Methylation of the Fourth Position of Cholestanol Is Not Required for Dauer Larva Formation

<p>Structural formulae and space-filling models of (A) cholestanol, (B) lophanol, and (C) 4αF-cholestanol. Abilities to support reproductive growth or dauer formation in the second generation are indicated. R, reproduction; D, dauer larva.</p

Depletion of Cholesterol Is Associated with a Decrease of Nonmethylated Sterols

<div><p>(A) Nematode-specific biosynthesis of 4-methylated sterols from exogenously added cholesterol. Open arrow shows the methylation at the fourth position. A vertical line indicates hydrophilic metabolites of cholesterol.</p> <p>(B) Cholesterol metabolism in the first (lanes 1 and 2) and the second (lanes 3–6) generations of worms derived from mothers fed with radioactive cholesterol. CE, cholesteryl esters; mS, methylated sterols (lophenol, 4-methylcholestenol); nmS, nonmethylated sterols (cholesterol, 7-dehydrocholesterol, lathosterol). The position of these compounds on TLC was determined by chromatography of cholesteryl stearate, lophenol, and cholesterol. E, eggs; L1, L1 larvae.</p></div

Mutant <i>daf-16</i> Worms Grown on Lophenol Form Defective Dauer Larvae

<div><p>(A) Low-magnification electron micrograph of lophenol-grown <i>daf-16</i>. The alae are defective although the striated layer (bracket) is visible. Note that the gut is not constricted and contains remnants of food.</p> <p>(B and C) High-magnification electron micrographs of lophenol-grown <i>daf-16</i> and wild-type dauer larvae. Arrowhead indicates an annular structure.</p></div

Growth on Lophenol Induces the Accumulation of DAF-16 in the Nuclei of Neurons in a DAF-12–Dependent Manner

<div><p>(A) When grown on cholesterol, the transgenic line DAF-16a::GFP/b^KO displays a diffuse staining in the cytoplasm and nuclei of many cells (only the pharynx region of an L3 larva is shown).</p> <p>(B) Staining of a larva of similar age by Hoechst. Note many nuclei in the pharynx.</p> <p>(C) The DAF-16a::GFP/b^KO line grown on lophenol shows strong staining of nuclei in neurons of the pharynx, tail, and ventral cord of a dauer larva.</p> <p>(D) An L3 larva of DAF-16a::GFP/b^KO in a <i>daf-12</i> null background grown on lophenol. Note the diffuse fluorescence in the pharynx cell similar to that shown in (A).</p></div

Depletion of Cholesterol Leads to Formation of Dauer-Like Larvae

<div><p>(A) For worms grown on plates without cholesterol, the first-generation worms (F1) laid fewer eggs than normal (133 ± 10 versus 210 ± 12) and more eggs failed to hatch. Filled ovals depict unhatched eggs; 17% of eggs laid by cholesterol-depleted worms failed to hatch in comparison to 0.02% of those laid by cholesterol-fed worms. The second generation of worms (F2) arrested after the completion of the L1-to-L2 molt.</p> <p>(B) Light micrograph of an arrested L2 larva.</p> <p>(C) Electron micrograph of the lateral cuticle of an arrested L2 larva 5 d after arrest, showing two cuticles. The outer cuticle resembles that of an L2, which has no alae, and the inner cuticle resembles the dauer cuticle with its distinctive striated layer (bracket) and an incomplete dauer ala.</p> <p>(D) Electron micrograph of an arrested L2 <i>daf-12</i> mutant grown without cholesterol. Arrows indicate vesicles beneath the cuticle which are not present in normal larvae.</p> <p>(E) Electron micrograph of a wild-type L2 larva grown with normal cholesterol.</p></div

Cross Talk between Two Signalling Pathways in the Process of Dauer Larva Formation

<p>Pheromone accumulated under the conditions of overcrowding or starvation induces the inhibition of gamravali production via the TGF-β pathway. Genes, mutants of which produced dauer larvae on lophenol, are shown in blue. Activated by the absence of gamravali, DAF-12 initiates the process of dauer larva production. One of its activities is to recruit DAF-16 into nuclei of neurons (shown in red). The insulin-like pathway has several physiological functions, among them the regulation of longevity and thermotolerance, and could be involved in the process of dauer formation by regulating the levels of gamravali via DAF-16.</p

Partial Purification of Gamravali

<div><p>(A) Lipidic extract of worms was separated by HPLC using a C₁₈ reverse-phase column. Retention times of (1) 7-dehydrocholesterol, (2) cholesterol/lathosterol, and (3) ecdysone/estradiol/testosterone are indicated with arrows.</p> <p>(B) Fractions of 2 min from the chromatography were assayed for the activity to rescue the formation of dauer larvae induced in the presence of lophenol.</p></div