Animal microRNAs (miRNAs) are gene regulatory factors that prevent the expression of specific messenger RNA targets by binding to their 3' untranslated region. The Caenorhabditis elegans lsy-6 miRNA (for lateral symmetry defective) is required for the left/right asymmetric expression of guanyl cyclase (gcy) genes in two chemosensory neurons termed ASE left (ASEL) and ASE right (ASER). The asymmetric expression of these putative chemoreceptors in turn correlates with the functional lateralization of the ASE neurons. Here we find that a mutation in the die-1 zinc-finger transcription factor disrupts both the chemosensory laterality and left/right asymmetric expression of chemoreceptor genes in the ASE neurons. die-1 controls chemosensory laterality by activating the expression of lsy-6 specifically in ASEL, but not in ASER, where die-1 expression is downregulated through two sites in its 3' untranslated region. These two sites are complementary to mir-273, a previously uncharacterized miRNA, whose expression is strongly biased towards ASER. Forced bilateral expression of mir-273 in ASEL and ASER causes a loss of asymmetric die-1 expression and ASE laterality. Thus, an inverse distribution of two sequentially acting miRNAs in two bilaterally symmetric neurons controls laterality of the nematode chemosensory system.

[Keywords: C. elegans; left/right asymmetry; transcription factor; homeobox] Supplemental material is available at http://www.genesdev.org.

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...

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