The main focus of my laboratory is to understand the gene regulatory control mechanisms that generate the astounding diversity of cell types in the nervous system. We study this problem by using two specific experimental advantages of the Caenorhabditis elegans model system: forward genetic mutant screens and transgenic reporter gene analysis. The ease with which cis-regulatory control regions can be analyzed through transgenic reporter analysis has allowed us to study the problem of neuronal differentiation from an angle not commonly used. The underlying basis of the functional and anatomical diversity of cell types in the nervous system is the differential expression of neuron-type-specific gene batteries, composed of terminal differentiation genes whose products define the specific properties of a mature neuron throughout its lifetime (e.g., neurotransmitter-synthesizing enzymes, transporters, receptors).
Thus, one way to understand the generation of neuronal diversity is a "bottom-up" approach that focuses on the terminal step of this process, that is, the regulation of terminal gene batteries. To this end, we have defined the molecular signatures of individual, terminally differentiated neuron types (Movie 1) and decoded the cis-regulatory control regions of terminal gene batteries through systematic mutational analysis of reporter genes in the context of transgenic animals. This mutational dissection revealed what we consider to be an important, far-reaching, and nonobvious principle: coregulation. That is, entire ensembles of terminal differentiation genes that define the terminal properties—thus, the identity and function—of a given neuron type share a common cis-regulatory signature. Through genetic analysis, we have identified phylogenetically conserved, neuron-type-specific trans-acting factors (or combinations thereof) that act through these cis-regulatory motifs. Loss of these factors leads to a loss of neuron-type-specific identity, whereas pan-neuronal features remain unaffected. We have termed these factors terminal selectors. Such a simple regulatory logic was not a given, because transcriptome profiling, conducted by us and others, revealed that individual, terminally differentiated neuron types express scores of transcription factors. Therefore, terminal gene batteries could have been envisioned to be under complex, piecemeal control, rather than being organized into relatively simple regulons.
Extending our studies to chordates, we have provided evidence that this regulatory principle may be conserved. Using mouse knockouts, we have shown that the terminal selector controlling dopaminergic neuron differentiation displays a similar function in mouse dopaminergic neurons and found that a terminal selector for cholinergic motoneurons is also functionally conserved in a simple chordate. We are using reverse genetic approaches in the mouse to further test the terminal selector concept.
While investigating how terminal selector gene function is linked to upstream developmental programs, we found that cis-regulatory regions of terminal selector loci read out transient regulatory states, characterized by a complex combination of transcriptional and signaling inputs that are specific for the lineage history of a cell. These regulatory events converge to transiently initiate terminal selectors, which then "lock in" the terminal state, often through autoregulatory feedback loops.
We have asked whether terminal selectors are not only required but also sufficient to drive specific neuronal differentiation programs. This is essentially a cellular reprogramming question, a topic that has garnered much recent attention. Misexpression of a terminal selector in mature animals results in the reprogramming of only a restricted number of neurons, mirroring the temporal and context dependency of many other prominent developmental control genes. Through genetic screens, we have identified mutants in which terminal selectors can now impose terminal differentiation programs much more broadly onto other cell types (Movie 2), demonstrating that the mechanistic basis of context dependency lies in the presence of inhibitory and likely chromatin-based mechanisms. We are continuing to pursue these approaches to understand the nature of the chromatin states that lock in terminal features of cell types and that need to be "cracked" to alter the identity of a cell type.
Another area of research in the lab that also relates to differentiation programs in the nervous system is based on two classic neurobiological observations that provide a fascinating conundrum. It has long been known that most nervous systems are largely bilaterally symmetric on a structural level, yet it is equally well known that nervous system function is strongly lateralized. How left/right asymmetry is superimposed on a bilateral state is poorly understood, in part because there are few molecular correlates to functional asymmetries in any nervous system. We have established a pair of structurally largely symmetric gustatory neurons in C. elegans as an entry point into this problem. These neurons express in a stereotyped, left/right asymmetric manner many members of a receptor-type guanylyl cyclase family that we found to be required for left/right asymmetric taste perception (Movie 3). The ability to easily visualize this functional asymmetry through gfp reporters that tag the rGC loci has provided us with a unique opportunity to ask how asymmetry is developmentally programmed. We have used a combination of classic mutant analysis and reporter gene studies to reveal the underlying molecular logic of this asymmetry. This analysis revealed a complex gene regulatory network, resulted in the identification of a miRNA in this network (at the time, this was the only miRNA shown to have a function in the nervous system), and revealed regulated chromatin compaction to be a critical component of this process as well.
Apart from understanding the "hard-wired" identity feature of individual neuron types, we also try to understand how environmental conditions, such as specific, widely perceived stressors, can induce neuron-type-specific changes in gene expression programs. For example, we have identified changes in the neurotransmitter identity of specific neuron types, induced by specific environmental conditions, and have begun to understand the mechanistic basis and physiological consequences of such genetic plasticity.
Lastly, we have been involved in developing, improving, and customizing methodologies to further exploit the specific advantages of C. elegans as a genetic model system. For example, we have pioneered the use of whole-genome sequencing (WGS) to pinpoint mutagen-induced molecular lesions, thereby shortcutting time-consuming positional cloning. We have developed customized software to make this approach widely accessible and developed a combined SNP-mapping/WGS strategy that we think represents the ultimate method for mutant identification in C. elegans.
This work was supported in part by grants from the National Institutes of Health.
As of March 4, 2016