First published online 11 March 2003
doi: 10.1242/jcs.00380
Modeling human peroxisome biogenesis disorders in the nematode Caenorhabditis elegans
Heather Thieringer1,*,
Britta Moellers2,
,
Gabriele Dodt2,
Wolf-H. Kunau2 and
Monica Driscoll1,
1 Department of Molecular Biology and Biochemistry, Rutgers University,
Piscataway, NJ 08554, USA
2 Institute für Physiologische Chemie, Ruhr-Universität Bochum,
D-44801 Bochum, Germany
* Present address: Department of Molecular Biology, Princeton University,
Princeton, NJ 08854, USA
Present address: Union Biometrica, Somerville, MA 02143, USA
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Fig. 1. GFP containing a PTS1 motif is imported into peroxisomes. Wild-type
nematodes were injected with the construct
pHSP16/2GFP—SKL to create the line,
Ex[pHSP-16/2GFP—SKL;pRF4(rol-6su1006)].
Transgenic animals were heat-shocked for 4 hours at 35°C and recovered at
20°C for 2 hours before photography. A distinctly punctate pattern was
observed in intestinal cells (A), in hypodermal cells (B) and in developing
embryos (C). Note that for photography we selected genetically mosaic animals
in which only a few cells harbored the transgene.
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Fig. 2. dsRNA interference directed against the C. elegans PEX-5 homolog
C36C6.6, prx-5, results in larval arrest. Young adult hermaphrodites
injected with dsRNA targeted to exon 6 of prx-5 laid eggs for a 12
hour period, 16 hours after injection. (A) RNAi knockdown of prx-5
results in developmental arrest at the L1/L2 stage. (B) Identical
magnification of a prx-5(RNAi) nematode 3 days after being laid, and
a time-matched wild-type worm. Bar, 10 µm. (C) After a 16 hour recovery
period postinjection, ten viable injected worms were placed on a plate for 12
hours to measure egg production. Egg production is reduced in
prx-5(RNAi) treated animals as compared to an unc-22(RNAi)
control injection.
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Fig. 3. Peroxisomal import is defective in prx-5(RNAi) nematodes.
Ex[pHSP16/2GFP-SKL] nematodes were injected with dsRNA
targeted to exon 6 of prx-5. Progeny were heat-shocked for 2 hours at
30°C and recovered for 1 hour. A disruption of peroxisomal localization of
the GFP—SKL is seen in (A), which results in the GFP remaining cytosolic
when compared with a control nematode in (B). In some animals we observed a
mosaic of GFP subcellular localization, in that both cells with cytosolic and
punctate GFP were observed in the same animal (C). Cells with GFP in all
panels are intestinal cells. Bars, 5 µm.
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Fig. 4. prx-5(RNAi)-arrested nematodes do not initiate postembryonic cell
divisions. Strain LT483 contains an integrated 2X rnr-1 promoter
driving expression of GFP. We injected this strain with dsRNA targeted to
prx-5 and examined the progeny. The animal in A and B has just two
faint cells that contain GFP. Eggs from this strain were hatched in the
absence of food to induce a post-hatching starvation-arrest (C,D).
prx-5(RNAi) animals and starvation-arrested animals of this strain
have similar GFP expression patterns, and in addition they lack
autofluorescence in their guts (A,C). (E) A young
prnr-1GFP L1 animal in which divided nuclei are apparent
by their more robust GFP signal. (F) An older prnr-1GFP L1
animal that contains numerous dividing nuclei. (G) A 4 day arrested
prx-5(RNAi). This animal has been arrested for 4 days, and does not
contain the numerous refractile structures found in a 4-day old
starvation-arrested worm (H). Bars, 10 µm.
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© The Company of Biologists Ltd 2003
