Figure 4

Evaluation of LBD function for FAX-1. Design and results of the experiments with LBD swaps among different NR2E LBDs and a deletion of the FAX-1 LBD. A. Schematic showing design of the genomic constructs created to test LBD function, as described in Methods. Percentages above LBD boxes indicate the percent identity of the swapped LBD to the C. elegans FAX-1 LBD. B. Results of movement assays. Figure shows forward movement rates for control wild-type, fax-1(gm83) fax-1(gm83) lin-15(n765) and FAX-1::INV FAX-1 strains, as well as each swap construct transgene. C. Rescue of the AVKR pathfinding defect in fax-1(gm83) mutants by LBD swap transgenes. The wild-type hermaphrodite shows a prominent single AVKR axon located in its proper position in the left bundle of the ventral nerve cord [56]. In the FAX-1:: INV FAX-1 negative control, the AVKR axon is missing from the ventral nerve cord due to misrouting [24]. The FAX-1::FAX-1 and FAX-1:: Δ LBD transgenic animals show the rescued wild-type anatomy. Rescue by FAX-1::Cb FAX-1, FAX-1::NHR-111, and FAX-1::NHR-67 transgenes was equivalent to the examples shown. The circular circuitry of FMRFamide-positive axons around the vulva is at the right side of each figure. All views are ventral views. D. Immunofluorescence demonstrating expression of fusion and deletion transgenes in fax-1(gm83) lin-15(n765) embryos. Left panels show Cy3 fluorescence detecting the FAX-1 DBD, right panels show matching DAPI staining of nuclei. The FAX-1::FAX-1 and FAX-1::Δ LBD embryos are a somewhat earlier stage (“late comma”), as compared to the FAX-1::NHR-111 and FAX-1::NHR-67 embryos (“two-fold elongation”). The anterior side of each embryo is oriented toward the top. Because of movement of elongation-stage embryos within the egg, orientation of each embryos varies.