Figure 4. Temporally Reiterated Expression of the
lin-42a Isoform Coincides with Molts (A) Gene models for the three major isoforms of
lin-42 annotated in WS227. Black boxes represent exons; gray boxes represent untranslated sequences. The position and size of the
ok2385 and
n1089 deletions and sequences used to construct
lin-42a and
lin-42b/c transcriptional fusion genes are represented. Accession numbers for the
lin-42 isoforms are as follows: a, NM_001027005; b, NM_001027006; and c, NM_001027007. Tennessen and colleagues previously described these three isoforms as
lin-42d,
lin-42c, and
lin-42a, respectively, and identified a fourth transcript (
lin-42b) by rapid amplification of complementary DNA (cDNA) ends (RACE) [23]. Nucleotide positions correspond to C. elegans chromosome II (
gij212645681). (B-E) Larvae were synchronized in starvation-induced L1-diapause and then fed and cultivated at 25C for the indicated times. (B) Representative fluorescence images show expression of the
lin-42ap::gfp-pest reporter in the pharynx (arrowhead) and lateral hypodermis (arrows). The larva imaged at 16 hr was molting and had a cuticle cap over the mouth. The asterisk indicates GFP expressed in AIY neurons from the cotransformation marker
ttx-3p::gfp. (C) Frequency of GFP expression in the hypodermis of Ex[
lin-42ap::gfp-pest] and Ex[
lin-42bp::gfp-pest] larvae throughout the L1 stage. By definition, time 0 corresponded to release from starvation and time 1.0 corresponded to ecdysis, for every larva observed. n = 12 for Ex[
lin-42ap::gfp-pest] larvae; n = 21 for Ex[
lin-42bp::gfp-pest] larvae. (D) Detection of
lin-42a and
lin-42b transcripts expressed in wild-type larvae by RT-PCR. Amplification of
mlt-10 cDNAs provided an internal benchmark for the first molt. Detection of
ama-1 transcripts controlled for sample quality and equivalent loading of PCR products. (E) Expression of
lin-42ap::gfp-pest cycles throughout larval development. The intensity of fluorescence in transgenic larvae was measured using the quantitative image analysis software Volocity. For each time point, values indicate the mean pixel intensity (6SEM) of 12-27 distinct objects (areas R 50
mm2) within the lateral hypodermis of 4-9 different larvae. Early and midstage larvae were selected from partly synchronized populations, based on gonad development and body size. Molting larvae were selected based on the presence of cuticle caps over the mouth. (F) Detection of LIN-42A (red) in hypodermal nuclei of late L4-stage animals by immunofluorescence microscopy using anti-LIN-42A/B primary antibodies. In aaaEx30[
lin-42a sur-5::gfp]
lin-42(
ok2385) larvae, nuclear fluorescence from SUR-5::GFP was also detected. Arrows mark examples of LIN-42A-positive hypodermal nuclei. Scale bars represent 10 um.