Elizabeth C. Bryda

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa1 (RP). So far, six loci (USH1A–USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A (ref. 2) for USH1B and the gene encoding harmonin3,4 for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10 (ref. 5). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G→C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, ΔM1281 and IVS51+5G→A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper6, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Positional cloning of jcpk/bpk locus of the mouse

By positional cloning techniques, we have identified the gene that is disrupted in the jcpk and bpk mouse models for polycystic kidney disease. This gene is the mouse homolog of the Drosophila Bicaudal C gene. Both of these mutations have been mapped to a very short stretch of Chromosome (Chr) 10. By sequencing the bicaudal C gene, Bicc1, in these models, it was found that the jcpk mutation results in a shortened and abnormal transcript, whereas the bpk mutation results in an abnormal 3′ coding region. In Drosophila, this gene encodes a protein known to influence developmental processes. The mammalian homolog contains three KH (K homology) domains and a SAM (sterile alpha motif) domain and is expressed in the developing embryo, indicating that it may be important in RNA-binding and/or protein interactions during embryogenesis.

The polycystic kidney disease-related proteins Bicc1 and SamCystin interact.

Mutations in either the Bicaudal-C or the Anks6 gene which encode the Bicc1 and SamCystin proteins respectively cause formation of renal cysts in rodent models of polycystic kidney disease, however their role in the mammalian kidney is unknown. Immunolocalization studies demonstrated that, unlike many other PKD-related proteins, SamCystin and Bicc1 do not localize to the primary cilia of cultured kidney cells. Epitope-tagged recombinant SamCystin and Bicc1 proteins were transiently transfected into inner medullary collecting duct (IMCD) cells and co-immunoprecipitated. The results showed that SamCystin self-associates, Bicc1 and SamCystin interact, the mutation responsible for PKD in the Han:SPRD-Cy rat disrupts the self-association of SamCystin but not the Bicc1-SamCystin interaction, and RNA may be an important component of the Bicc1-SamCystin complex. These studies provide the first evidence that Bicc1 and SamCystin interact at the protein level suggesting that they function in a common molecular pathway that when perturbed, is involved in cystogenesis.

Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur; it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

Differential susceptibility to colorectal cancer due to naturally occurring gut microbiota

Recent studies investigating the human microbiome have identified particular bacterial species that correlate with the presence of colorectal cancer. To evaluate the role of qualitatively different but naturally occurring gut microbiota and the relationship with colorectal cancer development, genetically identical embryos from the Polyposis in Rat Colon (Pirc) rat model of colorectal cancer were transferred into recipients of three different genetic backgrounds (F344/NHsd, LEW/SsNHsd, and Crl:SD). Tumor development in the pups was tracked longitudinally via colonoscopy, and end-stage tumor burden was determined. To confirm vertical transmission and identify associations between the gut microbiota and disease phenotype, the fecal microbiota was characterized in recipient dams 24 hours pre-partum, and in Pirc rat offspring prior to and during disease progression. Our data show that the gut microbiota varies between rat strains, with LEW/SsNHsd having a greater relative abundance of the bacteria Prevotella copri. The mature gut microbiota of pups resembled the profile of their dams, indicating that the dam is the primary determinant of the developing microbiota. Both male and female F344-Pirc rats harboring the Lewis microbiota had decreased tumor burden relative to genetically identical rats harboring F344 or SD microbiota. Significant negative correlations were detected between tumor burden and the relative abundance of specific taxa from samples taken at weaning and shortly thereafter, prior to observable adenoma development. Notably, this naturally occurring variation in the gut microbiota is associated with a significant difference in severity of colorectal cancer, and the abundance of certain taxa is associated with decreased tumor burden.

Bicc1 is a genetic determinant of osteoblastogenesis and bone mineral density.

Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture. While substantial progress has been made toward elucidating the genetic determinants of BMD, our understanding of the factors involved remains incomplete. Here, using a systems genetics approach in the mouse, we predicted that bicaudal C homolog 1 (Bicc1), which encodes an RNA-binding protein, is responsible for a BMD quantitative trait locus (QTL) located on murine chromosome 10. Consistent with this prediction, mice heterozygous for a null allele of Bicc1 had low BMD. We used a coexpression network-based approach to determine how Bicc1 influences BMD. Based on this analysis, we inferred that Bicc1 was involved in osteoblast differentiation and that polycystic kidney disease 2 (Pkd2) was a downstream target of Bicc1. Knock down of Bicc1 and Pkd2 impaired osteoblastogenesis, and Bicc1 deficiency-dependent osteoblast defects were rescued by Pkd2 overexpression. Last, in 2 human BMD genome-wide association (GWAS) meta-analyses, we identified SNPs in BICC1 and PKD2 that were associated with BMD. These results, in both mice and humans, identify Bicc1 as a genetic determinant of osteoblastogenesis and BMD and suggest that it does so by regulating Pkd2 transcript levels.

Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats

Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models.

Microbial modulation of behavior and stress responses in zebrafish larvae.

The influence of the microbiota on behavior and stress responses is poorly understood. Zebrafish larvae have unique characteristics that are advantageous for neuroimmune research, however, they are currently underutilized for such studies. Here, we used germ-free zebrafish to determine the effects of the microbiota on behavior and stress testing. The absence of a microbiota dramatically altered locomotor and anxiety-related behavior. Additionally, characteristic responses to an acute stressor were also obliterated in larvae lacking exposure to microbes. Lastly, treatment with the probiotic Lactobacillus plantarum was sufficient to attenuate anxiety-related behavior in conventionally-raised zebrafish larvae. These results underscore the importance of the microbiota in communicating to the CNS via the microbiome-gut-brain axis and set a foundation for using zebrafish larvae for neuroimmune research.

K Homology Domains of the Mouse Polycystic Kidney Disease-Related Protein, Bicaudal-C (Bicc1), Mediate RNA Binding in vitro

Background/Aims: The mouse Bicc1(mBicc1) gene is the orthologue of the DrosophilaBicaudal-C(Bic-C) gene. While the role of Bicc1 in the mouse is unknown, mutations in the mouse Bicc1 gene are associated with polycystic kidney disease (PKD). The mBicc1 protein contains three K homology (KH) domains. Evidence from other KH domain-containing proteins as well as studies involving both Drosophila and Xenopus Bic-C, suggest that this motif is important in interactions with RNA. Methods: RNA-binding assays were used to test whether mouse Bicc1 binds homoribopolymers in vitro. A series of constructs coding for different regions of the mBicc1 protein were used to determine which regions of the mBicc1 protein were important for in vitro RNA binding. Results: Mouse Bicc1 binds homoribopolymers in vitro and the third KH domain is necessary and sufficient for in vitro RNA binding. The mutation responsible for PKD in the jcpk mouse model results in a protein that is incapable of binding RNA in vitro. Conclusions: This study demonstrates that mouse Bicc1, a protein associated with PKD, has the ability to bind RNA in vitro. Disruption of this binding capability may be responsible for cyst formation in animals carrying mutations in the mBicc1 gene.

The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

The pathogenesis of inflammatory bowel diseases (IBD), Crohns disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with Helicobacter hepaticus triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4+ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.