Temporal control of cell-specific transgene expression in Caenorhabditis elegans

Abstract

Cell-specific promoters allow only spatial control of transgene expression in Caenorhabditis elegans. We describe a method, using cell-specific rescue of heat-shock factor-1 (hsf-1) mutants, that allows spatial and temporal regulation of transgene expression. We demonstrate the utility of this method for timed reporter gene expression and for temporal studies of gene function.

Figure 1.—

Temporal control of cell-specific transgene expression. In an animal lacking the normal heat-shock response due to loss of HSF-1 activity, hsf-1 is introduced in a desired subset of cells using a cell-specific promoter (pro). Since HSF-1 is present only in targeted cells, heat shock results in cell-specific transcription from heat-shock-responsive promoters (hsp). Therefore, any transgene under the control of a heat-shock promoter will be selectively expressed in the desired cells following a heat shock. Blue circles, HSF-1 protein. Green hexagons, protein of interest.

Figure 2.—

Spatiotemporal control of GFP expression using a heat-shock promoter. (A) Amphid sheath cell-specific labeling. Arrow, sheath cell body. Arrowhead, sheath cell process. Sheath cell-specific hsf-1 rescue was achieved by driving hsf-1 cDNA expression using a 5-kb promoter region of vap-1. GFP was under the control of the hsp-16.2 promoter. Transgenic animals with the genotype hsf-1(sy441); nsEx992 [P_vap-1hsf-1; P_hsp-16.2GFP; pRF4] were obtained by germline injection. The plasmid pRF4 encodes rol-6(su1006), a dominant marker used for selection of transgenic animals (Mello et al. 1991). Animals were incubated for 30 min at 34° and allowed to recover at 20° for 1 hr before imaging; an adult animal is shown. (B) DIC image of the animal in A. (C) Pharynx GFP expression. An adult animal expressing GFP in pharyngeal muscles after a 30-min heat shock. The genotype of the animal shown is hsf-1(sy441); nsEx1730 [P_myo-2hsf-1; P_hsp-16.2GFP; P_hsp-16.41GFP; pRF4]. (D) DIC image of the animal in C. (E) Ciliated neuron-specific GFP expression following heat shock. Animals in E–J have the genotype hsf-1(sy441); nsEx1129 [P_osm-6hsf-1; P_hsp-16.2GFP; pRF4]. In the animal in E, labeling is seen in four amphid neurons. (F) DIC image of the animal in E. (G) Heat-shock-induced GFP expression in ciliated neurons of the phasmid, a sensory organ in the tail, in an adult animal. (H) DIC image of the animal in G. (I) Neuronal GFP labeling in a twofold embryo. (J) DIC image of the animal in I. In all cases, anterior is at the top. Bar, 10 μm.

Figure 3.—

Cell-specific rescue of che-2(e1033) neuronal dye-filling defects. (A) Dye filling in an L4 animal of the genotype hsf-1(sy441); che-2(e1033); nsEx1555 [P_osm-6hsf-1; P_hsp-16.2che-2; pRF4] after a 30-min heat shock. Dye filling was performed 8 hr after heat shock by soaking for 20 min in 5 μg/ml DiI. Rescue was seen in all dye-filling amphid neurons. (B) DIC image of the animal in A. (C) Same as A, except that dye filling is restricted to neurons expressing sra-6. The genotype of the L4 animal shown is hsf-1(sy441); che-2(e1033); nsEx1552 [P_sra-6hsf-1; P_hsp-16.2che-2; pRF4]. (D) DIC image of the animal in C. Anterior, top. Arrows, neuronal cell bodies. Arrowheads, dendritic processes. Bar, 10 μm.