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First published online 21 May 2008
doi: 10.1242/dev.016972

Development 135, 2239-2249 (2008)
Published by The Company of Biologists 2008
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Genetic identification of HSD-1, a conserved steroidogenic enzyme that directs larval development in Caenorhabditis elegans

Dhaval S. Patel1, Lily L. Fang1, Danika K. Svy1, Gary Ruvkun2 and Weiqing Li1,*

1 Department of Biological Structure, University of Washington, Seattle, WA, USA.
2 Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA.


Figure 1
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  		Fig. 1. hsd-1 expression in the XXX cells globally regulates DAF-16 activity. (A-C) hsd-1 is exclusively expressed in the paired XXX cells of the head. (A) Phsd-1::GFP expression. (B) Psdf-9::RFP expression in the XXX cells. (C) Merged image showing that Phsd-1::GFP co-localizes with Psdf-9::RFP. (D-F) Level of DAF-16::GFP nuclear localization. Anterior of animal indicated by the arrows. (D) Diffuse (cytoplasmic) GFP in wild-type control L2 larva. Partially diffuse and punctate (nuclear) GFP (E) and almost entirely punctate GFP (F) in hsd-1(mg433); ncr-1(nr2022) L2d larvae.

 

Figure 2
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  		Fig. 2. Phenotypic analyses of hsd-1. (A) Brood sizes of N2, hsd-1, ncr-1 and ncr-2 single mutants, as well as hsd-1; ncr-1 and ncr-2; ncr-1 double mutants. The number of parent worms (n) is indicated above each column. Error bars: standard deviation (s.d.); asterisks indicate significantly reduced brood sizes compared with N2 (P<0.01) by Student's t-test. (B) Responses of different mutant/transgenic strains to two different concentrations of dauer pheromone extract: 4 µl/plate (light grey) and 8 µl/plate (dark grey). Total numbers scored are above the corresponding columns with the error bars representing the s.d. based on at least three replicates. Number in parentheses indicate average population density (worms/plate) ±s.d.

 

Figure 3
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  		Fig. 3. Population density in the ranges used in this report has minimal influence on dauer formation. In hsd-1(mg433); ncr-1(nr2022) worms (colored lines), sterol choice (not worm density) is the major effector of dauer formation at 25°C. hsd-1(mg433) (black line) single mutants, grown on standard cholesterol, display almost 100% dauer bypass at all population densities tested.

 

Figure 4
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  		Fig. 4. HSD-1 is involved in {Delta}4-dafachronic acid synthesis. (A) HSD-1 may function in the proposed biosynthetic pathway of {Delta}4- and {Delta}7-dafachronic acids [modified, with permission from Rottiers et al. (Rottiers et al., 2006Go)]. Predicted intermediates for biosynthesis of {Delta}7- and {Delta}4-dafachronic acids from cholesterol are indicated in the upper and lower branches of the pathway, respectively. The marked positions on the cholesterol structure (1-7) represent the standard numbering of the carbon atoms. 5-cholesten-3-one, in parentheses, is the presumed product, from cholesterol, after the first of the two-step 3β-HSD reactions. Shaded triangles and ovals indicate the sequential changes that HSD-1 is hypothesized to direct: triangles indicate 3β-dehydrogenase activity, ovals indicate isomerase activity. (B) The structures of ergosterol and β-sitosterol, the major fungal and plant sterols, respectively.

 

Figure 5
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  		Fig. 5. Effect of feeding different sterols on dauer arrest at 25°C. (A) hsd-1(mg433) dauers induced by pheromone at 4 µl/plate. (B) hsd-1(mg443); daf-36(k114) (light-gray bars) and hsd-1(mg433); daf-28(tm2308) (dark-gray bars). Total numbers scored are above the corresponding columns with the error bars representing the s.d. based on at least three replicates. Number in parentheses indicate average population density (worms/plate) ±s.d.

 

Figure 6
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  		Fig. 6. Effect of feeding sterols at different concentrations on dauer arrest at 25°C. Sterols were at 12.9 µM (1x), the standard concentration for cholesterol in NGM, unless indicated otherwise. (A) hsd-1(mg433); ncr-1(nr2022) with increasing sterol concentrations. (B) hsd-1(mg433); ncr-1(nr2022) with decreasing sterol concentrations. (C) ncr-2(nr2023); ncr-1(nr2022). Total numbers scored are above the corresponding columns with the error bars representing the s.d. based on at least three replicates. Number in parentheses indicate average population density (worms/plate) ±s.d.

 

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