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...

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Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (K d ‫؍‬ 4 ؋ 10 ؊10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His 6 )jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His 6 )jRXR showed that fulllength jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

During the clinical study and treatment of a group of patients with thyroid carcinoma, we have studied the effects of thyroid hormone on several metabolic systems under conditions in which the thyroid status of the patients could be controlled at will. Early in the tracer studies of iodoalbumin metabolism in these patients it was apparent that it was necessary to employ a mathematical approach somewhat different from published methods of compartment analysis. Accordingly a suitable mathematical approach was developed by one of the present authors (1). In this report, the practical difficulties encountered in application of the approach to experimental data and the theoretical implications of the approach are discussed.The kinetic analysis of the data of a single tracer study comprises the body of the report. Additional data from other tracer studies are included only insofar as they contribute to the development of the model chosen for analysis. METHODSGeneral. The methods of dialysis of the commercial 1131 labeled human serum albumin employed and details of the injection procedure, collection of blood and urine samples, quantitation of radioactivity in urine and plasma, determination of serum albumin and quantitation of total radioactivity removed in blood sampling have been given elsewhere (2). Stool collections were made only in the case of M. H. in Experiment III. The individual specimens were suspended in concentrated sodium hydroxide and distilled water to known volume, allowed to digest partly, and then agitated until a uniform pipettable mixture was obtained. The counting procedure was the same as that for urine and plasma (2). None of the patients studied showed albuminuria No chemical fractionation of label was done routinely on urine or plasma samples.

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Nuclear hormone receptors comprise a large family of zinc finger transcription factors, some with hydrophobic ligands, such as thyroid hormone, vitamin D, steroids, etc., and others for which no ligand has been found. Thyroid hormone receptors (TRs) generally are considered to be confined to the vertebrata that possess a thyroid gland. Tunicates represent the most primitive of the chordates, and there are data supporting a role for thyroid hormone in their metamorphosis, but no data are available on TRs in this genus; hence, we have studied Ciona intestinalis. Screening of a Ciona library with the DNA binding domain of Xenopus laevis TR (xTR) resulted in the isolation of a nuclear hormone receptor, C. intestinalis nuclear receptor 1 (CiNR1). CiNR1 is similar to TRs of more evolved species with a conserved DNA binding domain whereas the ligand binding domain shows poor homology to vertebrate sequences. The C-terminal part of CiNR1 spans Ϸ200 amino acids more than other TRs, lacks the AF2 transactivation domain, and is not able to bind triiodothyronine. Phylogenetically, CiNR1 appears to be close to the common ancestral gene of TRs. Expression of CiNR1 was limited to the developing embryo and the larval stage, which suggests a role during development and metamorphosis. In transfection experiments, CiNR1 down-regulated basal transcription of a reporter gene driven by the TR palindrome responsive element. When CiNR1 was cotransfected with chicken TR␣, it attenuated the normal thyroid hormone response in a dominant negative fashion. This attenuation required the C-terminal portion of the molecule.Nuclear receptors are zinc finger transcription factors that regulate gene expression by binding to specific cis-acting sequences in the promoter region (1-3) and include receptors for several hydrophobic ligands, such as steroids, retinoic acid, thyroid hormone, vitamin D, ecdysone, as well as a variety of receptors, called orphans, without a known ligand (4). To date, Ͼ150 different members of this superfamily, spanning a large diversity of species from nematode to human, have been described (5).From the evolutionary point of view, all nuclear receptors can be grouped into six subfamilies: (i) a large one containing thyroid receptors (TRs), retinoic acid receptors (RARs), vitamin D receptors, ecdysone receptors, and numerous orphan receptors; (ii) one clustering retinoid X receptors, COUP, HNF4, and other orphan receptors; (iii) one containing steroid hormone receptors; (iv) one the NGFB group of orphan receptors; (v) one containing FTZ-F1 orphan receptors; and (vi) the last one containing only the GCNF1 orphan receptor (6). Although the TR͞RAR and steroid receptor subfamilies are well characterized in vertebrates, little is known about their presence in lower species. Orphan receptors are found in most primitive animals, many of which have homologues or closely related genes in vertebrates (4). The common modular structure of nuclear receptors, their genomic organization, and their conservation in the respective d...

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...

A number of compounds such as 2,4-dinitrophenol,' salicylate, tetrachlorothyronine, and diphenylhydantoin have been shown to lower the protein-bound iodine level of the blood (1-5). In the case of DNP and salicylate, it was concluded that they acted in part via an inhibition of the pitutary TSH mechanism and partly by an accelerated loss of T4 from the circulation (2, 6). A likely site of action was, therefore, expected to involve binding of T4 to serum proteins.The ability of various analogs of thyroxine to bind to proteins of serum has been extensively studied with electrophoretic techniques. At least three serum proteins and perhaps four have been described which bind thyroxine (7). Less direct methods of studying this binding of the thyroid hormones consist of measurement of the "uptake" of triiodothyronine by erythrocytes (8) and the rate of dialysis of T4 through a cellophane membrane (9). By these techniques several compounds have been found that appear to alter the binding of T4 to serum proteins when studied by these techniques but if whole serum is used, the effect cannot be localized to specific proteins. Thus it has been suggested from dialysis studies that salicylates and DNP act by inhibiting T4 binding to serum proteins. Electrophoretic studies in barbital buffer of sera containing salicylate failed to show any influence on the distribution of T4 between TBG and albumin, nor was there any change in the T4 binding capacity measured under these conditions (2). Since then it has been shown that barbital buffer inhibits T4 binding to prealbumin (10) and Ingbar has stated that 1 The following abbreviations have been used: DNP = 2,4-dinitrophenol; PBI = protein-bound iodine, T4 = thyroxine; TBG = thyroxine-binding globulin of the interalpha zone in paper electrophoresis; TBPA = thyroxinebinding prealbumin; TCT=DL-tetrachlorothyronine; TSH =thyroid-stimulating hormone.salicylate could interfere with T4 binding in electrophoretic systems in buffers other than barbital (11). It was therefore desired to examine T4 binding in the presence of these four agents in a system where prealbumin binding of T4 could be observed. Furthermore, the possible correlation between the effect of inhibition of thyroxine binding to TBPA and to TBG by various drugs with the in vivo effect of these drugs on the PBI was studied. METHODSA single batch of pooled normal human serum obtained from the Baxter Laboratories was used in all the studies. Butanol solutions of thyroxine were evaporated to dryness in vacuo prior to the addition of serum. The mixtures were incubated at 4°C for at least 24 hours before use and were kept in the cold until used.Electrophoresis in 0.12 M ammonium carbonate buffer, pH 8.4, was performed at room temperature on 3.75 X 49 cm strips of Whatman no. 3 MM filter paper. The edges of the strips were coated with a thin border of paraffin. The strips were saturated with buffer and suspended horizontally between two plastic clamps in a closed system. The strips were equilibrated for at least 1 hour before the ...