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Null mutations in the C. elegans heterochronic gene lin-41 cause precocious expression of adult fates at larval stages. Increased lin-41 activity causes the opposite phenotype, reiteration of larval fates. let-7 mutations cause similar reiterated heterochronic phenotypes that are suppressed by lin-41 mutations, showing that lin-41 is negatively regulated by let-7. lin-41 negatively regulates the timing of LIN-29 adult specification transcription factor expression. lin-41 encodes an RBCC protein, and two elements in the lin-413'UTR are complementary to the 21 nucleotide let-7 regulatory RNA. A lin-41::GFP fusion gene is downregulated in the tissues affected by lin-41 at the time that the let-7 regulatory RNA is upregulated. We suggest that late larval activation of let-7 RNA expression downregulates LIN-41 to relieve inhibition of lin-29.

Tubby mice and individuals with Bardet-Biedl syndrome have defects in ciliated neuron function and obesity, suggesting an as-yet unknown metabolic signaling axis from ciliated neurons to fat storage tissues. Here we show coordinate regulation of Caenorhabditis elegans fat storage by orthologues of these genes acting in ciliated neurons and by a 3-ketoacyl-coA thiolase (encoded by kat-1) that acts in fat storage tissue. A genetic screen for markedly enhanced fat storage in tub-1 mutants led to the isolation only of kat-1 alleles, which impair fatty acid beta-oxidation. kat-1 acts in the intestine, the major C. elegans fat storage tissue, and is transcriptionally upregulated in animals with high fat storage. A genetic screen for synergistic increase in fat storage of a kat-1 mutant identified bbs-1. bbs-1 acts in 15 ciliated neurons that are poised to sense external and internal nutrient levels, supporting a model in which bbs-1 and tub-1 in ciliated neurons form part of an ancient, conserved neuroendocrine axis. This pathway also includes genes encoding intraflagellar transport proteins and cyclic nucleotide gated channels, demonstrating that C. elegans fat storage is under polygenic control.

The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged ∼1400 bp in size when using a ∼3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.

The large number of different types of neurons in both vertebrate and invertebrate nervous systems raises the question of how the development of a particular neuronal cell type is specified. To pursue this question, Desai et al. (1988;Desai and Horvitz 1989) initiated a genetic analysis of the HSN serotonergic motor neurons in Caenorhabditis elegans. This analysis identified genes that are required for a number of distinct aspects of HSN development and placed these genes in a pathway of gene action. Some of these genes encode types of proteins that have been shown to control neuronal development in other organisms. Examples of such genes are egl-5, which encodes a homeo domain protein (Wang et al. 1993); unc-6, which encodes a member of the netrin family {Serafini et al. 1994); unc-33, which encodes a protein that is related to CRMP-62, a chick collapsin response mediator protein (Goshima et al. 1995); and unc-86, which encodes a POU homeo domain protein (Finney and Ruvkun 1990). This correlation between the types of proteins that control HSN development and the types of proteins that control neuronal development in other organisms suggests that study of HSN development will reveal general principles of how neuronal cell type is specified.~Corresponding author.The POU gene unc-86 is required for the normal pattern of HSN axonal outgrowth, normal HSN nuclear morphology, and HSN serotonin expression (Desai et al. 1988). POU homeo domain proteins have been shown to be required for neuronal development in both Drosophila (Bhat et al. 1995;Yeo et al. 1995) and mammals (for review, see Sharp and Morgan 1996). Mutation of the C. elegans gene sere-4 (sex muscle defective) causes the same spectrum of defects in HSN development as that caused by mutation of unc-86 (Desai et al. 1988). Because POU homeo domain proteins seem likely to play an evolutionarily conserved role in neuronal development, we are interested in determining how the SEM-4 and UNC-86 proteins interact in controlling HSN development. To understand how sem-4 functions in HSN development as well as in the development of other cell types, we have performed a genetic and molecular analysis of sere-4.

Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes-germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defectsresemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1.Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system involving loss of memory and cognitive function. Amyloid plaques, whose major component is the ␤-amyloid, or A␤, peptide, are a neuropathological hallmark of AD. Dominant mutations in any of three genes, PS1, PS2, or APP, cause early-onset familial AD. PS1 and PS2 encode related proteins termed presenilins 1 and 2 (PS1 and PS2) (1-3), and APP encodes the amyloid precursor protein (APP), from which the A␤ peptide is generated by proteolytic processing (for review, see ref. 4).Three presenilin genes, spe-4 (5), sel-12 (6), and hop-1 (7), have been identified in the nematode Caenorhabditis elegans. Rescue experiments using transgenes have shown that human PS1 and PS2 can substitute for SEL-12, demonstrating that at least some aspects of presenilin function have been conserved from nematodes to mammals (8, 9). Experiments by Levitan and Greenwald (6) indicate that sel-12 acts as a positive regulator of Notch-pathway signaling mediated by the C. elegans Notch receptor homologs GLP-1 and LIN-12: loss-offunction mutations in sel-12 suppress lin-12 gain-of-function phenotypes and enhance lin-12 and glp-1 partial loss-offunction phenotypes. A similar interaction has been proposed to occur in mice: the lethal phenotype of PS1 knockout mice resembles that seen in Notch ligand and receptor knockouts (10, 11).sel-12 mutations do not cause strong Glp-1 or Lin-12 lossof-function phenotypes, suggesting that sel-12 might act redundantly with other presenilin genes (6). To examine the function of hop-1 and to test this hypothesis, we used a reverse genetic strategy to generate hop-1 and sel-12 deletion mutations. Our analysis of hop-1; sel-12 double mutant phenotypes indicates that hop-1 functions redundantly with sel-12 to promote Notch-pathway signaling in C. elegans. Thi...

Niemann-Pick type C (NP-C) disease is a progressive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in lysosomes [1]. NP-C patients show various defects including hepatosplenomegaly, ataxia, dystonia and dementia. Most cases of NP-C are associated with inactivating mutations of the NPC1 gene [2], which encodes a protein implicated in the retrograde transport of sterols and other cargo from lysosomes [3]. Furthermore, localization of the NPC1 protein to lysosomal/endosomal compartments is essential for proper transport [4]. To create a model of NP-C disease in a simple, genetically tractable organism, we generated deletion mutations in two Caenorhabditis elegans homologs of the human NPC1 gene, designated npc-1 and npc-2. Animals mutant for npc-1 developed slowly, laid eggs prematurely, and were hypersensitive to cholesterol deprivation. Furthermore, npc-1; npc-2 double-mutant animals inappropriately formed dauer larvae under favorable growth conditions. These phenotypes in C. elegans provide a model system for both genetic and chemical suppressor screening that could identify promising drug targets and leads for NP-C disease.

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