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1.
Figure 4

Figure 4. Gpc4 enhances the effects of full-length Wif1 in zebrafish embryos.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

Approximately 2 nL of mRNA of a given concentration was injected into one cell stage embryos, and embryos were scored at 28–30 hours post-fertilization. Images show representative examples of the penetrance of the short-tailed phenotype compared to an uninjected control. In top panels dorsal is up and anterior is to the left. Bar graphs show percentage of short-tailed embryos. The data is pooled from two independent experiments; frequencies were scored and their percentages were averaged. See Materials and Methods for information on RNA preparation.

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
2.
Figure 1

Figure 1. Zebrafish Wif1 inhibits D. melanogaster Wg.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

(A) Anterior margin of wild-type (WT) wing shows a dense array of sensory bristles (arrowhead). First longitudinal vein (L1, arrow) marks the anterior edge of the wing blade. (B) nub-Gal4-driven expression of UAS-wif1 eliminates anterior bristles (arrowhead) and disrupts L1 (arrow). (C) Anti-Sens staining along the presumptive wing margin in wild type late third instar wing disc. (D) dpp-Gal4-driven expression of UAS-wif1 in anterior cells of late third instar wing disc (marked green by anti-CiAct) eliminates anti-Sens staining locally and in adjacent posterior cells (arrow). In these and the remaining figures anterior is up. In adult wings distal is to the right, in wing discs ventral is to the right.

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
3.
Figure 6

Figure 6. Wif1 affects Hh activity in shf discs.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

(A–C) Wild-type wing discs, showing the regions with high levels (red bars) of CiAct (A) or Ptc (B), or showing the ventral accumulation of Hh-GFP in the posterior compartment after dorsal, ap-Gal4-driven expression of UAS-hh-GFP (C). (D–F) shf/Y wing discs show reductions in the width of domains expressing high levels of CiAct (D) or Ptc (E), and also show reduced ventral accumulation of Hh-GFP in the posterior compartment after dorsal ap-Gal4-driven expression of UAS-hh-GFP (F). (G–I) shf/Y wing discs with ap-Gal4-driven expression of UAS-wif1 have a broader domain of less intense anti-CiAct staining (G) and lower levels of anti-Ptc staining (H), but improve the ventral accumulation of Hh-GFP in the posterior compartment after ap-Gal4-driven expression of UAS-hh-GFP (I). Ventral Hh-GFP was more punctuate than in wild type discs (compare to C). To make it easier to see the differences in ventral Hh-GFP accumulation, levels were increased equally in the boxed regions in C, F, and I. Hh-GFP levels are quantified in Figure S9.

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
4.
Figure 5

Figure 5. The EGF-like domains are interchangeable between Wif1 and Shf during Wif1-dependent Wg inhibition.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

(A) Domain compositions of Wif1, Shf and the two chimeric constructs. Open boxes show the ‘WIF’ domain, filled boxes the EGF-like domains. (B–F) nub-Gal4-driven misexpression of respective transgenes. (B) UAS-WIFwif1-EGFshf strongly reduces the density of anterior wing margin bristles and interrupts L1. Arrow and arrowheads denote L1 or lack of thereof, respectively. (C) Co-expression of UAS-WIFwif1-EGFshf and UAS-dlp almost completely eliminates wing margin bristles and L1, and reduces the size of the wing. (D, E) Expression of either UAS-wif1 or UAS-WIFwif1-EGFshf similarly reduces ex-Wg levels on the surface of prospective margin cells (asterisks) and increases levels proximally. However, compared to UAS-wif1, UAS-WIFwif1-EGFshf expression does not increase ex-Wg as far proximally (compare red bars). (F, F′) Expression of two copies of UAS-WIFshf-EGFwif1 does not alter wing shape or size, and has no measurable effect on margin bristles (anterior margin details in F′).

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
5.
Figure 3

Figure 3. Dlp and Dally enhance the effects of Wif1 expression in Drosophila wings.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

(A–L) nub-Gal4 is used to drive transgene expression. (A) Wild-type wing. (B) Overexpression of UAS-dlp results in only slightly fewer bristles along the wing margin, no loss of L1 and no reduction in wing size. For a detailed comparison of bristle numbers, see Figure S3. (C) Expression of UAS-wif1 eliminates many bristles, interrupts L1 (arrow) and somewhat reduces wing size. (D) Co-expression of UAS-dlp and UAS-wif1 almost completely eliminates wing margin bristles and L1, and strongly reduces wing size. (E) Expression of UAS-wif1 causes much weaker wing margin defects in dlpA187/+ heterozygotes. (F) Overexpression of UAS-dally results in only very slightly fewer wing margin bristles and no obvious reductions in wing size. For detailed comparison of bristle numbers see Figure S3. (G) Combined expression of UAS-dally and UAS-wif1 almost completely eliminates wing margin bristles and L1, and further reduces wing size. (H) Expression of UAS-wif1 causes weaker wing margin defects in dally80/+ heterozygotes (e.g. more complete anterior L1; compare arrows in C and H). (I–L) EGF-depleted Wif1 is less effective at inhibiting Wg signaling and does not interact with Dlp. Control wings expressing pVal-UAS-wif1 show modest defects in margin development (I), which is synergistically enhanced by UAS-dlp; the arrow marks the interruption of L1 and the asterisks mark scalloping of the margin (J). pVal-UAS-wif1ΔEGF is less effective at reducing number of margin bristles than pVal-UAS-wif1 and its effects on wing margin development are not enhanced by co-expression of UAS-dlp (J). See Figure S3 for comparison of bristle numbers.

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
6.
Figure 2

Figure 2. Wif1 stabilizes Wg on Dlp-expressing cells in late third instar wing discs.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

Wing pouch regions of wing imaginal discs. (A) wg-lacZ expression along the prospective wing margin (asterisk) where prospective dorsal (D) and ventral (V) wing blade surfaces abut. (B) Extracellular Wg (ex-Wg) from the wg-expressing cells, which is high distally and lower proximally. (C) Pattern of nub-Gal4 expression, marked by UAS-GFP. In all subsequent panels, except for I-M, nub-Gal4 is used to drive UAS-transgene expression. (D) ex-Wg after expression of UAS-wif1. ex-Wg is higher on proximal cells than on distal ones (red bracket). (E) Anti-Dlp staining in wild-type wing disc. Dlp expression is downregulated in distal cells of the prospective wing margin (red bracket). (F) Anti-Dlp staining after UAS-wif1 expression. The width of the prospective wing margin region with reduced staining (red bracket) is narrowed compared to anti-Dlp staining in the wild-type disc in E. (G) ex-Wg staining after co-expression of UAS-wif1 and UAS-dlp. ex-Wg is increased at the wing margin (asterisk). (H) ex-Wg staining after co-expression of UAS-wif1 and UAS-dlp RNAi is similar to that in the wild-type disc in E. (I) Posterior expression of UAS-dlp (using hh-Gal4). ex-Wg accumulates in the posterior compartment. (J-M) Flpout actin-Gal4 (ac) clones marked with UAS-GFP (green). (J) High ex-Wg levels inside and, to a lesser extent, outside clones expressing: UAS-armS10, UAS-wif1, and UAS-dlp. (K) Low, largely unchanged ex-Wg levels inside clones expressing UAS-armS10 and UAS-dlp. (L) Reduced ex-Wg levels in clones expressing UAS-wif1 and UAS-armS10. zWIF1 increases ex-Wg outside the clone. (M) Reduced ex-Wg levels in clones expressing ArmS10. (N) After expression of pVal-UAS-wif1, ex-Wg staining is high proximally and low along the wing margin (asterisk). (O) Expression of pVal-UAS-wif1ΔEGF does not lead to a strong increase in proximal ex-Wg, and does not decrease ex-Wg along the wing margin (asterisk).

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.
7.
Figure 7

Figure 7. Vertebrate WIF domain regulates long-range Hh signaling.. From: The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling.

(A) Wild type wing showing the positions of the first through fifth longitudinal veins (L1–L5) and the position of the A/P compartment boundary (dashed line). (B) Reduced L3–L4 spacing in shf mutant wing (B) is not improved upon expression of UAS-shfΔWIF (B′). (C, D) Posterior, en-Gal4-driven expression of UAS-WIFwif1-EGFshf (C) or UAS-WIFshf-EGFwif1 (D) in shfx33/Y wings. UAS-WIFwif1-EGFshf strongly improved and UAS-WIFshf-EGFwif1 weakly improved L3–L4 spacing. (E) Comparison of L3–L4 spacing in wild type, shf2, and shfx33/Y with en-Gal4-driven UAS-construct expression. To compensate for differences in overall wing size, we normalized the L3–L4 distance to the distance between the anterior and posterior margins (red bars). In all but one case we presented the experimental normalized L3–L4 distances as percentages of the wild type normalized distances. However, since expression of UAS-WIFwif1-EGFshf reduced the size of the posterior compartment, and thus the distance between the anterior and posterior margins, we compared the normalized L3–L4 distance in shfx33 UAS-WIFwif1-EGFshf wings to the normalized L3–L4 distance in non-shf UAS-WIFwif1-EGFshf siblings (n = 31). Bars denote standard deviation. Two-tailed Student's t test showed no significant differences between UAS-WIFshf-EGFwif1 and UAS-shfΔEGF, or between shf2 and shf2, UAS-shfΔWIF. Differences between the other conditions were significant (p<0.0001). (F–I) Anti-Ptc staining in wild type (F), shfx33/Y (G), and shfx33/Y wing discs with ap-Gal4-driven expression of UAS-WIFwif1-EGFshf (H) or UAS-WIFshf-EGFwif1 (I). The improvement in the width of Ptc expression in shfx33 discs was stronger after expression of UAS-WIFwif1-EGFshf than UAS-WIFshf-EGFwif1. (J, K) shfx33/Y wing discs with ap-Gal4-driven expression of UAS-hh-GFP and UAS-WIFwif1-EGFshf (J) or UAS-hh-GFP and UAS-WIFshf-EGFwif1 (K). UAS-WIFwif1-EGFshf strongly improved the ventral accumulation of Hh-GFP, while the improvement with UAS-WIFshf-EGFwif1 was more modest (quantifications in Figure S9).

Andrei Avanesov, et al. PLoS Genet. 2012 February;8(2):e1002503.

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