Defense Date

2015

Document Type

Thesis

Degree Name

Master of Science

Department

Human Genetics

First Advisor

James Lister, Ph.D

Second Advisor

Rita Shiang, Ph.D

Third Advisor

Sarah Elsea, Ph.D

Abstract

Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal rai1 expression as early as zero hours post fertilization in wild type embryos. Using end-point PCR, we found that in fact there is maternal rai1 expression is detectable as early as 2 hours post fertilization (hpf) in wild type zebrafish embryos. Furthermore, we quantified rai1 expression using qPCR and found that rai1 expression declines significantly after 6 hpf. We hypothesize that a down regulation of rai1 or loss of rai1 will lead to morphological phenotypes, especially if that loss of rai1 function occurs during the earliest stages of zebrafish embryogenesis. Using a rai1morpholino oligonucleotide (MO), we found a loss of rai1 expression did not induce a morphological phenotype in in wild type embryos; furthermore, we also found that a loss of maternal rai1 expression did not induce a morphological phenotype as well. Utilizing a mutant rai1 zebrafish line, we found that both rai1 +/fh370 progeny nor rai1 fh370/fh370 progeny exhibited a morphological phenotype and that downstream targets such as bdnf were not affected by a reduction or complete loss of rai1. Prior research has shown that retinoic acid (RA) can induce rai1 expression. We hypothesize that RA can induce expression of rai1 during zebrafish embryogenesis. Using wild type fish and a rai1 in situ hybridization probe, we found that RA treatment at 25 hpf induced expression of rai1. The construction of a rai1 overexpression vector used for overexpression studies was started. Further development of GFP expression vector and zebrafish rai1 antibody are needed to determine if the morpholino is reducing rai1 protein expression.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-7-2015