Donald G. Moerman

Functional genomics of the cilium, a sensory organelle

Cilia and flagella play important roles in many physiological processes, including cell and fluid movement, sensory perception, and development. The biogenesis and maintenance of cilia depend on intraflagellar transport (IFT), a motility process that operates bidirectionally along the ciliary axoneme. Disruption in IFT and cilia function causes several human disorders, including polycystic kidneys, retinal dystrophy, neurosensory impairment, and Bardet-Biedl syndrome (BBS). To uncover new ciliary components, including IFT proteins, we compared C. elegans ciliated neuronal and nonciliated cells through serial analysis of gene expression (SAGE) and screened for genes potentially regulated by the ciliogenic transcription factor, DAF-19. Using these complementary approaches, we identified numerous candidate ciliary genes and confirmed the ciliated-cell-specific expression of 14 novel genes. One of these, C27H5.7a, encodes a ciliary protein that undergoes IFT. As with other IFT proteins, its ciliary localization and transport is disrupted by mutations in IFT and bbs genes. Furthermore, we demonstrate that the ciliary structural defect of C. elegans dyf-13(mn396) mutants is caused by a mutation in C27H5.7a. Together, our findings help define a ciliary transcriptome and suggest that DYF-13, an evolutionarily conserved protein, is a novel core IFT component required for cilia function.

C. elegans PAT-6/Actopaxin Plays a Critical Role in the Assembly of Integrin Adhesion Complexes In Vivo

BACKGROUNDnThe novel focal adhesion protein actopaxin includes tandem unconventional calponin homology (CH) domains and a less well-conserved N-terminal stretch. Dominant-negative studies have implicated actopaxin in focal adhesion formation.nnnRESULTSnPAT-6/actopaxin, the sole actopaxin homolog in C. elegans, is located in body wall muscle attachments that are in vivo homologs of focal adhesions. We show using pat-6 protein null alleles that PAT-6/actopaxin has critical nonredundant roles during attachment maturation. It is required to recruit UNC-89 and myofilaments to newly forming attachments, and also to reposition the attachments so that they form the highly ordered array of dense body and M line attachments that are characteristic of mature muscle cells. PAT-6/actopaxin is not required for the deposition of UNC-52/perlecan in the basal lamina, nor for the initiation of attachment assembly, including the clustering of integrin into foci and the recruitment of attachment proteins PAT-4/ILK, UNC-112, and DEB-1/vinculin from the cytosol. PAT-6/actopaxin, PAT-4/ILK, and UNC-112 are each required for the same steps during attachment assembly in vivo, consistent with the notion that they work together in multiprotein complex. Supporting this idea, PAT-4/ILK can simultaneously bind to PAT-6/actopaxin and UNC-112, forming a ternary complex, in yeast three-hybrid assays. Finally, we show that both calponin homology domains are required for PAT-6/actopaxins critical functions during attachment assembly in vivo.nnnCONCLUSIONSnWe show directly by loss-of-function genetics that PAT-6/actopaxin plays essential roles during the maturation of integrin-mediated muscle attachments in vivo.

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics

BackgroundThe recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes.ResultsBy screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5.ConclusionOur comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Towards a mutation in every gene in Caenorhabditis elegans

The combined efforts of the Caenorhabditis elegans Knockout Consortium and individuals within the worm community are moving us closer to the goal of identifying mutations in every gene in the nematode C. elegans. At present, we count about 7000 deletion alleles that fall within 5500 genes. The principal method used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, the Moerman group has incorporated array comparative genome hybridization (aCGH) to detect deletions across the entire coding genome. Other methods used to detect mutant alleles in C. elegans include targeting induced local lesion in genomes (TILLING), transposon tagging, using either Tc1 or Mos1 and resequencing. These combined strategies have improved the overall throughput of the gene-knockout labs, and have broadened the types of mutations that we, and others, can identify. In this review, we will discuss these different approaches.

C. elegans Deletion Mutant Screening

The methods used by the Caenorhabditis elegans Gene Knockout Consortium are conceptually simple. One does a chemical mutagenesis of wild-type C. elegans, and then screens the progeny of the mutagenized animals, in small mixed groups, using polymerase chain reaction (PCR) to identify populations with animals where a portion of DNA bounded by the PCR primers has been deleted. Animals from such populations are then selected and grown clonally to recover a pure genetic strain. We categorize the steps needed to do this as follows: (1) mutagenesis and DNA template preparation, (2) PCR detection of deletions, (3) sibling selection, and (4) deletion stabilization. These are discussed in detail in this chapter.

16 Muscle: Structure, Function, and Development

I. INTRODUCTION Muscle cells are characterized by a filament-lattice structure that generates contractile force in a regulated manner. Caenorhabditis elegans contains several groups of muscles with diverse functions. The most numerous (95 in adults) are striated (multiple sarcomere) muscles used for locomotion of the animal. Eighty one of these body-wall muscles are present at birth, with the remainder added early during postembryonic development (Sulston and Horvitz 1977; Sulston et al. 1983). A variety of nonstriated (single-sarcomere) muscles are formed during embryogenesis. These muscles are used for diverse functions: pharyngeal pumping (20 pharyngeal muscle cells), intestinal contraction (2 intestinal muscles), and defecation control (1 anal sphincter and 1 anal depressor). During postembryonic development, the hermaphrodite adds a set of muscles for fertilization and egg laying (8 vulval and 8 uterine muscles and the contractile gonadal sheath), whereas the male adds a specialized set of 41 muscles to be used in mating. Anatomical simplicity has been a significant factor in the development of C. elegans as a model organism for studies of muscle. Due to the predominance of the body-wall muscle class, this class has been the most extensively analyzed; thus, much of the information in this chapter focuses on body-wall muscle. Muscle has been a fertile ground for molecular genetic studies with C. elegans. Although muscle is essential for viability (see, e.g., Waterston 1989), partial muscle function is sufficient for growth under laboratory conditions. This allowed the isolation of many mutations affecting muscle (see, e.g., Brenner 1974; Epstein and Thomson 1974;...