Figure 1.
Anatomical expression patterns of fktf-1β::gfp::fktf-1b.
DIC and fluorescence images of transgenic S. stercoralis larvae. Each DIC image is a separate individual. All scale bars = 10 µm. (A,B) Transgenic first-stage larvae with GFP expression in the procorpus (arrow) of the pharynx. (C,D) Expression of the GFP::FKTF-1b(wt) transgene in the hypodermis of an L1. (E,F) Transgenic L3i expressing the GFP::FKTF-1b(wt) fusion protein in the hypodermis and a narrow band in the pharynx (arrow). (G) Sites of gfp expression under the direction of the fktf-1β promoter in 125 transgenic first-stage larvae from five or more experiments expressing gfp reporters under the direction of fktf-1β promoter. Due to variations in transformation rates between experiments, all transgenic larvae were pooled to quantify expression patterns. χ2 test, P = 2.9E-14.
Figure 2.
Intra-cellular localization of GFP::FKTF-1b phosphorylation mutants.
DIC and fluorescence images of representative hypodermal cells of transgenic S. stercoralis L1s. In all images, the nucleus is identified with an arrow. All scale bars = 10 µm. (A,B) L1 expressing pPV244(S238E/T240E), the “phospho-mimetic” GFP::FKTF-1b with fluorescence in the cytoplasm. (C,D) L1 expressing pPV234, GFP::FKTF-1b(wt), with fluorescence in both the cytoplasm and the nucleus. (E,F) L1 with strong nuclear localization of the FKTF-1b(4A) fusion protein. (G) Percentages of hypodermal cells in transgenic larvae with intra-cellular localization of GFP classified as “cytoplasmic”, “cytoplasmic/nuclear”, or “nuclear”. Results include transgenic larvae from at least two separate microinjection experiments per transgene. Phospho-mimetic: n = 47 GFP+ cells in 11 larvae. GFP::FKTF-1b: n = 29 GFP+ cells in 8 larvae. Phospho-null: n = 37 GFP+ cells in 8 larvae. χ2 test P = 1.23E-10.
Figure 3.
S. stercoralis transgenic L1s 24 hours post-hatch.
Representative images of S. stercoralis wild-type and transgenic larvae at 24 hours post-hatch. Each DIC image is a separate individual. All scale bars = 10 µm. By 24 hours, the wild-type (A) and GFP::FKTF-1b(wt) (B) larvae have grown to comparable lengths with similar numbers of intestinal granules. (C) GFP::FKTF-1b(dom-neg)–expressing larvae are a similar length but show reduced intestinal granularity. (D) GFP::FKTF-1b(dom-rep)–expressing animals exhibit almost a complete loss of intestinal architecture and do not survive past 24 hours. Comparable levels of transgene expression were seen in the transgenic animals at 24 hours (E–G). (H–K) Higher magnification images of intestinal cells near primordial gonad (arrow) showing fewer storage granules in the GFP::FKTF-1b(dom-neg) (J) and the loss of cell integrity in GFP::FKTF-1b(dom-rep)– (K) expressing larvae at 24 hours. (L) The presence of storage granules in the intestinal cells of wild-type and transgenic larvae was scored+if the majority of cells near the primordial gonad (arrow) contained granules. (M) Wild-type and transgenic larvae were scored for overall intestinal defects including structural damage and abnormal morphology. Larvae expressing either of the dominant-interfering transgenes exhibited intestinal defects at all timepoints (see Table S2 for counts). (N) All larvae were measured from the tip of the buccal cavity to the tip of the tail using the ImageJ program [27]. A minimum of 10 larvae per category per timepoint, with the exception of GFP::FKTF-1b(wild-type) L3i where n = 8, were examined and measured (Table S2). At 1 hour post-hatch, larvae expressing either of the dominant-interfering transgenes were slightly shorter than the wild-type larvae (P<0.05). At 24 hours post-hatch, only the GFP::FKTF-1b(dominant-repressor) transgene–expressing larvae were significantly shorter than the wild-type (P<0.01). The transgenic L3i expressing the GFP::FKTF-1b(dom-neg) transgene were not significantly shorter than the wild-type L3is (P>0.05).
Figure 4.
Third-stage S. stercoralis larvae expressing dominant interfering transgenes.
DIC and fluorescence images of transgenic L3s [n = 11 for GFP::FKTF-1b(dominant-negative), n = 8 for GFP::FKTF-1b(wild-type)]. All scale bars = 10 µm. Except where noted, each DIC image is a separate individual. (A,B) An L3i expressing the GFP::FKTF-1b(wt) transgene. (C,D) An L3 expressing the GFP::FKTF-1b(dominant-negative) transgene. (E) Higher magnification image of the tail of the L3 in (C) showing an L4 tail inside the characteristic L3i cuticle (arrow). Bacterial mass in the posterior intestine (white triangle) indicating failure of intestine to become radially constricted and blocked. (F) A second transgenic L3 showing the free-living L4 tail inside the L3i cuticle. (G) Wild-type L3i tail for comparison to transgenic tails. (H) A third transgenic L3 expressing the dominant-negative transgene with an elongated rhabditiform pharynx, with an intestine that is neither radially constricted nor blocked. (I) Grinder-like structure (black triangle) at the pharynx-intestine junction of (H). This structure is absent in wild-type L3is.