Mutation in Bmpr1b Leads to Optic Disc Coloboma and Ventral Retinal Gliosis in Mice.

Purpose: The clinical phenotype of retinal gliosis occurs in different forms; here, we characterize one novel genetic feature, (i.e., signaling via BMP-receptor 1b). Methods: Mouse mutants were generated within a recessive ENU mutagenesis screen; the underlying mutation was identified by linkage analysis and Sanger sequencing. The eye phenotype was characterized by fundoscopy, optical coherence tomography, optokinetic drum, electroretinography, and visual evoked potentials, by histology, immunohistology, and electron-microscopy. Results: The mutation affects intron 10 of the Bmpr1b gene, which is causative for skipping of exon 10. The expression levels of pSMAD1/5/8 were reduced in the mutant retina. The loss of BMPR1B-mediated signaling leads to optic nerve coloboma, gliosis in the optic nerve head and ventral retina, defective optic nerve axons, and irregular retinal vessels. The ventral retinal gliosis is proliferative and hypertrophic, which is concomitant with neuronal delamination and the reduction of retinal ganglion cells (RGCs); it is dominated by activated astrocytes overexpressing PAX2 and SOX2 but not PAX6, indicating that they may retain properties of gliogenic precursor cells. The expression pattern of PAX2 in the optic nerve head and ventral retina is altered during embryonic development. These events finally result in reduced electrical transmission of the retina and optic nerve and significantly reduced visual acuity. Conclusions: Our study demonstrates that BMPR1B is necessary for the development of the optic nerve and ventral retina. This study could also indicate a new mechanism in the formation of retinal gliosis; it opens new routes for its treatment eventually preventing scar formation in the retina.

The First Scube3 Mutant Mouse Line with Pleiotropic Phenotypic Alterations.

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

Generation and Standardized, Systemic Phenotypic Analysis of Pou3f3L423P Mutant Mice.

Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.

Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels.

Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in hematological parameters.

Research on hematological disorders relies on suitable animal models. We retrospectively evaluated the use of the hematological parameters hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT) in the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project as parameters for the generation of novel animal models for human diseases. The analysis was carried out on more than 16,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the levels of the chosen parameters. Identification of animals exhibiting altered values and transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters MCV, RBC, and PLT. Analysis of the causative mutation was started in selected lines, thereby revealing a novel mutation in the transferrin receptor gene (Tfrc) in one line. Thus, novel phenotype-driven mouse models were established to analyze the genetic components of hematological disorders.

N-ethyl-N-nitrosourea mutagenesis produced a small number of mice with altered plasma electrolyte levels.

BACKGROUND: Clinical chemical blood analysis including plasma electrolytes is routinely carried out for the diagnosis of various organ diseases. Phenotype-driven N-ethyl-N-nitrosourea (ENU) mouse mutagenesis projects used plasma electrolytes as parameters for the generation of novel animal models for human diseases. METHODS: Here, we retrospectively evaluated the use of the plasma electrolytes calcium, chloride, inorganic phosphorus, potassium and sodium in the Munich ENU mouse mutagenesis project where clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in various plasma parameter levels. RESULTS: We identified a small number of animals consistently exhibiting altered plasma electrolyte values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for the parameters calcium and potassium. Published data from other phenotype-driven ENU projects also included only a small number of mutant lines which were generated according to altered plasma electrolyte levels. CONCLUSION: Thus, use of plasma electrolytes detected few mouse mutants in ENU projects compared to other clinical chemical blood parameters.

Generation of N-ethyl-N-nitrosourea-induced mouse mutants with deviations in plasma enzyme activities as novel organ-specific disease models.

Measurement of plasma enzyme activities is part of routine medical examination protocols and provides valuable parameters for the diagnosis of various organ diseases. In the phenotype-driven Munich N-ethyl-N-nitrosourea (ENU) mouse mutagenesis project, clinical chemical blood analysis was carried out on more than 20,000 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, alpha-amylase and creatine kinase. We identified a large number of animals that consistently exhibited altered plasma enzyme activities. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of mutant lines for each parameter. Breeding experiments in selected lines detected the linkage of the causative mutations to defined chromosomal regions. Subsequently, identification of the mutated genes was successfully carried out in chosen lines, resulting in a novel alkaline phosphatase liver/bone/kidney (Alpl) alteration in one line and the strong indication for a dystrophin (Dmd) alteration in another line. The mouse mutants with abnormal plasma enzyme activities recovered in the Munich ENU project are novel tools for the systematic dissection of the pathogenesis of organ diseases.