Figure 2. Alternative splicing of UNC-62 generates intestine-specific unc-62(7a) and neuronal/hypodermal-specific unc-62(7b) isoforms.
(A) Strains expressing isoform-specific reporters for unc-62(7a) and unc-62(7b) show stage- and tissue-specific expression. (top) Beginning with the transgenic fosmid described in Figure 1C, stop codons were inserted into unc-62 exons 7a or 7b by site-directed mutagenesis to obtain isoform-specific reporters. During this process, a kanamycin resistance cassette was inserted into the eighth intron of unc-62. (bottom) Alternative isoforms of UNC-62 show tissue-specific expression in adults. (left) UNC-62:GFP is observed in intestinal, neuronal, and hypodermal cells. (center) UNC-62(7a):GFP is highly expressed in the intestine in the L4 larval stage and young adults, but is not visible in other tissues. (right) UNC-62(7b):GFP is not observed in the intestine, but is expressed in the hypodermis (not shown), the ventral nerve cord, and other neurons. Strains were imaged in a glo-4(ok623) background to limit gut autofluorescence. (B) UNC-62(7a):GFP expression decreases between day 1 and day 8 of adulthood. Strain contains glo-4(ok623) to reduce gut autofluorescence, and expression was quantified only in the first pair of intestinal nuclei (dotted red circles). (C) Quantification of UNC-62(7a):GFP as shown in (B). Bars indicate mean fluorescence (in arbitrary units) observed from populations of at least 32 worms measured at different days of adulthood, with error bars indicating standard error of the mean. Day 1 expression was significantly higher compared to expression in days 3, 8, or 12 (p<10−4 by Student's t-test) in two independent experiments (see Figure S2).