Myosin 6 Is Required for Iris Development and Normal Function of the Outer Retina

PURPOSE. To determine the molecular basis and the pathologic consequences of a chemically induced mutation in the translational vision research models 89 (tvrm89) mouse model with ERG defects. METHODS. Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened for behavioral abnormalities and defects in retinal function by ERGs. The chromosomal position for the recessive tvrm89 mutation was determined in a genome-wide linkage analysis. The critical region was refined, and candidate genes were screened by direct sequencing. The tvrm89 phenotype was characterized by circling behavior, in vivo ocular imaging, detailed ERG-based studies of the retina and RPE, and histological analysis of these structures. RESULTS. The tvrm89 mutation was localized to a region on chromosome 9 containing Myo6. Sequencing identified a TC point mutation in the codon for amino acid 480 in Myo6 that converts a leucine to a proline. This mutation does not confer a loss of protein expression levels; however, mice homozygous for the Myo6 tvrm89 mutation display an abnormal iris shape and attenuation of both strobe-flash ERGs and direct-current ERGs by 4 age weeks, neither of which is associated with photoreceptor loss. CONCLUSIONS. The tvrm89 phenotype mimics that reported for Myosin6-null mice, suggesting that the mutation confers a loss of myosin 6 protein function. The observation that homozygous Myo6 tvrm89 mice display reduced ERG a-wave and b-wave components, as well as components of the ERG attributed to RPE function, indicates that myosin 6 is necessary for the generation of proper responses of the outer retina to light

Role of CysE in Production of an Extracellular Signaling Molecule in Providencia stuartii and Escherichia coli: Loss of cysE Enhances Biofilm Formation in Escherichia coli

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-l-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-l-serine [NAS], O-acetyl-l-threonine, and N-acetyl-l-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4- to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal

Myosin 6 is required for iris development and normal function of the outer retina.

PURPOSE: To determine the molecular basis and the pathologic consequences of a chemically induced mutation in the translational vision research models 89 (tvrm89) mouse model with ERG defects. METHODS: Mice from a G3 N-ethyl-N-nitrosourea mutagenesis program were screened for behavioral abnormalities and defects in retinal function by ERGs. The chromosomal position for the recessive tvrm89 mutation was determined in a genome-wide linkage analysis. The critical region was refined, and candidate genes were screened by direct sequencing. The tvrm89 phenotype was characterized by circling behavior, in vivo ocular imaging, detailed ERG-based studies of the retina and RPE, and histological analysis of these structures. RESULTS: The tvrm89 mutation was localized to a region on chromosome 9 containing Myo6. Sequencing identified a T→C point mutation in the codon for amino acid 480 in Myo6 that converts a leucine to a proline. This mutation does not confer a loss of protein expression levels; however, mice homozygous for the Myo6(tvrm89) mutation display an abnormal iris shape and attenuation of both strobe-flash ERGs and direct-current ERGs by 4 age weeks, neither of which is associated with photoreceptor loss. CONCLUSIONS: The tvrm89 phenotype mimics that reported for Myosin6-null mice, suggesting that the mutation confers a loss of myosin 6 protein function. The observation that homozygous Myo6(tvrm89) mice display reduced ERG a-wave and b-wave components, as well as components of the ERG attributed to RPE function, indicates that myosin 6 is necessary for the generation of proper responses of the outer retina to light. Invest Ophthalmol Vis Sci. 2013 Nov 1; 54(12):7223-33

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation.

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB

Light-Evoked Responses of the Retinal Pigment Epithelium: Changes Accompanying Photoreceptor Loss in the Mouse

Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration (PrphRd2/+), which was crossed onto a Nyxnob background to eliminate the b-wave and most other postreceptoral ERG components. On this background, PrphRd2/+ mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in PrphRd2/+ mice than predicted by rod photoreceptor activity (RmP3) or anatomical analysis. At 4 mo of age, the RPE in PrphRd2/+ mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease