The elucidation of the architecture of gene regulatory networks that control cell-type specific gene expression programs represents a major challenge in developmental biology. We describe here a cell fate decision between two alternative neuronal fates and the architecture of a gene regulatory network that controls this cell fate decision. The two Caenorhabditis elegans taste receptor neurons ''ASE left'' (ASEL) and ''ASE right'' (ASER) share many bilaterally symmetric features, but each cell expresses a distinct set of chemoreceptors that endow the gustatory system with the capacity to sense and discriminate specific environmental inputs. We show that these left͞right asymmetric fates develop from a precursor state in which both ASE neurons express equivalent features. This hybrid precursor state is unstable and transitions into the stable ASEL or ASER terminal end state. Although this transition is spatially stereotyped in wild-type animals, mutant analysis reveals that each cell has the potential to transition into either the ASEL or ASER stable end state. The stability and irreversibility of the terminal differentiated state is ensured by the interactions of two microRNAs (miRNAs) and their transcription factor targets in a double-negative feedback loop. Simple feedback loops are found as common motifs in many gene regulatory networks, but the loop described here is unusually complex and involves miRNAs. The interaction of miRNAs in double-negative feedback loops may not only be a means for miRNAs to regulate their own expression but may also represent a general paradigm for how terminal cell fates are selected and stabilized.left͞right asymmetry ͉ bistable ͉ network motif ͉ regulatory RNA ͉ cellular diversification N ervous systems are characterized by a striking degree of cellular diversity. The molecular correlates to morphological and functional diversity of nervous systems are neuron-type specific gene expression programs. The experimental accessibility of the nematode Caenorhabditis elegans offers the opportunity to (i) determine the nature of neuron-type specific gene expression programs on a single-cell level and (ii) to genetically dissect the mechanisms that establish and maintain these singlecell specific programs. The two main gustatory neurons of C. elegans, ASE left (ASEL) and ASE right (ASER), display a particularly intricate level of neuronal diversity. Although bilaterally symmetric in many different regards (cell position, axodendritic morphology, synaptic connectivity, and molecular features), each neuron expresses a distinct spectrum of putative chemoreceptors, a feature that the worm requires to navigate through complex sensory environments (1, 2). The ASE neurons therefore not only provide a model to study sensory neuron fate diversification but also to study neuronal laterality, a common but poorly understood feature of many nervous systems.To elucidate the nature of the gene regulatory program that diversifies ASEL and ASER, we have isolated mutants in which ASE asymmetry is disrupted...

Functional left/right asymmetry (''laterality'') is a fundamental feature of many nervous systems, but only very few molecular correlates to functional laterality are known. At least two classes of chemosensory neurons in the nematode Caenorhabditis elegans are functionally lateralized. The gustatory neurons ASE left (ASEL) and ASE right (ASER) are two bilaterally symmetric neurons that sense distinct chemosensory cues and express a distinct set of four known chemoreceptors of the guanylyl cyclase (gcy) gene family. To examine the extent of lateralization of gcy gene expression patterns in the ASE neurons, we have undertaken a genomewide analysis of all gcy genes. We report the existence of a total of 27 gcy genes encoding receptor-type guanylyl cyclases and of 7 gcy genes encoding soluble guanylyl cyclases in the complete genome sequence of C. elegans. We describe the expression pattern of all previously uncharacterized receptor-type guanylyl cyclases and find them to be highly biased but not exclusively restricted to the nervous system. We find that .41% (11/27) of all receptor-type guanylyl cyclases are expressed in the ASE gustatory neurons and that one-third of all gcy genes (9/27) are expressed in a lateral, left/right asymmetric manner in the ASE neurons. The expression of all laterally expressed gcy genes is under the control of a gene regulatory network composed of several transcription factors and miRNAs. The complement of gcy genes in the related nematode C. briggsae differs from C. elegans as evidenced by differences in chromosomal localization, number of gcy genes, and expression patterns. Differences in gcy expression patterns in the ASE neurons of C. briggsae arise from a difference in cisregulatory elements and trans-acting factors that control ASE laterality. In sum, our results indicate the existence of a surprising multitude of putative chemoreceptors in the gustatory ASE neurons and suggest the existence of a substantial degree of laterality in gustatory signaling mechanisms in nematodes.

SUMMARY Background Even though functional lateralization is a predominant feature of many nervous systems, it is poorly understood how lateralized neural function is linked to lateralized gene activity. A bilaterally symmetric pair of gustatory neurons in the nematode C. elegans, ASEL and ASER, serves as a model to study the genetic basis of functional lateralization as this pair senses a number of chemicals in a left/right asymmetric manner. The extent of functional lateralization of the ASE neurons and genes responsible for the left/right asymmetric activity of ASEL/R are unknown. Results We show here that a large panel of salt ions is sensed in a left/right asymmetric manner, as demonstrated by behavioral assays, imaging of neural activity with a genetically encoded calcium sensor and by genetic manipulations that alter the fate of either ASEL or ASER. We show that lateralized salt responses allow the worm to discriminate between distinct salt cues. To identify molecules that may be involved in sensing salt ions and/or transmitting such sensory information, we examined the chemotaxis behavior of animals harboring mutations in eight different receptor-type, transmembrane guanylyl cyclases (encoded by gcy genes), which are expressed in either ASEL (gcy-6, gcy-7, gcy-14), ASER (gcy-1, gcy-4, gcy-5, gcy-22) or ASEL and ASER (gcy-19). Disruption of a ASER-expressed gcy gene, gcy-22, resulted in a broad chemotaxis defect to nearly all salts sensed by ASER, as well as to a left/right-asymmetrically sensed amino acid. In contrast, disruption of other gcy genes resulted in highly salt ion-specific chemosensory defects. Furthermore, we show that not only the cyclase domain, but also the extracellular domain of GCY proteins is important for their activity in salt sensation. Conclusions Our findings broaden our understanding of lateralities in neural function, provide insights into how this laterality is molecularly encoded and reveal an unusually diverse spectrum of signaling molecules involved in gustatory signal transduction.