From the outside looking in, the brain looks rather homogenous. It has folds and creases, some protruding lobes, but really only a handful of features that make it unique upon gross inspection. Taking a closer look (depending on the species) reveals hundreds to thousands, or even one hundred billion neurons that help make up the brain. Taking an even deeper dive, unfolds a richness of cell types that gives the brain an immense amount of diversity. The mechanisms governing cell-type diversity in the brain is poorly understood, but incredibly important. Understanding the genetic programs that make neurons different may help elucidate what went wrong when neurons (and brain structures) become pathologic.
Spearheading the research that investigates the molecular mechanisms governing neuronal cell-type diversity is Oliver Hobert. Professor Hobert has appointments in biological sciences and molecular biophysics at Columbia University, and has the privilege of being a Howard Hughes Medical Institute investigator. Dr. Hobert’s lab uses Caenorhabditis elegans (C. elegans) to take a “bottom-up” approach to elucidate the genetic programs responsible for cell type diversification in the brain. The “Bottom-up” approach is an attempt to define sequences of DNA (AKA the “gene battery”) that specify anatomical and functional properties of cells, and then dissects the regulatory elements governing the transcription of neighboring genes (AKA “cis-regulatory elements”). Ultimately, C. elegans provides a genetically tractable model with well described neuroanatomy to test hypotheses regarding the development of different neuronal cell types. Hopefully, the molecular and analytical tools used in C. elegans, can be used to investigate these developmental mechanisms in other species.
Recently, the Hobert lab published a paper in Neuron titled: “Diversification of C. elegans Motor Neuron Identity via Selective Effector Gene Repressor”, which elucidates the mechanisms governing C. elegans motor neuron diversification. In the paper, they point out that C. elegans motor neurons are cholinergic and GABAergic, but can be further subdivided. For example, the cholinergic neurons can be divided into six classes based on their features (Fig 1a). In the end, they sought to uncover the mechanisms governing cholinergic motor neurons (MNs) diversification in the ventral nerve cord (VNC) of C. elegans.
Previous studies showed that 5/6 classes of MNs in the VNC shared the unc-3 transcription factor (Fig 1b). Additional studies showed that the loss of or misexpression of unc-3 lead to the loss of specific MN subtypes. Therefore, the unc-3 transcription factor was not only shared among 5/6 MNs, but also specified MN subtypes, which is a little paradoxical. How can a shared transcription factor also specify MN subtype? The Hobert lab sought to answer this question by testing two models. They hypothesized that either the unc-3 transcription factor requires class specific co-factors to activate class-specific features (co-activator model upper panels of Fig 1d) or unc-3 is capable of activating all features, including class-specific features, but is prevented from doing so via class-specific repressor proteins (repressor model lower panels of Fig 1d). If the activator model were true, loss of unc-3 would lead to loss of class-specific features. If the repressor model were true, loss of unc-3 would lead to ectopic expression of class-specific features.
To test the two models, they screened C. elegans mutants to identify alleles in which MN class-specific effector genes are either misexpressed (supporting repressor model) or lost in specific MN classes (supporting the co-activator model). An example of how the data was collected and analyzed is shown in figure 2. Using the genetic screen, the ot721 allele was found and unc-129 (an effector gene) was ectopically expressed. Figure 2a shows the expression pattern of unc-129 with GFP in both the wild-type and mutant ot721 C. elegans. In the mutant ot721 phenotype there is green protein found in VA and VB MNs, which indicates ectopic expression. Expression patterns for the mutants are shown in fig 2b. Further analysis showed that the mutant ot721 allele corresponded to a previously undescribed zinc finger transcription factor encoding gene bnc-1 (fig 2c). When bnc-1 is expressed in the mutant ot721 C. elegans, the phenotype was rescued.
In the end, they found that MN diversification was a result of class-specific repressor proteins that prevents unc-3 from activating subsets of class-specific effector genes. Furthermore, all the reported repressors are phylogenetically conserved. Therefore, the proposed mechanism for MN diversification in C. elegans may constitute a broadly applicable principle of neuronal identity diversification across species.
To learn more about the tools used to investigate the genetic programs that lead to neuronal diversification join Dr. Hobert and the rest of UCSD neurograduate program at 4pm Tuesday (05/29/18) at the CNCB Seminar Room at UCSD.
Elischa Sanders is a first-year Neuroscience Ph.D. student, currently working in Eiman Azim’s lab