokine, stem cell factor, EGF, FGF, PDGF, TGF-beta, VEGF, insulin, CSF, TNF, HGF, and IFN growth and differentiation factor families in addition to multiple comitogens and (2) during tumorigenic conversion mouse oval cells undergo alterations which result in both alterations in GDF responsiveness and the autocrine/paracrine production of multiple GDFs.

The molecular biology of polycystic kidney disease.

In recent years there have been a number of developments in polycystic kidney disease (PKD) research. The genes associated with the predominant forms of autosomal dominant PKD have been cloned, and the gene associated with a mouse model for autosomal recessive PKD has been identified and characterized. Other studies have yielded new information regarding the role of the epidermal growth factor receptor gene in promoting renal cyst formation. In this review article we summarize recent published data on the molecular genetics of autosomal dominant and autosomal recessive PKD and provide a working model of how multiple genes participate in the PKD disease pathway.

The tetratricopeptide repeat containing Tg737 gene is a liver neoplasia tumor suppressor gene.

The Tg737 gene was investigated for gross alterations in a series of rodent/human liver tumors and human tumorigenic cell lines. The Tg737 gene was found to be altered in approximately 40% of the rodent chemically-induced liver tumors, 40% of the human liver tumors, and in liver, kidney and pancreatic human tumor cell lines. Ectopic re-expression of the Tg737 gene in a Tg737 deleted mouse liver tumor cell line resulted in suppression of tumorigenic growth, without altering in vitro cell culture growth. Treatment of mice which are either homozygous normal or heterozygous deleted at the Tg737 locus with the carcinogen diethylnitrosamine resulted in an increase in preneoplastic foci formation in the Tg737 heterozygous deleted mice. Ectopic expression of the Tg737 gene results in multinucleated cells, loss of Tg737 gene expression results in the proliferation of liver stem cells (oval cells) without concomitant differentiation, and reexpression of the Tg737 gene reestablished responsiveness to external differentiation factors. We believe this is the first report demonstrating tumor suppression activity for a tetratricopeptide repeat gene family member and provides insights into the function of this family of genes in mammalian cells.