N2 larvae were fed bacteria expressing dsRNA corresponding to the indicated genes, or control bacteria not expressing dsRNA of a worm gene (A). Panels A–D show the anterior, whereas (E) shows the mid-body of a larva. Black arrowheads mark unshed cuticle. White arrowhead (C) indicates the buccal capsule. Nomarski optics.

(A) Expression of mlt-8p::gfp-pest in an early L1 larva, a larva molting from the L1 to the L2 stage, and an early L2 stage larva. Black arrows indicate cuticle separated from the body of the molting larva. The particular larva shown at L2 was fluorescent before the L1/L2 molt.
(B) Ex[mlt-8p::gfp-pest] (dashed line) or Ex[mlt-10p::gfp-pest] (solid line) larvae were examined for fluorescence and for molting from late in the L1 stage until early adulthood. Graph shows the percent of worms that were fluorescent over time, on a scale normalized to the molting cycle of each worm under observation (see Materials and Methods).
(C) Cycles of fluorescence observed in the hypodermis and seam cells of Ex[mlt-9p::gfp-pest] (dashed line) or Ex[mlt-11p::gfp-pest] (solid line) larvae.
(D) mlt-10 messenger RNA detected by Northern analysis; ribosomal RNA stained with ethidium-bromide provides a loading control.

(1) Endocrine and possibly neuroendocrine cues trigger molting in C. elegans, 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 mlt-8, mlt-9, mlt-10, mlt-11, acn-1, and nas-37 in the hypodermis, as well as xrn-2 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 gfp fusion to the corresponding gene was expressed in epithelial cells. † indicates that the gene is required for expression of mlt-10p::gfp-pest in the hypodermis.

Expression of GFP (A,C,D,G) or GFP-PEST (B,E,F) from the promoters of the indicated genes.
(A) Fluorescence from qua-1p::gfp in the hypodermis and specialized epithelia.
(B) Fluorescence from nas-37p::gfp-pest in the seam cells and hypodermis of a late L4 stage larva.
(C) Fluorescence from mlt-9p::gfp in the seam cells and hypodermis of a late L3 stage larva.
(D) Fluorescence from xrn-2p::gfp 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.
(E) Fluorescence from acn-1p::gfp-pest in the seam cells and hypodermis of a late L1 stage larva.
(F) Fluorescence from mlt-11p::gfp-pest in the seam cells and hypodermis of a late L1 stage larva.
(G) Fluorescence from xrn-2p::gfp 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

(A) Ex[mlt-8p::gfp-pest] or Ex[mlt-10p::gfp-pest] larvae were fed bacteria expressing dsRNA for each gene indicated. Graph shows the percent of animals that were fluorescent before a defective molt, normalized to the percent of control larvae that were fluorescent before molting from the same stage. The number of larvae observed is shown in parenthesis. Note that RNAi of mlt-8 or acn-1 typically prevented completion of the L2/L3 molt, whereas RNAi of qua-1, fbn-1, or mlt-9 interfered most often with the L3/L4 or L4/A molts. RNAi of nhr-23 blocked the L3/L4 or L4/A molts in Ex[mlt-10p::gfp-pest] larvae, but prevented completion of the L2/L3 molt in most Ex[mlt-8p::gfp-pest] larvae. In control Ex[mlt-10p::gfp-pest] larvae, fluorescence was observed in 95% (n = 56), 100% (n = 43), or 94% (n = 48) of, respectively, L2, L3, or L4 stage animals. In control Ex[mlt-8p::gfp-pest] larvae, fluorescence was observed in 74% (n = 57) or 70% (n = 36) of L2 stage, and 90% (n = 49) of L4 stage animals. Pair-wise chi-square tests indicate that the decreased fraction of nhr-23(RNAi) or acn-1(RNAi) larvae that express mlt-8p::gfp-pest, and of nhr-23(RNAi), acn-1(RNAi), or mlt-8(RNAi) larvae that express mlt-10p::gfp-pest, relative to control animals, is significant, with p ≤ 0.001 in all cases.
(B) Ex[mlt-10p::gfp-pest] larvae were fed bacteria expressing dsRNA for each gene indicated, or control bacteria not expressing dsRNA of a worm gene. Graph shows the percent of larvae that were fluorescent late in the late L2, L3, and L4 stage, normalized to control animals, with values representing the weighted average of two independent experiments. ‡ indicates that larvae failed to develop to the stage of observation. Table S5 contains the raw data contributing to this figure.