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-Usher syndrome type I (USH1) is a hereditary sensorineural deafness combined with retinitis pigmentosa and is the most frequent cause of hereditary deafness-blindness in humans. Planar cell polarity (PCP) describes the proper orientation of cells within a sheet during development through convergent extension. Usher genes are related to the machinery that develops this polarity. Vangl2 is a known core-PCP gene in the conserved PCP pathway, with a mutant form known as Looptail. Pcdh15 mutants crossed with Looptail mice should show an enhanced mutant phenotype if there is molecular interaction between the two pathways during the establishment of PCP within the mouse auditory sensory organ. It was hypothesized that the Pcdh15 mutants would show defects in hair cell patterning orientation indicative of PCP machinery defects. The inner ears of mice mutant for pcdh15 (one of the genes responsible for USH1), Looptail, and double heterozygous mutants were analyzed using immunostaining and confocal microscopy to determine any disruption in PCP. Images of pcdh15 mutant inner ears revealed little hair cell misorientation in the organ of Corti, but more severe misorientation within the vestibular organs. This mutant phenotype was enhanced in the double heterozygous mutants. Since the vestibular organs are related to balance, behavioral tests such as rotor rod, swim test, and a tail-hanging test were used to determine the severity of any vestibular malfunction. Little or no conclusions can be drawn from behavioral test data at this point, but preliminary data shows a possible change in proper cell patterning, orientation, and association of the kinocilium with the stereocilia bundle in the cochlea of Pcdh15 mutants that is enhanced by the Looptail mutation.
-The cochlea duct of the mammalian inner ear contains the auditory sense organ, the organ of Corti.
-Planar cell polarity (PCP) refers to the coordinated orientation of cells along an axis parallel to the plane of the cell sheet. In vertebrates, the inner ear sensory organs display distinctive forms of PCP.
-One of the inner ear PCP characteristics is the coordinated positioning of a primary cilium eccentrically in every sensory hair cell within each organ.
-The inner ear, therefore, provides an opportunity to explore the cellular role of primary cilia in PCP signaling.
-The intrinsic polarity of hair cells is marked by the position of the kinocilium and the polarity of the stereociliary bundle.
-Usher syndrome type I (USH1) is a hereditary sensorineural deafness combined with retinitis pigmentosa and is the most frequent cause of hereditary deafness-blindness in humans.
-There are three clinical subtypes, USH1-3, according to the severity and onset of the hearing impairment, vestibular dysfunction, and retinitis pigmentosa.
-Each USH sybtype is genetically heterogeneous, and at least 12 chromosomal loci are assigned to USH.
-USH1F is caused by a cadherin-related protein protocadherin 15.
-Looptail is a mutant form of the known core-PCP gene Vangl2.
-Looptail heterozygous mice crossed with pcdh15 usher mutant mice will show an enhanced phenotype if the two are both within the same molecular pathway.
-Mouse models for USH1 and USH2D linked USH1 and USH2D genes to the development of hair cell stereociliary bundles.
-USH gene studies has implicated a common genetic pathway for ciliary genes and USH genes in the determination of intrinsic cellular polarity.
-The stereocilia phenotype in USH1 mutant mice includes the replacement of the V-shaped stereociliary bundle by clusters of stereocilia, the presence of circularly shaped steroecilia, and the loss of graded heights of stereocila, and the reduction of lateral links between stereocilia.
Pcdh15 Mice:
-Pcdh15 mice used in these experiments arise from a natural point insertion of an adenosine at position 4521 in the coding region of the pcdh15 gene. The colony was started from a breeding pair obtained from the Jackson Laboratory.
Looptail Mice:
-Looptail mice used in these experiments arise from a transition point mutation changing a G to an A at position 1391. This colony was also started from a breeding pair obtained from the Jackson Laboratory.
Phenotypic Tests:
-Mouse inner ears were fixed in 4% PFA and then the auditory sensory organs were stained with conjugated Alexa 488-phalloidin and rhodemine-goat anti-rabbit as a secondary antibody to the rabbit-derived arl13b kinocilium antibody.
-Confocal imaging was performed on a Zeiss LSM Meta confocal microscope.
Behavioral Tests:
-Mice were video-recorded swimming. Severity of vestibular defect was determined by the ability of the mice to swim normally, swim normally with the aid of the edge of the container, or not swim normally at all.
-Mice were held by their tails for twenty seconds and video-recorded. Severity of vestibular defect was determined by behavior such as whipping of the head and clasping of the hind paws.
Pcdh15 mutants show hair cell misorientation in the cochlea and vestibule
Pcdh15 mutants show dissociation of the kinocilia from the stereociliary bundle in the cochlea
Pcdh15 mutants show disrupted cell patterning in the cochlea
Pcdh15 mutants show abnormal behavior in swimming tests
Pcdh15 mutants show abnormal behavior in tail- hanging tests
-Pcdh15 appears to be a core PCP mutant rather than a ciliary mutant, because it causes defects in cell patterning and orientation rather than improper formation of the stereociliary bundle.
-Further support for a PCP role of Pcdh15 is shown by the enhancement of the mutant phenotype when crossed with Looptail, the known core PCP gene mutant.
-In order to gain more statistical significance for quantified data, more samples need to be analyzed using confocal microscopy.
-Data from behavioral tests is difficult to quantify currently and methods for less subjective analysis need to be determined.
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No.52005873 and the Student Inquiry Research Experience award from the Office of Undergraduate Studies, Emory College.
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