The levels of <i>lin-14</i> mRNA derived from the wild-type and the <i>lin-14(n536n540)</i> mutant allele bearing a 3′UTR deletion are similar.

<p>Shown are Northern blots of embryos and L4 staged <i>lin-14(n536n540)/szT1</i> heterozygotes, using probes from the <i>lin-14</i> (top), histone (middle) and vitellogenin genes (bottom). The levels of 3.5-kb wild-type <i>lin-14</i> and 2.9-kb <i>lin-14(n536n540)</i> mRNA are almost equal at both the embryonic and L4 stages. Histone mRNA is used as a control for even loading. Vitellogenin mRNA, encoding yolk polypeptides, which is most abundant during oogenesis, is shown to indicate animal stages.</p

Temporal analyses of <i>lin-14</i> mRNA, protein, and <i>lin-4</i> miRNA levels in wild-type and <i>lin-14(n355n679)</i> mutant animals.

<p>(A, C) Quantification of <i>lin-14</i> mRNA, LIN-14 protein and <i>lin-4</i> miRNA from early L1 (0 hours post-feeding) to early L2 (24 hours post-feeding) in wild-type (A) and <i>lin-14(n355n679)</i> mutant animals (C). All results are shown relative to the level at the 0 hour time point. Error bars represent SEM for two independent experiments. (B, D) Representative immunoblots showing the abundance of LIN-14 protein in wild-type (B) and <i>lin-14(n355n679)</i> mutant animals (D) over 24 hours of development. Actin serves as a control for the normalization of LIN-14.</p

Functional Genomic Analysis of <em>C. elegans</em> Molting

<div><p>Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in <em>C. elegans</em> through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development.</p> </div

Models of EAK-4, SDF-9, and EAK-6 Function

<div><p>(A) Cell autonomous signaling in XXX. A schematic of one XXX cell is shown. EAK proteins function in parallel with AKT-1 and in the same pathway as AKT-2 to promote nondauer development by potentiating DAF-9/CYP27A1 function either directly or indirectly. DAF-9/CYP27A1 synthesizes 3-keto-7(5α)-cholestenoic acid, a ligand that promotes reproductive development by inhibiting the nuclear hormone receptor DAF-12 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b091" target="_blank">91</a>]. DAF-16/FoxO is denoted with dashed lines, since DAF-16A does not appear to be expressed in XXX. It is not known whether other DAF-16/FoxO isoforms or DAF-2/InsR are expressed in XXX.</p> <p>(B) Nonautonomous signaling from XXX. A schematic of the anterior portion of an animal is shown with the head pointing left. The pharynx is also shown. XXX cells are denoted by red ovals, and DAF-16/FoxO-expressing cells are denoted by green ovals. EAK proteins in the XXX cells generate signals that regulate the synthesis or secretion of a hormone that inhibits DAF-16/FoxO function in other cells.</p></div

Expression of AKT-1 in XXX is Sufficient to Rescue the 25 °C Dauer Phenotype of an <i>eak-4;akt-1</i> Double Mutant

<p><i>eak-4(mg326);akt-1(mg306)</i> double mutant animals carry a transgene containing the <i>akt-1</i> genomic region and 3' untranslated region under the control of the <i>eak-4</i> promoter. Three independent lines rescue <i>eak-4(mg326);akt-1(mg306)</i> dauer arrest at 25 °C. Error bars indicate standard deviation. This experiment was performed twice. Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals scored.</p

Expression of Molting Gene <i>gfp</i> Fusion Genes

<div><p>Expression of GFP (A,C,D,G) or GFP-PEST (B,E,F) from the promoters of the indicated genes.</p> <p>(A) Fluorescence from <i>qua-1p::gfp</i> in the hypodermis and specialized epithelia.</p> <p>(B) Fluorescence from <i>nas-37p::gfp-pest</i> in the seam cells and hypodermis of a late L4 stage larva.</p> <p>(C) Fluorescence from <i>mlt-9p::gfp</i> in the seam cells and hypodermis of a late L3 stage larva.</p> <p>(D) Fluorescence from <i>xrn-2p::gfp</i> in the pharyngeal myoepithelium (P) of a late L1 stage larva. Only the head of the worm is shown. The less intense fluorescence anterior to the posterior bulb of the pharynx likely corresponds to neurons.</p> <p>(E) Fluorescence from <i>acn-1p::gfp-pest</i> in the seam cells and hypodermis of a late L1 stage larva.</p> <p>(F) Fluorescence from <i>mlt-11p::gfp-pest</i> in the seam cells and hypodermis of a late L1 stage larva.</p> <p>(G) Fluorescence from <i>xrn-2p::gfp</i> in an adult worm, showing the intestine, a neuronal projection along the ventral cord, and a sensory neuron. The anterior of the worm faces right in all pictures</p></div

A Model for Molting of <i>C. elegans</i>

<p>(1) Endocrine and possibly neuroendocrine cues trigger molting in <i>C. elegans,</i> stimulating epithelial cells to remodel the exoskeleton near the end of each larval stage. (2) Transcriptional cascades involving NHRs alter gene expression in response to the endocrine cue. In particular, NHR-23 directly or indirectly activates expression of many genes, including <i>mlt-8, mlt-9, mlt-10, mlt-11, acn-1,</i> and <i>nas-37</i> in the hypodermis, as well as <i>xrn-2</i> in the pharyngeal myoepithelium. (3) Factors downstream of NHR-23, including MLT-8 and ACN-1, amplify the signal to molt. Signaling via transmembrane proteins likely stimulates release of the old cuticle. (4) Extracellular matrix proteins and secreted enzymes identified in our screen contribute to the new cuticle or regulate release of the old one. We expect precise regulation of these transmembrane proteins and secreted enzymes to accompany the molt. In theory, intercellular signaling might coordinate events in different epithelial cells, the muscle, and the intestine. We further expect secreted signals to provide feedback on the status of the molt to endocrine regulators. The Hint domain protein QUA-1 is a good candidate for a signal secreted from the hypodermis that might amplify a cue for ecdysis, signal to adjacent tissues, or provide feedback. Green shading indicates that a <i>gfp</i> fusion to the corresponding gene was expressed in epithelial cells. † indicates that the gene is required for expression of <i>mlt-10p::gfp-pest</i> in the hypodermis<i>.</i></p

A Functional DAF-16A::GFP Fusion Protein Is Not Expressed in XXX

<p>Wild-type animals harboring integrated DAF-16A::GFP and <i>sdf-9p</i>::RFP arrays were analyzed by confocal microscopy. Representative photographs of a single animal are shown. The animal is oriented anterior left and dorsal up. Merging of GFP and RFP images reveals no colocalization of fluorescent proteins.</p

EAK-4, SDF-9, and EAK-6 Localize to the Plasma Membrane of the XXX Cells

<div><p>(A) <i>eak-4, sdf-9,</i> and <i>eak-6</i> promoters drive expression in the same cells. Wild-type animals harboring an extrachromosomal array with <i>eakp::GFP</i> and <i>sdf-9p::RFP</i> constructs were analyzed by fluorescence microscopy. Representative photographs are shown. Animals are oriented anterior left and dorsal up.</p> <p>(B) EAK-4::GFP, SDF-9::GFP, and EAK-6::GFP fusion proteins localize to the plasma membrane of XXX. Animals harboring EAK::GFP translational fusion constructs and an integrated <i>sdf-9p::RFP</i> array were analyzed using fluorescence microscopy. Representative photographs of a XXX cell are shown.</p> <p>(C) Mutation of the invariant glycine in the <i>N</i>-myristoylation motif of EAK-4 abrogates plasma membrane localization. Animals harboring either a wild-type EAK-4::GFP construct or an EAK-4::GFP construct with the glycine at position 2 mutated to alanine (G2A) were analyzed using fluorescence microscopy.</p></div

Dauer Formation Phenotypes of <i>eak</i> Mutants

<p><i>eak</i> single mutants, <i>eak-x;eak-y</i> double mutants, or <i>eak;akt-1</i> double mutants were assayed for dauer arrest at (A) 25 °C and (B) 27 °C. (C) <i>eak</i> 27 °C dauer arrest phenotypes are suppressed by a mutation in <i>daf-16/FoxO</i>. (D) <i>akt-1, eak-4,</i> and <i>eak-6</i> 27 °C dauer arrest phenotypes are suppressed by RNAi of <i>daf-16/FoxO</i> and <i>daf-12/NHR</i>. All error bars indicate standard deviation. All experiments were performed three times. Refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals scored. <i>eak-5</i> is allelic to the synthetic dauer formation gene <i>sdf-9</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-b062" target="_blank">62</a>] and is referred to as <i>sdf-9</i> throughout the paper. Mutant alleles used were <i>daf-2(e1370), akt-1(mg306), eak-4(mg348), sdf-9(mg337)</i> and <i>sdf-9(ut187), eak-6(mg329),</i> and <i>daf-16(mgDf47)</i>. <i>sdf-9(ut187)</i> was used to construct the <i>eak-4;sdf-9, eak-6;sdf-9,</i> and <i>daf-16;sdf-9</i> double mutants. Multiple alleles of <i>eak-4</i> and <i>sdf-9</i> yielded phenotypes similar to those shown. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020099#pgen-0020099-st001" target="_blank">Table S1</a> for numbers of animals assayed.</p