Preserving the Precious Gift of Sight

Milan Jamrich, PhD

LAWRENCE RESEARCH PROJECT

Molecular and Cellular Biology

Baylor College of Medicine

Houston, TX

 

Dr. Jamrich’s Research Project

Function of Rx in the Specification, Differentiation and Survival of Vertebrate Retinal Cells

Current Research Interests

The goal of this project is to identify genes and developmental processes that are responsible for development and survival of vertebrate retinal cells. Identification of these genes and molecular processes will lead to the better understanding of eye diseases. During eye development the undifferentiated cells of the retina develop into a layered array of cell types with specific capabilities. These include the light sensitive photoreceptor cells, the bipolar interneuron cells, and the ganglion cells that transmit the information from the eye to the brain. The retinal gene Rx, initially isolated in Dr. Jamrich’s laboratory, plays a critical role in the vertebrate eye development and is also expressed in adult retinal cells including Muller glial cells and photoreceptor cells. There is a possibility that Rx genes might play a role in the survival of photoreceptor cells, and Dr. Jamrich has decided to investigate the role of Rx in adult retinal cells.

Progress in 2016

Dr. Jamrich has investigated the role of mouse Mrx gene in the survival of retinal cells. He has conditionally eliminated Rx expression in postmitotic retinal cells of mice using the Cre-floxP recombination system and monitored the survival of retinal cell types during the lifespan of the animal. There was no change found in the morphology or survival of retinal cells after the post-natal day 4 all the way to 6 months old animals. This strongly suggests that Mrx has no influence on the survival of the photoreceptors in adult mice. If Mrx were required for the survival or morphology of rod cells, a reduction of photoreceptor cells in comparison to the wild type retina after post-natal day 4 would have been observed. This was not the case.

Plans for 2017

Dr. Jamrich will identify the direct target genes of Mrx during retinal development by large-scale chromatin immunoprecipitation combined with sequencing. He will initially perform ChIP-sequencing on isolated E12.5 eyes from the Rx/WBP/FLAG knock in strain of mouse that he made during the previous funding period. This stage was selected because at this time point Rx is abundantly expressed in the embryonic eye and the eyes can be easily isolated. Chromatin immunoprecipitation will be performed. Isolated DNA fragments will be sequenced and potential Rx target genes will be identified using duplicate sets of ChIP-Seq experiments.

Progress in 2015

Dr. Jamrich made a mouse line in which the Rx protein is tagged with FLAG tags. Using antibodies against the FLAG tag, he was able to demonstrate expression of the tagged Rx protein in sections of embryonic eyes. Furthermore, he was able to purify the tagged Rx protein from embryonic extracts using the anti-FLAG antibodies. As a next step he has used the anti-FLAG antibodies to identify the direct target genes of Rx (targetome) by large-scale chromatic immunoprecipitation (ChIP) combined with sequencing. This was successful and he has identified several novel Rx target genes. He has begun to analyze the protein-protein interaction experiments, but has not yet been able to complete the Mass Spectroscopy experiments.

Progress in 2014

The goal of this project is to identify genes and developmental processes that are responsible for development and survival of vertebrate retinal cells. Identification of these genes and molecular processes will lead to the better understanding of eye diseases. Dr. Jamrich’s laboratory made genetically modified mice that allow determining whether there is a direct binding between Rx and other proteins. He has generated the N-tagged Rx by inserting the 3XFLAG and SBP sequences immediately downstream of the Rx ATG initiation codon using gene targeting. He made heterozygous ES cells that were injected into mouse blastocysts and has obtained several chimeric mice that contained the mutant, pigmented cells that contain the tagged Rx locus. As the FLAG antibodies recognize the flagged-Rx protein, Dr. Jamrich’s laboratory is now in process of identifying Rx interacting proteins.

Progress in 2013

To test the possibility that Rx acts during retinal development by interacting with other known transcriptional regulators, Dr. Jamrich analyzed genetic interactions between Rx and other transcription factors known to be involved in early steps of retinal formation. In these experiments, he took advantage of the fact that while Rx-deficient embryos have no eyes, the Rx heterozygous animals have no obvious eye phenotype. Dr. Jamrich crossed Rx heterozygous mice to mice that were heterozygous for mutations in other transcription factors. He argued that if two transcription factors interact, then the double heterozygous animals would have a more pronounced phenotype than their siblings that are heterozygous for only one of the two transcription factors. Using this approach, he found genetic evidence that Rx interacts with the transcription factor Lhx2.

Progress in 2012

To test the possibility that Rx acts during retinal development by interacting with other known transcriptional regulators, Dr. Jamrich began to analyze genetic interactions between Rx and other transcription factors known to be involved in early steps of retinal formation. In these experiments, he took advantage of the fact that while Rx-deficient embryos have no eyes, the Rx heterozygous animals have no obvious eye phenotype. Dr. Jamrich crossed Rx heterozygous mice to mice that were heterozygous for mutations in other transcription factors. Using this approach, he found genetic evidence that Rx interacts with the transcription factor Lhx2. While the mode of action of Lhx2 is not known in detail, it has been shown that this gene is required for the specification and the morphogenesis of the retinal field. There is strong genetic evidence that Rx and Lhx2 interact.

The goal of this project is to identify genes and developmental processes that are responsible for development and survival of vertebrate retinal cells.  Identification of these genes processes will lead to the better understanding of eye diseases, resulting in new diagnostic procedures and treatments of eye diseases.

During eye development, the initially undifferentiated cells of the retina develop into a layered array of cell types with specific capabilities. The retinal gene Rx, initially isolated in our laboratory, plays a critical role, as retinal cells do not form in species in which this gene does not function correctly. How Rx functions at the molecular level is not known at present. It is the aim of this study to determine the mode of action of Rx gene during formation and survival of retinal cells.

Dr. Jamrich’s genetic experiments suggest that the transcription factor Rx interacts with the transcription factor Lhx2. It is his laboratory’s goal to determine the reason for abnormal development of the optic cup.

In principle, there could be three reasons for the abnormal morphology of the optic cup. First, the ventral and dorsal region of the optic cup may not be properly specified. To determine whether the dorsal and ventral region of the cup is properly specified, they will monitor expression of the ventral and dorsal marker genes like Tbx5, Pax6, Pax2 and Vax2.

If the ventral and dorsal region of the optic cup is properly specified, then there are two other possible explanations: the reduced rate of proliferation in the ventral region and the increased rate of apoptosis in the ventral optic cup. To determine whether proliferation or apoptosis are affected in Rx/Lhx2 heterozygous mice, Dr. Jamrich will analyze cell proliferation by using antibodies against phosphohistone H3. These experiments will show which molecular events are affected in Rx/Lhx2 heterozygous animals and will allow the design of further experiments addressing the function of the Rx gene in development and survival of retinal cells.

Expression of the Pax6 – lacZ reporter in the head of an E16.5 wild type mouse
embryo

Progress in 2011

The goal of the project during 2011 was to determine whether the two human RAX genes could rescue eye formation in mice lacking the mouse Rx gene. We made transgenic mice using two RAX genes. While our transgenic mouse line was carrying the human RAX1 gene, this gene was not expressed in the transgenic wild type mouse. RAX2 was expressed in the transgenic mouse, but this gene was not able to rescue eye formation in Rx deficient mice.

Vertebrate retinal formation is a complex process that requires the formation of the anterior neural plate as well as the specification and differentiation of retinal cells. This requires the interplay of several genes essential for eye formation.

Dr. Jamrich’s laboratory has shown previously that Rx, a paired like homeobox gene, has a critical role in vertebrate eye formation, as mice missing Rx function do not develop eyes. Furthermore, their preliminary evidence suggests that Rx also has a role in the survival of differentiated retinal cells. Since Rx gene appear to be the key gene in vertebrate eye formation, Dr. Jamrich is examining the molecular network that mediates Rx function in the specification, differentiation and survival of vertebrate retinal cells.

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Mission of RRF

The mission of the Retina Research Foundation is to reduce retinal blindness worldwide by funding programs in research and education. As a public charity, RRF raises funds from the private sector and the investment of its endowment funds.

RRF’s 48th Anniversary

October 1, 2017