Figure S3.
madd-4's Post-Embryonic Expression Pattern. A. Shown is the
madd-4 genomic locus (with intron 9 highlighed in blue) along with the four constructs used to investigate
madd-4's expression pattern. The expression pattern from chromosomally-integrated versions the transcriptional reporter pPR721(madd-4Bp::YFP) (i.e. trIs79) and pPR731(madd-4Bp::RFP) (i.e. trIs80) is presented in Figure 3B and 3A, respectively, of the main text. In (B) through (D), the expression pattern from extra-chromosomal arrays of the transcriptional reporters pPR681(madd-4Ap::RFP) (on the left) and an integrated version of pPR731(madd-4Bp::RFP) (i.e. trIs80) (on the right) is presented. The expression pattern of an extra-chromosomal array of pPR725, which is a fosmid clone carrying a functional (see Figure 1K) bi-cistronically-tagged
madd-4 locus within the
madd-4 fosmid WMR0626cA02 is similar to that generated by pPR731(madd-4Bp::RFP). On the pPR725 schematic, the small purple box to the left of the red box (that represents coding sequence for mCherry) indicates a nuclear localization signal. See Tursun et al. (2009) for more details on bi-cistronic reporters. B-D. An analysis of the expression pattern of madd-4Ap::RFP (on the left) and madd-4Bp::RFP (on the right). The animals on the left habour an extrachromosomal transgenic array that contains pPR681(madd-4Ap::RFP) and a YFP marker for the array that is expressed in the pharynx (black asterisks). The animals on the right harbour the trIs80 integrated transgenic array that contains the pPR731(madd-4Bp::RFP) construct. In addition, all of the animals shown have the trIs8 chromosomally integrated transgenic array. trIs8 drives expression of YFP in all 26 gabaergic neurons from the
unc-25 promoter, including the 19 motor neurons in adults (and the six in young larvae) (Jin et al., 1999), and CFP in only select cholinergic motor neurons (from the
unc-129 neural specific promoter) (Colavita et al., 1998), and RFP in the excretory canal (marked with white asterisks). These are markers that allow us to unambiguously identify many of the neurons that express RFP from the
madd-4 promoters. 'B' shows typical L1 hatchlings. The RFP channel on the left in B (i.e. madd-4Ap::RFP) is increased in relative intensity in order to visualize the RFP-expressing cells. 'C' shows typical L2 larvae. 'D' shows typical adults. The white arrowheads indicate overlap between RFP-expressing cells, and CFP or YFP-expressing cells. In contrast to madd-4Bp::RFP expression, which is consistently high in all classes of commissural motor neurons in all larval stages and adults, animals carrying madd-4Ap::RFP express low levels of RFP in the first larval stage in both gabaergic and cholinergic motor neurons. madd-4Ap::RFP expression then increases dramatically after the L1 stage, but only in cholinergic cells. The scale bar represents 25 μm. This figure is related to Figure 3.