The extent of gene regulation in cell differentiation is poorly understood. We previously used saturation mutagenesis to identify 18 genes that are needed for the development and function of a single type of sensory neuron--the touch receptor neuron for gentle touch in Caenorhabditis elegans. One of these genes, mec-3, encodes a transcription factor that controls touch receptor differentiation. By culturing and isolating wild-type and mec-3 mutant cells from embryos and applying their amplified RNA to DNA microarrays, here we have identified genes that are known to be expressed in touch receptors, a previously uncloned gene (mec-17) that is needed for maintaining touch receptor differentiation, and more than 50 previously unknown mec-3-dependent genes. These genes are randomly distributed in the genome and under-represented both for genes that are co-expressed in operons and for multiple members of gene families. Using regions 5' of the start codon of the first 20 genes, we have also identified an over-represented heptanucleotide, AATGCAT, that is needed for the expression of touch receptor genes.

Summary Because microtubules perform many essential functions in neurons, delineating unique roles attributable to these organelles presents a formidable challenge. Microtubules endow neurons with shape and structure and are required for developmental processes including neurite outgrowth [1], intracellular transport [2], and synaptic formation and plasticity [3, 4]; microtubules in sensory neurons may be required for the above processes in addition to a specific sensory function. In Caenorhabditis elegans six touch receptor neurons (TRNs) sense gentle touch [5] and uniquely contain 15-protofilament microtubules [6]. Disruption of these microtubules by loss of either the MEC-7 β-tubulin [7] or MEC-12 a-tubulin [8] or by growth in 1 mM colchicine causes touch insensitivity [5, 6], altered distribution of the touch transduction channel, and a general reduction in protein levels. We show that the effect on touch sensitivity can be separated from the others; microtubule depolymerization in mature TRNs causes touch insensitivity but does not result in protein distribution and production defects. In addition, the mec-12(e1605) mutation selectively causes touch insensitivity without affecting microtubule formation and other cellular processes. Touching e1605 animals produces a reduced mechanoreceptor current that inactivates more rapidly than in wild type, suggesting a specific role of the microtubules in mechanotransduction.

Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans , the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to regulate cellular functions.

The nematode Caenorhabditis elegans was the first organism for which touch insensitive mutants were obtained. The study of the genes defective in these mutants has led to the identification of components of a mechanosensory complex needed for specific cells to sense gentle touch to the body. Multiple approaches using genetics, cell biology, biochemistry, and electrophysiology have characterized a channel complex, containing two DEG/ENaC pore-forming subunits and several other proteins, that transduces the touch response. Other mechanical responses, sensed by other cells using a variety of other components, are less well understood in C. elegans. Many of these other senses may use TRP channels, although DEG/ENaC channels have also been implicated.

Selective protein degradation is a key regulator of neuronal development and synaptogenesis. Complexes that target proteins for degradation often contain F-box proteins. Here we characterize MEC-15, an F-box protein with WD repeats, which is required for the development and function of Caenorhabditis elegans touch receptor neurons (TRNs). Mutations in mec-15 produce defects in TRN touch sensitivity, chemical synapse formation, and cell-body morphology. All mec-15 mutant phenotypes are enhanced by mutations in a MAP kinase pathway composed of the MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAPK PMK-3. A mutation of the rpm-1 gene, which encodes an E3 ubiquitin ligase that negatively regulates this pathway to promote synaptogenesis, suppresses only the mec-15 cell-body defect. Thus, MEC-15 acts in parallel with RPM-1, implicating a second protein degradation pathway in TRN development. In addition, all mec-15 phenotypes can be dominantly suppressed by mutations in mec-7, which encodes a b-tubulin, and dominantly enhanced by mutations in mec-12, which encodes an a-tubulin. Since mec-15 phenotypes depend on the relative levels of these tubulins, MEC-15 may target proteins whose function is affected by these levels.