Microphthalmia Gene Expression in the Vitiligo Mutant Mouse and its Effect on Neural Retinal Adhesion and Photoreceptor Cell Death

Hdl Handle:
http://hdl.handle.net/10675.2/344239
Title:
Microphthalmia Gene Expression in the Vitiligo Mutant Mouse and its Effect on Neural Retinal Adhesion and Photoreceptor Cell Death
Authors:
Bora, Naba
Abstract:
The murine microphthalmia gene (Mitf) encodes a basic-helix-loop-helix transcription factor known to regulate transcription of genes encoding proteins of the pigmentation pathway. It is also thought to promote pigment cell survival and development. One of the least severe M itf mutations occurs in the vitiligo mouse (M itfvit) which has a G to A point mutation in the region of M itf encoding the first a-helix. This mutant has a normal eye size unlike many of the other mutant alleles and exhibits uneven pigmentation of the retinal pigment epithelium (RPE) and slow, progressive photoreceptor cell (PC) loss. Events leading to the retinal degeneration have not been elucidated. The purpose of the present study was three-fold. First, to determine whether apoptosis was the underlying mechanism of photoreceptor cell death in M itf’1. Second, to assess the adhesion between neural retina and RPE of the M itf1 allele since retinal detachment is known to induce PC cell apoptosis. Third, to establish the normal spatio-temporal expression o f the M itf gene in wild-type mice and to determine whether this pattern was disrupted in vitiligo mutant mice. To quantitate the level of PC apoptosis, the TUNEL assay was used to label cleaved chromatin, a hallmark of apoptosis. Results indicated that the number of apoptotic PCs was significantly higher in M it f 1 during the initial stages o f the degeneration and remained higher than wild-type mice. These findings were confirmed by transmission electron microscopy. To address RPE/neural retina adhesiveness, a peeling experiment was performed in which the RPE was examined for the amount of pigment adherent to it as a direct assessment of strength o f adhesion. Scanning electron microscopy was used to examine the level of damage sustained during the separation process. Results indicated that adhesion o f the neural retina in M itf* was diminished greatly in vivo compared to wild-type. In addition, using an in vitro RPE/neural retina recombination assay, it was shown that, surprisingly, mutant RPE could attach similarly to control and mutant retinas at an early age, but not at later ages. Finally, to quantify Mitf expression, RT-PCR was performed and demonstrated a transient elevation of Mitf between E10.5-E13.5. in the Mitfil mutant. In situ hybridization analysis of wild-type mice localized Mitf to the neuroepithelium during onset of optic vesicle formation (E9.0-9.5) and subsequently to the RPE during optic cup formation (E10.0-E11.5). The findings of this third study provided evidence that the Mitf gene is expressed early during ocular development underscoring its importance in specifying the RPE. Its specific expression in the RPE also provides molecular evidence that the primary site of the Mitf" defect is the RPE, a notion that had been hypothesized by several groups but not demonstrated. These studies suggest that the abnormal RPE defects are due to developmental changes as reflected by the transient elevation of Mitf expression in the RPE early in its formation. These defects render the RPE unable to form a normal interaction with the neural retina which eventually detaches from the RPE leading to loss of photoreceptor cells by apoptosis.
Affiliation:
Department of Cellular Biology and Anatomy
Issue Date:
Dec-1997
URI:
http://hdl.handle.net/10675.2/344239
Additional Links:
http://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304463594?accountid=12365
Type:
Dissertation
Appears in Collections:
Department of Cellular Biology and Anatomy Theses and Dissertations; Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorBora, Nabaen
dc.date.accessioned2015-02-06T20:19:25Z-
dc.date.available2015-02-06T20:19:25Z-
dc.date.issued1997-12-
dc.identifier.urihttp://hdl.handle.net/10675.2/344239-
dc.description.abstractThe murine microphthalmia gene (Mitf) encodes a basic-helix-loop-helix transcription factor known to regulate transcription of genes encoding proteins of the pigmentation pathway. It is also thought to promote pigment cell survival and development. One of the least severe M itf mutations occurs in the vitiligo mouse (M itfvit) which has a G to A point mutation in the region of M itf encoding the first a-helix. This mutant has a normal eye size unlike many of the other mutant alleles and exhibits uneven pigmentation of the retinal pigment epithelium (RPE) and slow, progressive photoreceptor cell (PC) loss. Events leading to the retinal degeneration have not been elucidated. The purpose of the present study was three-fold. First, to determine whether apoptosis was the underlying mechanism of photoreceptor cell death in M itf’1. Second, to assess the adhesion between neural retina and RPE of the M itf1 allele since retinal detachment is known to induce PC cell apoptosis. Third, to establish the normal spatio-temporal expression o f the M itf gene in wild-type mice and to determine whether this pattern was disrupted in vitiligo mutant mice. To quantitate the level of PC apoptosis, the TUNEL assay was used to label cleaved chromatin, a hallmark of apoptosis. Results indicated that the number of apoptotic PCs was significantly higher in M it f 1 during the initial stages o f the degeneration and remained higher than wild-type mice. These findings were confirmed by transmission electron microscopy. To address RPE/neural retina adhesiveness, a peeling experiment was performed in which the RPE was examined for the amount of pigment adherent to it as a direct assessment of strength o f adhesion. Scanning electron microscopy was used to examine the level of damage sustained during the separation process. Results indicated that adhesion o f the neural retina in M itf* was diminished greatly in vivo compared to wild-type. In addition, using an in vitro RPE/neural retina recombination assay, it was shown that, surprisingly, mutant RPE could attach similarly to control and mutant retinas at an early age, but not at later ages. Finally, to quantify Mitf expression, RT-PCR was performed and demonstrated a transient elevation of Mitf between E10.5-E13.5. in the Mitfil mutant. In situ hybridization analysis of wild-type mice localized Mitf to the neuroepithelium during onset of optic vesicle formation (E9.0-9.5) and subsequently to the RPE during optic cup formation (E10.0-E11.5). The findings of this third study provided evidence that the Mitf gene is expressed early during ocular development underscoring its importance in specifying the RPE. Its specific expression in the RPE also provides molecular evidence that the primary site of the Mitf" defect is the RPE, a notion that had been hypothesized by several groups but not demonstrated. These studies suggest that the abnormal RPE defects are due to developmental changes as reflected by the transient elevation of Mitf expression in the RPE early in its formation. These defects render the RPE unable to form a normal interaction with the neural retina which eventually detaches from the RPE leading to loss of photoreceptor cells by apoptosis.en
dc.relation.urlhttp://ezproxy.augusta.edu/login?url=http://search.proquest.com/docview/304463594?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectRetinal degenerationen
dc.subjectCell Deathen
dc.subjectapoptosisen
dc.subjectNeural-retinal pigment epithelium adhesionen
dc.subjectMitf alleleen
dc.titleMicrophthalmia Gene Expression in the Vitiligo Mutant Mouse and its Effect on Neural Retinal Adhesion and Photoreceptor Cell Deathen
dc.typeDissertationen
dc.contributor.departmentDepartment of Cellular Biology and Anatomyen
dc.description.advisorSmith, Sylviaen
dc.description.committeeCaldwell, Ruth; Liou, Gregory; Marcus, Dennis; Schoenlein, Patricia; Wrenn, Roberten
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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