Summer Snowfall Workshop : Scattering Properties of Realistic Frozen Hydrometeors from Simulations and Observations, as well as Defining a New Standard for Scattering Databases

Peer reviewe

The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling

Electric Actuator Demo

Author Abstract: The problem, as defined by Bimba, is to create a portable demonstration case for two actuators that can be easily brought to various clients as a sales tool. Bimba’s customer preferences and priorities have ranked portability, affordability, functionality, and a professional look as their highest requests. The team’s solution is to provide a case with wheels and a handle that keeps this weight at a minimum while having a working system of the B27 and RS9 actuator with their associated servo motors within the case. The actuators will be able to function within the box to demonstrate various motion paths. A program will be written to create these motion paths using a toggle on the case’s dashboard

Wif1 affects Hh activity in <i>shf</i> discs.

<p>(A–C) Wild-type wing discs, showing the regions with high levels (red bars) of Ci^Act (A) or Ptc (B), or showing the ventral accumulation of Hh-GFP in the posterior compartment after dorsal, <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (C). (D–F) <i>shf</i>/<i>Y</i> wing discs show reductions in the width of domains expressing high levels of Ci^Act (D) or Ptc (E), and also show reduced ventral accumulation of Hh-GFP in the posterior compartment after dorsal <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (F). (G–I) <i>shf</i>/<i>Y</i> wing discs with <i>ap-Gal4</i>-driven expression of <i>UAS-wif1</i> have a broader domain of less intense anti-Ci^Act staining (G) and lower levels of anti-Ptc staining (H), but improve the ventral accumulation of Hh-GFP in the posterior compartment after <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> (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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s009" target="_blank">Figure S9</a>.</p

The EGF-like domains are interchangeable between Wif1 and Shf during Wif1-dependent Wg inhibition.

<p>(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) <i>nub-Gal4</i>-driven misexpression of respective transgenes. (B) <i>UAS-WIF^wif1-EGF^shf</i> 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 <i>UAS-WIF^wif1-EGF^shf</i> and <i>UAS-dlp</i> almost completely eliminates wing margin bristles and L1, and reduces the size of the wing. (D, E) Expression of either <i>UAS-wif1</i> or <i>UAS-WIF^wif1-EGF^shf</i> similarly reduces ex-Wg levels on the surface of prospective margin cells (asterisks) and increases levels proximally. However, compared to <i>UAS-wif1</i>, <i>UAS-WIF^wif1-EGF^shf</i> expression does not increase ex-Wg as far proximally (compare red bars). (F, F′) Expression of two copies of <i>UAS-WIF^shf-EGF^wif1</i> does not alter wing shape or size, and has no measurable effect on margin bristles (anterior margin details in F′).</p

Wif1 stabilizes Wg on Dlp-expressing cells in late third instar wing discs.

<p>Wing pouch regions of wing imaginal discs. (A) <i>wg-lacZ</i> 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 <i>wg</i>-expressing cells, which is high distally and lower proximally. (C) Pattern of <i>nub-Gal4</i> expression, marked by <i>UAS-GFP</i>. In all subsequent panels, except for I-M, <i>nub-Gal4</i> is used to drive UAS-transgene expression. (D) ex-Wg after expression of <i>UAS-wif1</i>. 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 <i>UAS-wif1</i> 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 <i>UAS-wif1</i> and <i>UAS-dlp</i>. ex-Wg is increased at the wing margin (asterisk). (H) ex-Wg staining after co-expression of <i>UAS-wif1</i> and <i>UAS-dlp RNAi</i> is similar to that in the wild-type disc in E. (I) Posterior expression of <i>UAS-dlp</i> (using <i>hh-Gal4</i>). ex-Wg accumulates in the posterior compartment. (J-M) Flpout <i>actin-Gal4</i> (<i>ac</i>) clones marked with <i>UAS-GFP</i> (green). (J) High ex-Wg levels inside and, to a lesser extent, outside clones expressing: <i>UAS-arm^S10</i>, <i>UAS-wif1</i>, and <i>UAS-dlp</i>. (K) Low, largely unchanged ex-Wg levels inside clones expressing <i>UAS-arm^S10</i> and <i>UAS-dlp</i>. (L) Reduced ex-Wg levels in clones expressing <i>UAS-wif1</i> and <i>UAS-arm^S10</i>. zWIF1 increases ex-Wg outside the clone. (M) Reduced ex-Wg levels in clones expressing Arm^S10. (N) After expression of <i>pVal-UAS-wif1</i>, ex-Wg staining is high proximally and low along the wing margin (asterisk). (O) Expression of <i>pVal-UAS-wif1</i>Δ<i>EGF</i> does not lead to a strong increase in proximal ex-Wg, and does not decrease ex-Wg along the wing margin (asterisk).</p

Dlp and Dally enhance the effects of Wif1 expression in <i>Drosophila</i> wings.

<p>(A–L) <i>nub-Gal4</i> is used to drive transgene expression. (A) Wild-type wing. (B) Overexpression of <i>UAS-dlp</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a>. (C) Expression of <i>UAS-wif1</i> eliminates many bristles, interrupts L1 (arrow) and somewhat reduces wing size. (D) Co-expression of <i>UAS-dlp</i> and <i>UAS-wif1</i> almost completely eliminates wing margin bristles and L1, and strongly reduces wing size. (E) Expression of <i>UAS-wif1</i> causes much weaker wing margin defects in <i>dlp^A187/+</i> heterozygotes. (F) Overexpression of <i>UAS-dally</i> results in only very slightly fewer wing margin bristles and no obvious reductions in wing size. For detailed comparison of bristle numbers see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a>. (G) Combined expression of <i>UAS-dally</i> and <i>UAS-wif1</i> almost completely eliminates wing margin bristles and L1, and further reduces wing size. (H) Expression of <i>UAS-wif1</i> causes weaker wing margin defects in <i>dally⁸⁰</i>/+ 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 <i>pVal-UAS-wif1</i> show modest defects in margin development (I), which is synergistically enhanced by <i>UAS-dlp</i>; the arrow marks the interruption of L1 and the asterisks mark scalloping of the margin (J). <i>pVal-UAS-wif1</i>Δ<i>EGF</i> is less effective at reducing number of margin bristles than <i>pVal-UAS-wif1</i> and its effects on wing margin development are not enhanced by co-expression of <i>UAS-dlp</i> (J). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s003" target="_blank">Figure S3</a> for comparison of bristle numbers.</p

Zebrafish Wif1 inhibits <i>D. melanogaster</i> Wg.

<p>(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) <i>nub-Gal4</i>-driven expression of <i>UAS-wif1</i> 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) <i>dpp-Gal4</i>-driven expression of <i>UAS-wif1</i> in anterior cells of late third instar wing disc (marked green by anti-Ci^Act) 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.</p

Vertebrate WIF domain regulates long-range Hh signaling.

<p>(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 <i>shf</i> mutant wing (B) is not improved upon expression of <i>UAS-shf</i>Δ<i>WIF</i> (B′). (C, D) Posterior, <i>en-Gal4</i>-driven expression of <i>UAS-WIF^wif1-EGF^shf</i> (C) or <i>UAS-WIF^shf-EGF^wif1</i> (D) in <i>shf^x33</i>/<i>Y</i> wings. <i>UAS-WIF^wif1-EGF^shf</i> strongly improved and <i>UAS-WIF^shf-EGF^wif1</i> weakly improved L3–L4 spacing. (E) Comparison of L3–L4 spacing in wild type, <i>shf²</i>, and <i>shf^x33/Y</i> with <i>en-Gal4</i>-driven <i>UAS-construct</i> 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 <i>UAS-WIF^wif1-EGF^shf</i> 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 <i>shf^x33 UAS-WIF^wif1-EGF^shf wings</i> to the normalized L3–L4 distance in non-<i>shf UAS-WIF^wif1-EGF^shf</i> siblings (<i>n = 31</i>). Bars denote standard deviation. Two-tailed Student's t test showed no significant differences between <i>UAS-WIF^shf-EGF^wif1</i> and <i>UAS-shfΔEGF</i>, or between <i>shf²</i> and <i>shf²</i>, <i>UAS-shfΔWIF</i>. Differences between the other conditions were significant (<i>p</i><0.0001). (F–I) Anti-Ptc staining in wild type (F), <i>shf^x33</i>/<i>Y</i> (G), and <i>shf^x33</i>/<i>Y</i> wing discs with <i>ap-Gal4</i>-driven expression of <i>UAS-WIF^wif1-EGF^shf</i> (H) or <i>UAS-WIF^shf-EGF^wif1</i> (I). The improvement in the width of Ptc expression in <i>shf^x33</i> discs was stronger after expression of <i>UAS-WIF^wif1-EGF^shf</i> than <i>UAS-WIF^shf-EGF^wif1</i>. (J, K) <i>shf^x33</i>/<i>Y</i> wing discs with <i>ap-Gal4</i>-driven expression of <i>UAS-hh-GFP</i> and <i>UAS-WIF^wif1-EGF^shf</i> (J) or <i>UAS-hh-GFP</i> and <i>UAS-WIF^shf-EGF^wif1</i> (K). <i>UAS-WIF^wif1-EGF^shf</i> strongly improved the ventral accumulation of Hh-GFP, while the improvement with <i>UAS-WIF^shf-EGF^wif1</i> was more modest (quantifications in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#pgen.1002503.s009" target="_blank">Figure S9</a>).</p

Gpc4 enhances the effects of full-length Wif1 in zebrafish embryos.

<p>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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002503#s4" target="_blank">Materials and Methods</a> for information on RNA preparation.</p