[
European Worm Meeting,
2006]
The highly conserved C. elegans DAF-19 protein, a regulatory factor X (RFX) transcription factor was originally identified as a key regulator for cilia development (Swoboda et al. 2000). Cilia are specialized subcellular organelles that in C. elegans are present on the endings of sensory neuron dendrites. They are functional compartments that mediate signal reception and transduction from the environment. In order to be able to receive many different signals from the environment, cell type specific expression of signal transduction and cilia structural genes have evolved. This resulted in, for example, rod-like cilia, which are involved in the perception of soluble chemotaxis compounds. By contrast, the wing-like cilia are involved in smell perception. However, how different cilia subtypes develop is hardly known. Recent data suggests the involvement of developmental cascades in the promotion of cell specific neuron cilia subtypes (Lanjuin and Sengupta 2004). In this cascade well-conserved transcription factors through possibly multiple transcription events are thought to specify the identity of sensory neuron cilia subtypes. This is similar to the neuron cell (sub)type specification cascade. We are interested whether DAF-19 is involved in this cilia subtype specification cascade. This would imply that DAF-19 does not only play a role in general ciliogenesis, but also plays a role in cilia subtype specification. In a genome-wide search for candidate genes with a DAF-19 recognition site in the promoter region, the X-box, many transcription factors were identified (Blacque et al. 2005; Efimenko et al. 2005). We are currently establishing the expression patterns of more than 20 selected transcription factors, for three of which we could already demonstrate to be expressed in ciliated neurons. In addition we will establish if those transcription factors, expressed in ciliated sensory neurons, are transcriptionally dependent on DAF-19 and determine if they play a role in cilia subtype specification.
[
International Worm Meeting,
2007]
The C. elegans protein DAF-19, a Regulatory Factor X (RFX) transcription factor (TF), is a key regulator for cilia development (Swoboda et al. 2000). Via its recognition site in promoters of target genes, the X-box, DAF-19 directly controls the expression of genes that are important for ciliary structure and function. Cilia are specialized sub-cellular organelles that in C. elegans are present only on the endings of sensory neuron dendrites. In genome-wide searches for candidate genes with X-box promoter motifs, hundreds of genes were predicted to be DAF-19 dependent, and as a consequence many new ciliary genes could be identified (Blacque et al. 2005; Efimenko et al. 2005; Chen et al. 2006). Despite its successful application, the accuracy of the X-box prediction method for ciliary genes is still worth improving, especially since most candidate genes chosen for experimental verification, encode protein domains frequently found in other, already described ciliary genes. Moreover, a number of genes found in those studies were not expressed at all in ciliated neurons (so called false positives). To significantly improve the X-box prediction method that can distinguish true direct DAF-19 targets from false positives, we selected all TFs that have a promoter X-box motif. We consider this a more unbiased group of genes, since TFs are not directly involved in ciliary structure and function. Of 50 selected TFs we analyzed more than 30 TF::GFP fusions in transgenic worms. One third turned out to be expressed in ciliated neurons in a DAF-19 dependent manner. These results, together with information from previously experimentally proven direct DAF-19 targets and cross species comparisons with C. briggsae and C. remanei, allowed us to establish a high-accuracy X-box prediction method. In addition, we could clearly split X-box promoter motifs into two groups. Group I X-boxes have near-palindromic sequences, locate close to gene starts, are conserved across species, and direct gene expression in most or all ciliated neurons. Group II X-boxes have less palindromic sequences, are less conserved across species and direct gene expression in only one or a few ciliated neurons. We are currently creating a system in which we can not only predict a candidate genes DAF-19 dependence with high precision, but also whether its expression pattern will be in all or only a subset of the ciliated neuron class. Moreover, prelimenary results show that other cis-regulatory motifs, together with the X-box, are essential for the proper expression of DAF-19 dependent genes. Our work will help to find all the genes necessary for structurally and functionally correct sensory neuron cilia.
[
European Worm Meeting,
2008]
The highly conserved Caenorhabditis elegans RFX-type transcription factor. DAF-19 was originally identified as a key regulator of cilia development. (Swoboda et al. 2000). Cilia are specialized sub-cellular organelles that. in C. elegans are present exclusively at the endings of sensory neuron. dendrites. Loss of DAF-19 function leads to complete loss of all ciliated. structures in the worm. Cilia act as sensory antennae that control e.g. the. animal''s chemotactic movements in response to chemicals from the. environment. To sense many different chemical stimuli, the worm has a. variety of cilia with distinct neuron specific morphologies. Cilia with a. cylindrical morphology detect hydrophilic molecules, while adjacent wing. cilia have a fan-like structure and detect volatile odorants. How these. distinct structures and sensory modalities develop is to a large extent. unknown. For example, it is not known, which pathway determines that one. olfactory neuron develops a cylindrical ciliary structure, whereas another. forms fan-like cilia. It was hypothesized that these developmental. specifications may be accomplished by highly conserved transcription factor. cascades (Lanjuin et al. 2004; Mukhopadhyay et al. 2007).. We are interested in whether DAF-19 is not only involved in general. ciliogenesis, but also plays a role in cilia subtype specification. In. genome-wide searches for candidate direct target genes with a DAF-19. recognition site in the promoter region, the X-box, many transcription. factors were identified (Blacque et al. 2005; Efimenko et al. 2005; Chen et. al. 2006). A sizeable group of these are nuclear hormone receptors (NHR).. In C. elegans, NHR compose a large class of transcription factors (284),. some of which have been shown to function in development. We are currently. establishing expression patterns of those NHR, which are candidate direct. DAF-19 targets, including determining their DAF-19 dependence. So far we. could already demonstrate that four NHR show DAF-19 dependent expression in. ciliated sensory neurons (CSN). Interestingly, these four NHR are DAF-19. dependent in only a subset of CSN. We are continuing to analyze the. expression patterns and DAF-19 dependence of all "X-box selected" NHR and. in addition investigating their possible role in cilia subtype. specification.