SACKLER SPECIAL FEATURE: PERSPECTIVE Regulatory logic of neuronal diversity: Terminal selector genes and selector motifs Oliver Hobert1 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032 Edited by Michael S. Levine, University of California, Berkeley, CA, and approved September 8, 2008 (received for review June 24, 2008) Individual neuronal cell types are defined by the expression of unique batteries of terminal differentiation genes. The elucidation of the cis-regulatory architecture of several distinct, single neuron type-specific gene batteries in Caenorhabditis elegans has revealed a strikingly simple cis-regulatory logic, in which small cis-regulatory motifs are activated in postmitotic neurons by autoregulating transcription factors (TFs). Loss of the TFs results in the loss of the identity of the individual neuron type. I propose to term these TFs ‘‘terminal selector genes’’ and their cognate cis-regulatory target sites ‘‘terminal selector motifs.’’ Terminal selector genes assign individual neuronal identities by directly controlling the expression of downstream, terminal differentiation genes and act in specific regulatory network configurations. The simplicity of the cis-regulatory logic on which the terminal selector gene concept is based may contribute to the evolvability of neuronal diversity. Caenorhabditis elegans ͉ neuronal differentiation ͉ gene regulation ͉ transcription factors ͉ cis-regulatory motifs pproximately a century ago, ‘‘neuron type,’’ which is more frequently AIY Interneurons Ramon y Cajal (1) discovered used in vertebrates) (2). Gene expres- The identity of the AIY class of inter- and described in detail the an- sion data exist for many individual neu- neurons, which is composed of two atomical complexity of cell ron types, and cis-regulatory control bilaterally symmetric interneurons in- Atypes in nervous systems. His work regions can be easily dissected through volved in processing sensory information framed the fundamental question of mutational analysis of reporter genes and in behavioral plasticity (7, 8), is how such cellular complexity is gener- expressed in transgenic animals. controlled by a similarly simple regula- ated during development. The molecular I will first briefly describe several ex- tory logic. A broad battery of genes that correlates to the anatomical diversity of amples in which the regulatory program defines AIY identity shares a small cis- neuron types in a mature nervous sys- of individual neuron types has been elu- regulatory motif, called the AIY motif tem are neuron-type specific gene bat- cidated in C. elegans in substantial detail (Table 1), which, like the ASE motif, is teries. The composition of neuron (Table 1) (3) and will then discuss com- required and sufficient to instruct gene type-specific gene batteries is highly mon principles that may apply across combinatorial. That is, the differentiated phylogeny. expression in AIY (9). Like the ASE properties of individual neuron types motif, the AIY motif commonly occurs are usually not defined by the unique ASE Gustatory Neurons in single copy in the vicinity of AIY- expression of specific gene products, but The ASE class of sensory neurons is expressed genes. The AIY motif is syn- rather by the unique combination of composed of two bilaterally symmetric ergistically activated by a dimer of two genes that may each be more broadly sensory neurons. Their transcriptome homeodomain proteins, the TTX-3 LIM expressed. The question of how neuro- was determined by SAGE analysis (4). homeodomain protein and the CEH-10 nal diversity is generated, that is, how The analysis of the regulatory regions of Paired-type homeodomain protein (9). individual neurons execute distinct and ASE-expressed genes, performed by re- Each of these factors is expressed in a unique differentiation programs, can porter gene analysis in transgenic few neurons, but their expression exclu- therefore be essentially boiled down to worms, revealed a strikingly simple cis- sively overlaps in the AIY neuron class, the question of how such combinatorial regulatory logic. ASE-expressed genes thereby making them, as is the case for gene expression mechanisms are en- contain in their proximity a small cis- CEH-1, a unique identifier for AIY coded in the genome, on both the level regulatory motif of Ϸ12 bp, called the identity (10). In either ttx-3 or ceh-10 of cis-regulatory control elements and ASE motif (Table 1), which is absolutely null mutants, the identity of the mature the trans-acting factors that read these required as well as sufficient by itself to AIY interneuron is completely lost. As control elements. drive gene expression in the ASE neu- in the case of che-1 and the ASE neuron One strategy to tackle this problem is rons (4). This motif is bound and acti- class, the AIY interneurons are gener- to use a bottom-up approach in which vated by a zinc finger transcription ated and still express pan-neuronal fea- one first defines the nuts-and-bolts gene factor (TF), CHE-1, that is exclusively batteries that define the specific ana- expressed in the ASE sensory neurons. tomical and functional properties of a In the absence of the che-1 gene, ASE This paper results from the Arthur M. Sackler Colloquium of neuron and then dissects the cis- motif-containing genes fail to be acti- the National Academy of Sciences, ‘‘Gene Networks in An- imal Development and Evolution,’’ held February 15–16, regulatory control elements of these vated, resulting in a complete loss of the 2008, at the Arnold and Mabel Beckman Center of the genes. Such an approach is particularly identity of the mature ASE sensory neu- National Academies of Sciences and Engineering in Irvine, feasible in the experimentally easily ron class (4–6). Panneuronal features CA. The complete program and audio files of most presen- amenable and neuroanatomically well are unaffected and no specific alterna- tations are available on the NAS web site at http:// characterized model organism Caeno- tive fate appears to be executed upon www.nasonline.org/SACKLER_Gene_Networks. rhabditis elegans, which contains a ner- loss of che-1, indicating that ASE is Author contributions: O.H. wrote the paper. vous system of 302 neurons that fall into stuck in an indeterminate ground neuro- The author declares no conflict of interest. 118 anatomically precisely defined neu- nal state. Moreover, CHE-1 is sufficient This article is a PNAS Direct Submission. ron classes (the term ‘‘neuron class’’ is to induce ASE neuron fate if expressed 1E-mail: [email protected]. used here interchangeably with the term in other sensory neurons (6). © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806070105 PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20067–20071 Downloaded by guest on September 29, 2021 Table 1. Examples of neuronal terminal selector genes in C. elegans Sufficiency Terminal selector gene Neuron class Selector motif of motif CHE-1 zinc finger transcription ASE sensory neurons ASE motif Yes factor TTX-3/CEH-10 LIM/Prd AIY interneurons AIY motif Yes homeodomain dimer AST-1 ETS-type transcription All dopaminergic neurons DA motif Yes factor MEC-3/UNC-86 LIM/POU Mechanosensory neurons Yes homeodomain dimer UNC-30 Prd-type GABAergic ventral cord ND homeodomain motorneurons See text for references. ND, not determined. tures in ttx-3 or ceh-10 mutants and have (Table 1) (17, 18). The identity of all ago, which describes genes that are re- therefore ‘‘only’’ lost their neuron type- dopaminergic neurons in the worm are quired to determine the identity of a specific identity. Ectopic coexpression of specified by an ETS domain TF that specific developmental field or organ. ttx-3 and ceh-10 can transform neurons acts through a defined cis-regulatory The original selector gene definition into an AIY-like fate, but, notably, only motif, the DA motif, that is a required included genes that turned out to act in specific cellular contexts (10). and sufficient determinant for gene ex- relatively early in developmental path- pression in all DA neuron types (N. ways (21), and the definition included Touch Sensory Neurons Flames and O.H., unpublished data). A no specific mechanism of action. By ter- In the most classic example of regula- more detailed phenotypic characteriza- minal selector gene I mean to indicate tory logic of neuronal specification in C. tion of animals carrying mutations in that the gene is not only required to elegans, the identity of a group of six other neuron type-specific TFs, e.g., determine the identity of a specific neu- mechanosensory neurons is determined ttx-1 (AFD neurons), odr-7 (AWA neu- ron type but that it does so by directly by the combinatorial activity of two ho- rons), lim-4 (AWB neurons), and ceh-36 regulating the expression of terminal meobox genes mec-3 and unc-86 (11– (AWC neurons) (3, 19), and the identifi- differentiation genes [such terminal dif- 13). A heterodimer of MEC-3 and cation of their direct target gene batter- ferentiation genes were coined ‘‘realiza- UNC-86 acts synergistically to directly ies may reveal that those factors also tor’’ genes by Garcia-Bellido (20)]. A control the expression of mechanosen- broadly control neuron type-specific terminal differentiation gene battery sory-specific, terminal differentiation gene batteries through simple cis- defines the stable, unique properties of genes through a conserved cis- regulatory motifs. a specific, postmitotic neuron type, is regulatory motif that is required and maintained throughout the life of a neu- sufficient for touch neuron expression Terminal Selector Genes and Terminal ron, and encompasses genes such as (Table 1) (14, 15). This cis-regulatory Selector Motifs neurotransmitter receptors, ion chan- motif was subsequently found to be The examples described above and listed nels, neurotransmitter synthesis pathway present in many other touch-neuron in Table 1 reveal a common underlying genes, structural protein, etc.