“The brain is a world consisting of a number of unexplored continents and great stretches of unknown territory.”

Santiago Ramón y Cajal

How are the many distinct cell types that characterize a mature nervous system genetically specified? How stable is neuronal identity over the life time of an animal? For a terminally differentiating neuron these questions boil down to a gene regulatory question: how is the expression of the distinct batteries of genes that define the terminal, functional properties of distinct neuron type induced and maintained? How are changes in gene expression, and hence changes in neuronal phenotypes, triggered? We study these questions in the context of the nematode C. elegans, and we have begun to uncover what appear to some simple, phylogenetically conserved principles that underlie the generation of diverse neuronal identities.

Our entry point in understanding the generation of neuronal diversity has been a “bottom-up” approach that focuses on the terminal step of this process, the regulation of terminal gene batteries. “Terminal gene batteries” include genes that define, for example, the neurotransmitter phenotype of a neuron, its resident ion channels or neuropeptides. To this end, we have defined the molecular signatures of individual, terminally differentiated neuron types 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 analysis through the entire C. elegans nervous system, we found that master-regulatory terminal selector can be found in essentially most, if not all neuron classes that we have examined so far.

Movie 1: Our playground – the nervous system of the nematode C. elegans. From Gendrel, M., Atlas, E., and Hobert, O. 2016. eLife.

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.

Movie 2: Cell fate conversion: A gonad full of neurons. From Tursun, B., Patel, T., Kratsios, P., and Hobert, O. 2011. Science.

Apart from understanding the “hard-wired” identity feature of individual neuron types, we also try to understand how extrinsic and intrinsic conditions can modify phenotypic features of individual neuron types. For example, we have asked how a hormonally controlled diapause arrest stage can modify many different molecular, anatomical and functional features of indiviual neuron types, or how the sexual identity of an animal reprograms neuronal features during sexual maturation of the animal. In each of these cases, we ask how very broad signals or states can be translated into highly neuron-type specific alterations in neuronal phenotypes.

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.