CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR͞RZR͞RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNAmediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.molting ͉ development N uclear hormone receptors (NHRs) form a large superfamily of transcription factors that are important for the regulation of cell metabolism, development, and reproduction (1-3). The typical receptor consists of six domains. The most conserved domain is the DNA-binding domain (DBD) and is characteristic for the NHR superfamily. It is formed by two zinc finger motifs that are conserved through evolution (4-6). A second, less conserved domain is the ligand-binding domain, located in the C terminus of the molecule and involved in binding of small hydrophobic molecules, hormones of the steroid class, thyroid hormone, retinoic acid stereoisomers, farnesoids, prostaglandins, and terpenoids (2). The generally accepted classification of NHRs is based on homology in both the DBD and the ligandbinding domain and the specific ligands (if known), which activate the receptors. The majority of NHRs have no known ligands and usually are referred to as orphan receptors.The NHRs are present in a majority of Metazoan species higher than diploblasts. NHRs apparently evolved from a single gene present in a Metazoan ancestor before the divergence of diploblastic species (4-6). There are about 70 NHRs in vertebrates, 22 in Drosophila We previously have cloned the C. elegans orphan NHR CHR3 (19), which is a homologue of Drosophila DHR3 (20), Manduca sexta MHR3 (21), and mammalian ROR͞RZR͞Rev ErbA (22, 23). For clarity, we refer to it in this paper by using both its common and gene names, CHR3 (nhr-23). The gene is classified as NR1F4 in a unified nomenclature system for the NHR superfamily (24). We previously showed that CHR3 (nhr-23) is expressed in the epidermis of C. elegans throughout development and that it was required for proper molting (16).Molting is a complex of developmental processes characteristic for a clad Ecdysozoa, which includes arthropods, tardigrades, onychophorans, nematodes, nematomorphs, kinorhynchs, and priapulids (25). At each molt, there is production of the new outer-body cover, the exoskeleton, and the old part is shed. Molting represents...

NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in C. elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical co-regulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

CHR3 is a Caenorhabditis elegans orphan nuclear hormone receptor highly homologous to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. Related vertebrate factors include RORalpha/RZRalpha, RZRbeta and RevErb. Gel-shift studies show that CHR3 can bind the DR5-type hormone response sequence. CHR3 is a nuclear protein present in all blastomeres during early embryogenesis. During morphogenesis, both CHR3 protein and zygotically active reporter genes are detectable in epidermal cells and their precursors. Inhibition of the gene encoding CHR3 results in several larval defects associated with abnormal epidermal cell function, including molting and body size regulation, suggesting that CHR3 is an essential epidermal factor required for proper postembryonic development.

The large family of steroid/thyroid hormone receptor (STR) genes has been extensively studied in vertebrates and insects but little information is available on it in more primitive organisms. All members possess a DNA binding domain of zinc fingers of the C2, C2 type. We have used the polymerase chain reaction with degenerate oligonucleotide primers covering this region to clone three distinct members of this family from the nematode Caenorhabditis elegans. All three belong to the retinoic acid receptor (RAR), thyroid hormone receptor subfamily of genes. The cDNA of one of these clones shows such a high homology to DHR3, an early ecdysone response gene found in Drosophila, and MHR3, identified in Manduca sexta, that we have termed it CHR3. Furthermore, the C-terminal portion of the deduced protein sequence shows a box containing eight identical amino acids among CHR3, DHR3, and MHR3 suggesting an identical specific ligand for these proteins. CNR8 shows homology to NAK1, and CNR14 has homology to both the RAR-yl gene and to another ecdysone response gene, E78A. Neither of the latter two cDNAs is a clear homologue of any known gene and each is distinctive. All of these genes are expressed varyingly in both larval and adult stages of nematode development as shown by Northern blot analyses. These data demonstrate that the STR family of genes is represented in a nematode whose ancestor appeared well before the branching that gave rise to the Arthropoda and Chordata.More than 200 different cDNAs of the steroid/thyroid hormone receptor (STR) gene family have been cloned and sequenced from a variety of organisms. Many of these sequences are clear homologues-i.e., the thyroid hormone receptor (TR) genes from rat, chicken, mouse, human, and frog code for a highly similar protein that binds thyroid hormone. However, the term homologue can be ambiguous; in addition to the above, it can also mean evolutionarily related, as are all of these STR genes whether they bind steroids, retinoids, or thyroid hormone or have no known ligands. Our interest has been to discern the evolutionary history of this gene family. The family has been variously divided into 3-10 different groups (1-3). Sequences are available mostly from vertebrates and arthropods, although one sequence is known from an echinoderm (4). All of these genes can be divided into five or six domains denoted A-F (2, 3) (see Fig. 4 A). The A/B domain is most N terminal and is poorly conserved. The C domain is the zinc finger DNA binding domain and is so highly conserved as to identify the superfamily (5). The sequence of the distal part of the first zinc finger, termed the P box, distinguishes the three main subfamilies of the STR (2). The D, E, and F domains contain the activating domain, a dimerization domain, and the ligand binding domain (2, 3, 6, 7). We have searched for related genes in several invertebrate phyla and report here the identification and cDNA sequences of three genes from Caenorhabditis elegans. t The sequences of the P box of the cDNAs we have...

Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism.