New advances in high throughput molecular analysis are enabling researchers to investigate how the hundreds of different neuronal cell types arise and develop in the brain. While we have known about the basic rules underlying the development of the nervous system for some time, these techniques are providing insights into the genetic control of the development of individual cell types. 

Research published in IBRO journal Neuroscience explores neuronal development in the retina, which is an outpost of the brain within the eye. The study focuses on retinal ganglion cells (RGCs), the output neurons of the retina, which transmit visual information from the eye to the rest of the brain. It investigates which genes change their levels of activity as RGCs specialize into several types and mature. The study, conducted in mice, also addresses an intriguing question – to what extent is the maturation of RGCs controlled by a mouse’s environment. Nature vs nurture, in other words. The research has implications not only for our understanding of neural development at a cellular level, but also how we might treat the most common cause of irreversible blindness in the world – glaucoma.

The research, conducted by researchers in the US, appeared in a special issue of Neuroscience honoring German neuroscientist Professor Friedrich Bonhoeffer, who in the 1970s and 1980s pioneered research on nervous system development. Step forward a few decades and in this new study, researchers at Harvard University and University of California, Berkeley (UC Berkeley) used high-throughput single-cell RNA sequencing to understand the complex pattern of gene expression that controls RGC development. 

Single-cell RNA sequencing allows researchers to find out which genes are expressed at single-cell resolution for tens of thousands of cells in parallel and at low cost. To measure gene expression, the researchers analyzed the amounts of different types of messenger RNA (mRNA) produced by genes in mouse RGCs at different moments in time while they matured. RGCs were taken from mice at six different times – three when they were inside the embryo and three after birth. These snapshots of cellular development revealed which genes turned on and off as the cells became specialized and matured, elaborating their structures and becoming functionally active.

Dr. Karthik Shekhar

This sequencing provided an inventory of 1707 genes whose expression changes as RGCs develop, with some of these thought to control the distinctive properties of different RGC types. “The early stages appear to be enriched for transcription factors, proteins that bind to specific regions of the DNA and regulate other genes,” says Dr. Karthik Shekhar, Professor in Chemical and Biomolecular Engineering at UC Berkeley. “Later on, there is an enrichment for genes encoding cell surface proteins. These are the proteins that, among other things, allow particular cells to make synaptic contacts with a specific subset of other cells.” Genes that code for neurotransmitters also became active later in cell development. 

Mice have roughly 45 different types of RGCs, each attuned to a small subset of features in the visual scene, such as objects moving in a particular direction or the edges of objects. “Retinal ganglion cells parse the visual scene into parallel representations,” says Dr. Irene Whitney, co-first author and Senior Director of Commercial Strategy at Honeycomb Biotechnologies. “Then higher up in the brain that all gets compiled back together.” Some of the genes identified were expressed in many different retinal ganglion cell types, whereas others were only expressed in one, or a small number of types. Whitney conducted this study as a postdoctoral associate in the laboratory of Dr Joshua R. Sanes at Harvard University. Sanes and Shekhar are the joint senior authors in this study.

Dr. Irene Whitney

Then there is the question of the extent to which light affects these processes. “I think intuitively, one might imagine that it does require some sort of external cues to develop that much diversity in retinal ganglion cells,” says Whitney. To find out, the researchers cut off light to the mouse RGCs in three ways. In one, mice were housed in darkness after they were born and in the two others, mice were genetically modified so they did not have photoreceptors or bipolar neurons, cells which connect photoreceptors to RGCs. The researchers found that all three means of removing light did not affect the specialization of the ganglion cells. 

“All 45 different flavors of RGCs were present in the correct proportions in retinae that have been deprived of light in three different ways,” says Shekhar. “Often there is a bias among scientists that negative results are disappointing. But here the negative result is actually quite significant because we are talking about 45 different ganglion cell types and we have found that their development is largely reproducible, even with such seemingly extreme deprivation of early visual input.” 

Salwan Butrus

There were, however, many subtle gene expression changes to the RGCs in the mice with visual deprivation. “Subtle alterations of developmental programs can still have sharp changes in cell properties,” says Shekhar. Exactly how those changes alter the function of RGCs would require further research. Another unanswered question is how the gene expression changes during RGC development are orchestrated in the DNA. “We don’t understand what changes to the DNA are occurring that are telling the cell to make more of this RNA at this time, less of this RNA at this time,” says Salwan Butrus, a doctoral student in Shekhar’s lab, and a co-first author of the paper.

The research has important clinical implications. In glaucoma, a disease that impacts more than 80 million people worldwide, RGCs die leading to progressive and irreversible vision loss. Therapies that can protect RGCs from dying or regenerate dead RGCs do not currently exist. In the future, the genes found to control the development of these cells could potentially be used to reprogram replacement RGCs, restoring vision. 

Shekhar says publishing with Neuroscience, including the review system, was a smooth process: “I think it’s always good when the criticisms of the paper are constructive, which helped us improve the analysis.” The fact that the study appeared in a special issue in honor of Friedrich Bonhoeffer was also fitting. “Friedrich Bonhoeffer was one of the giants of neural development who actually thought about these questions classically, so we were particularly pleased to submit this study in his honor.”

This article was written by Dr. Andy Ridgway.

About Neuroscience

Established in 1976, Neuroscience is the flagship journal of IBRO. The journal features papers describing the results of original research on any aspect of the scientific study of the nervous system. Papers of any length are considered for publication provided that they report significant, new, and carefully confirmed findings with full experimental details. Together with IBRO Neuroscience Reports, IBRO’s open access journal, Neuroscience plays a crucial role in supporting the organization’s global neuroscience activities, as ​​proceeds from both journals support more than 90% of IBRO’s initiatives.

Learn more about Neuroscience.