John L.R. Rubenstein

John Rubenstein grew up surrounded by science. His father, Edward, was a professor of medicine at Stanford University, whose research interests included clotting disorders and nonprotein amino acids, and who was elected to the National Academy of Medicine. In this environment, Rubenstein later came to know some of his father’s colleagues, which included scientists such as Linus Pauling and Arthur Kornberg,. Rubenstein’s mother who encouraged his scientific interests. His sister and his son both died from birth defects; this inspired him to pursue a research topic that could improve the lives of other children. 

Following in his father’s footsteps, John attended Stanford for his undergraduate degree, where he was able to work with a number of scientists, on a number of different projects. During his undergraduate years, his research projects included studying how the immune system responded to heart transplantation with the physician-scientists Norman Shumway and Randall Morris; researching DNA replication with Arthur Kornberg and William Wickner; as well as projects supervised by the scientists A. T. Ganesan, David Clayton and Douglas Brutlag, that looked at bacteriophage replication and mitochondrial DNA. These projects gave him a well-rounded view of how research is done, as well as what potential projects were available for him to concentrate on. 

During his sophomore year at Stanford, Rubenstein decided to focus on gene regulation in the developing brain, after attending a lecture during his sophomore year as at Stanford. “I saw that it was an important frontier in science,” Rubenstein said. 

However, after talking with a neuroscientist John Nicholls, who was also a family friend, Rubenstein decided to wait to study brain development, due to the fact that the field was not yet advanced enough to study his intended problem. “He told me to wait, that it was premature to study gene regulation in the brain,” Rubenstein said. 

After finishing his undergraduate degree, Rubenstein decided to enroll in an MD/PhD program at Stanford. “I was hoping that the scientific work that I did would be informed by my medical training, to be successful in helping people,” Rubenstein said. “That was one of my goals.” 

As an MD/Ph.D. student, Rubenstein’s research focused on the biophysics of cell membranes, which included studying the dynamics of lipids and proteins, as well as biogenesis. This work was conducted under the guidance of Harden McConnell and James Rothman, and also benefited from the advice and expertise of Hiroto Okayama and Paul Berg. “It was all excellent foundational scientific training,” Rubenstein said. 

Rubenstein then did his postdoctoral training at the Pasteur Institute in Paris, where he worked on developing tools to study developmental biology with Francois Jacob, Jean Francois Nicholas and Josh Sanes. At first, his work had nothing to do with studying the development of the brain, but the tools they developed would later become useful such as antisense RNA. “It was there that we applied retroviral lineage analyses to developmental biology,” Rubenstein said. This technique was first used to do lineage analysis for skin cells, but soon thereafter, Rubenstein turned his attention to neurodevelopment. 

After his postdoctoral training, Rubenstein elected to further his medical training, by doing a residency in adult and child psychiatry at Stanford. During his residency, he turned his attention to studying the development of the mammalian forebrain with mentorship from Roland Ciaranello. Over a period of two years, Rubenstein and his collaborators developed a technique to identify genes that were differentially regulated in the embryonic and adult forebrain.

This technique, called subtractive hybridization, used antisense RNA to identify RNA strands that were expressed in one area of the developing brain, but not another. “It took a long time to get that procedure working properly, but it worked great,” Rubenstein said. 

This led to the discovery of the gene Dlx2, which would form the foundation of much of his later work. “Our experiments showed us that Dlx2 is expressed in a very specific subset of regions in the developing forebrain,” Rubenstein said. This work was greatly aided by collaboration with Luis Puelles.

Rubenstein later identified a second gene, called Tbr1, that was also central to forebrain development. In follow-up experiments, they discovered that the excitatory neurons and the inhibitory neurons were being produced in two separate parts of the brain, that corresponded with  Tbr1 and Dlx2 expression, respectively. “That  was a fundamental discovery,” Rubenstein said. 

As Rubenstein and his collaborators eventually discovered, Dlx2 and Tbr1 are both transcription factors that turn on genes responsible for neuronal cell type specification. Dlx2 directs the specification of inhibitory neurons in the basal ganglia, while Tbr1 directs the specification of excitatory neurons in the cortex. 

Following the discovery that inhibitory and excitatory neurons are produced in two separate parts of the developing forebrain, this raised the question of how these neurons ended up in other parts of the fully developed forebrain. In later experiments, Rubenstein and his collaborators were able to show that a subset of inhibitory neurons migrate from the basal ganglia to the hippocampus and cerebral cortex, during early development. “That’s how we discovered that the basal ganglia was the source of most inhibitory neurons in the cortex and the hippocampus, at least in mice,” Rubenstein said. 

Meanwhile, Rubenstein and his collaborators sought to understand how Dlx2 and Tbr1 direct the development of excitatory and inhibitory neurons. This included identifying the DNA sequences that these transcription factors bind to, called enhancer regions, which then leads to the gene expression that directs the specification of different neuronal cell types. Currently, Rubenstein and his collaborators are taking a systems-wide approach to elucidate gene regulatory networks that orchestrate cell fate specification and differentiation in the developing mammalian forebrain. 

In a 2003 paper, he proposed with the neuroscientist Michael Merzenich a model for autism that hypothesizes that in a subset of individuals, cognitive function is abnormal due to an altered ratio of excitatory and inhibitory (E/I) activity. “Signal to noise detection in the cortex is important for the efficiency of cognitive functions,” Rubenstein said. If there is too much noise, the signal gets overpowered, while not enough signal means that it risks getting lost in the noise. The E/I ratio hypothesis remains just an idea, but it has been influential in framing the experimental approaches and clinical trials of many labs. 

Rubenstein wants to emphasize that all of his work was made possible by collaborations with over 100 trainees and many other Professors. He is particularly proud of the successes of his trainees after they have left his lab and gone onto their own careers.