Caenorhabditis elegans sine oculis/SIX-type homeobox genes act as homeotic switches to define neuronal subtype identities.

The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification.

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.

Molecular topography of an entire nervous system.

We have produced gene expression profiles of all 302 neurons of the C. elegans nervous system that match the single-cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses distinct codes of ∼23 neuropeptide genes and ∼36 neuropeptide receptors, delineating a complex and expansive "wireless" signaling network. To demonstrate the utility of this comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression and (2) reveal adhesion proteins with potential roles in process placement and synaptic specificity. Our expression data are available at https://cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity, and function throughout the C. elegans nervous system.

The Prop1-like homeobox gene unc-42 specifies the identity of synaptically connected neurons.

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as 'circuit organizer transcription factors' to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment.

Unique homeobox codes delineate all the neuron classes of C. elegans.

It is not known at present whether neuronal cell-type diversity-defined by cell-type-specific anatomical, biophysical, functional and molecular signatures-can be reduced to relatively simple molecular descriptors of neuronal identity1. Here we show, through examination of the expression of all of the conserved homeodomain proteins encoded by the Caenorhabditis elegans genome2, that the complete set of 118 neuron classes of C. elegans can be described individually by unique combinations of the expression of homeodomain proteins, thereby providing-to our knowledge-the simplest currently known descriptor of neuronal diversity. Computational and genetic loss-of-function analyses corroborate the notion that homeodomain proteins not only provide unique descriptors of neuron type, but also have a critical role in specifying neuronal identity. We speculate that the pervasive use of homeobox genes in defining unique neuronal identities reflects the evolutionary history of neuronal cell-type specification.

Alternative polyadenylation produces multiple 3' untranslated regions of odorant receptor mRNAs in mouse olfactory sensory neurons.

BACKGROUND: Odorant receptor genes constitute the largest gene family in mammalian genomes and this family has been extensively studied in several species, but to date far less attention has been paid to the characterization of their mRNA 3' untranslated regions (3'UTRs). Given the increasing importance of UTRs in the understanding of RNA metabolism, and the growing interest in alternative polyadenylation especially in the nervous system, we aimed at identifying the alternative isoforms of odorant receptor mRNAs generated through 3'UTR variation. RESULTS: We implemented a dedicated pipeline using IsoSCM instead of Cufflinks to analyze RNA-Seq data from whole olfactory mucosa of adult mice and obtained an extensive description of the 3'UTR isoforms of odorant receptor mRNAs. To validate our bioinformatics approach, we exhaustively analyzed the 3'UTR isoforms produced from 2 pilot genes, using molecular approaches including northern blot and RNA ligation mediated polyadenylation test. Comparison between datasets further validated the pipeline and confirmed the alternative polyadenylation patterns of odorant receptors. Qualitative and quantitative analyses of the annotated 3' regions demonstrate that 1) Odorant receptor 3'UTRs are longer than previously described in the literature; 2) More than 77% of odorant receptor mRNAs are subject to alternative polyadenylation, hence generating at least 2 detectable 3'UTR isoforms; 3) Splicing events in 3'UTRs are restricted to a limited subset of odorant receptor genes; and 4) Comparison between male and female data shows no sex-specific differences in odorant receptor 3'UTR isoforms. CONCLUSIONS: We demonstrated for the first time that odorant receptor genes are extensively subject to alternative polyadenylation. This ground-breaking change to the landscape of 3'UTR isoforms of Olfr mRNAs opens new avenues for investigating their respective functions, especially during the differentiation of olfactory sensory neurons.