Study First to Define Factors Determining Retinal Cell Type

“This is the first time determinant factors have been defined for any retinal cell type,” says senior author Xiuqian Mu, MD, PhD, associate professor of ophthalmology.

Because the study is based on the neural retina as a model system, Mu’s findings may have broader implications. They may lend insight into genetic mechanisms that form diverse cell types throughout the central nervous system (CNS).

“What we learned may be able to be applied or extended to fundamental processes in other parts of the CNS,” Mu says.

Also, the study may help focus and direct efforts to develop stem cell therapies for retinal disease.

The researchers sought to define how retinal progenitor cells — the forerunner of all seven retinal cell types — decide to adopt an eventual fate as retinal ganglion cells.

“We found a clear and definitive answer,” says Mu, also a faculty member in biochemistry and neuroscience and genetics, genomics and bioinformatics programs.

Mu and his team discovered that two gene-regulating proteins suffice: the POU domain, class 4, transcription factor 2 (Pou4f2) and the insulin gene enhancer protein 1 (Isl1).

Like all transcription factors, these proteins bind to DNA and dictate which genes are turned on, or expressed. 

“Our study shows these two factors not only specify, or determine, the retinal ganglion cell fate, they activate the whole gene expression program required for progenitor cells to differentiate into ganglion cells,” Mu says.

In their quest to identify the genes involved in the generation of a cell type, Mu and his team tested their ideas in the developing retinas of mouse embryos.

After identifying Pou4f2 and Isl1 as likely candidates to specify ganglion cell fate, they used genetic tools to modify the structure of the mouse genome.

They knew that the Atoh7 gene is required, but not sufficient, for retinal ganglion cells to form. “So we used a genetic trick to take away Atoh7 and express the two candidate genes at the same time,” he explains. 

To achieve this, the researchers created two mouse alleles or gene variants in their artificial binary knockin-transgenic system.

We asked: “Can these two genes generate retinal ganglion cells? Are they capable of specifying the cell fate?” Mu says. “The answer was yes.”

The retinal ganglion cells generated in the experiments “are largely normal in gene expression, survive to postnatal stages and are physiologically functional,” Mu adds.

Unlike this work, previous studies have identified transcription factors by analyzing loss and gain of function. “But those studies often fail to reveal the specific roles those factors play in the development of cell types,” says Mu.

Understanding the genetic mechanisms that form and maintain retinal ganglion cells may lead to clues about why they die, Mu notes. Death of these cells is involved in retinal diseases — including glaucoma — that cause vision loss and blindness.

These diseases affect millions, Mu notes.

His findings may help scientists develop cell-based treatments by guiding efforts to efficiently generate retinal ganglion cells from stem cells.

This is especially important because retinal cells — or the vast majority of cell types in the central nervous system — can’t regenerate. “Once they are specified, they are fixed for life,” Mu explains. “If they are damaged, we can’t grow or replace them naturally.”

The paper, “Two Transcription Factors, Pou4f2 and Isl1, are Sufficient to Specify the Retinal Ganglion Cell Fate,” has been published as a PNAS Plus article by the Proceedings of the National Academy of Sciences. This type of article is longer and more detailed than the standard papers the journal publishes.

Other UB co-authors are:

From the Department of Ophthalmology:

• First author Fuguo Wu, PhD, research scientist
• Renzhong Li, PhD, former postdoctoral associate

• Malcolm M. Slaughter, PhD, professor

From UB’s Neuroscience Program:

• second author Tadeusz J. Kaczynski, PhD student
• Santhosh Sethuramanujam, PhD ’14
• Varsha Jain, former visiting scholar

The study was funded by the National Eye Institute, the Whitehall Foundation and a Research to Prevent Blindness unrestricted grant to the Department of Ophthalmology.