Hh sequestration decreases wing size in both species and relative eyespot size in <i>J. coenia</i>.

<p> Forewing height in <i>B. anynana</i> (A) and <i>J. coenia</i> (B) and forewing area in <i>J. coenia</i> (C) are smaller in 5E1-injected butterflies compared with NS1-injected controls. Relative eyespot size, e.g., the diameter of the black and gold rings of the Cu1 eyespot on both ventral (vent) and dorsal (dors) surfaces of <i>J. coenia</i> is also smaller in 5E1-injected individuals (D-F; mean trait values are displayed for a wing height of 16 mm). GLM analyses use sex as a grouping variable but here sexes are plotted together. (G) Regression of black disc diameter of Cu1 dorsal eyespot on wing height for <i>B. anynana</i> (left) and <i>J. coenia</i> (right). In <i>J. coenia</i>, 5E1-injected individuals (red dots) display significantly smaller Cu1 eyespots relative to NS1-injected individuals (green dots) of comparable size, while <i>B. anynana</i> eyespots are not significantly smaller for an individual of a given size.</p

F statistics and p-values for GLM analysis testing for differences in wing compartment size in <i>B. anynana</i> across treatments.

<p>Individuals were injected with either 5E1 antibody or NS1 control media as larvae. Treatment and sex were used as fixed factors and wing height was used as a covariate. Treatment and sex were not significant across analyses.</p

Measurements taken of adult <i>B. anynana</i> and <i>J. coenia</i> wings.

<p>Wing height and wing area (<i>J. coenia</i> only) and a series of eyespot trait diameters for the M1 and Cu1 eyespots were measured on both ventral (left) and dorsal surfaces (right). R5, R4, M1, M2, M3, Cu1, Cu1+Pc, and 1A+2A refer to the wing compartments that were individually measured in <i>B. anynana</i> wings only. Their combined height defined the <i>B. anynana</i> wing height. w: white center; b: black disc; g: gold ring; in: inner ring (from the distal border of the black patch to the proximal border of the orange patch).</p

Differential expression of Hh signaling pathway members in <i>Junonia coenia</i> and <i>Bicyclus anynana</i> larval hindwings.

<p>Summary of mRNA (italics) and protein expression data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Keys1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Saenko1" target="_blank">[9]</a>. <i>hedgehog</i> (<i>hh</i>) and its receptor <i>patched</i> (<i>ptc</i>) are expressed in primitive patterns throughout the posterior wing compartment and in a narrow anterior domain abutting that compartment, respectively. These two genes are also expressed in novel domains in <i>J. coenia</i> but not in <i>B. anynana</i>: flanking the developing eyespot centers, and in the centers, respectively. The other depicted genes share a similar expression pattern between <i>J. coenia</i> and <i>B. anynana</i>.</p

F statistics and p-values for GLM analysis testing for differences in eyespot trait sizes across treatments.

<p>Individuals were injected with either 5E1 antibody or NS1 control media as larvae. All significant differences correspond to smaller trait sizes in the 5E1-injected individuals. GLM analyses were performed with sex (always significant across traits with females usually displaying a larger trait size than males, not shown); and interaction between line and sex (not significant in all analyses; not shown). *p<0.05, **p<0.01, ***p<0.001.</p

F statistics and p-values for GLM analysis testing for differences in eyespot trait sizes across treatments using wing size as a covariate.

<p>Individuals were injected with either 5E1 antibody or NS1 control media as larvae. All significant differences correspond to smaller trait sizes in the 5E1-injected individuals. GLM analyses were performed with sex (always significant across traits with females usually displaying a larger trait size than males; not shown); interaction between line and sex (not significant in all analyses; not shown); and wing size as a covariate. *p<0.05.</p

Injections of 5E1 antibody reduce the levels of <i>en/inv</i> transcripts one day later in both <i>B. anynana</i> and <i>J. coenia</i>.

<p>(A) PCR amplification of <i>en/inv</i> (top) and the house-keeping gene <i>EF1a</i> (bottom) from the same samples after injection of either 5E1 antibody or NS1 vehicle. Samples 1–3: <i>B. anynana</i> NS1; 4–6: <i>B. anynana</i> 5E1; 7–8: <i>J. coenia</i> NS1; 9–10: <i>J. coenia</i> 5E1. (B) Quantification of brightness levels of <i>en/inv</i> PCR amplification relative to brightness levels of the <i>EF1a</i> housekeeping gene (averages from data in A). Asterisk (*) indicates a significant difference in <i>en/inv</i> relative levels between 5E1 and NS1 injections.</p

Western blots and similarity of Sonic-Hh and butterfly Hh sequences suggest that 5E1 antibody recognizes Hh in butterflies.

<p>(A) Alignment of sequences corresponding to the Sonic-Hh peptide used to make the 5E1 monoclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Ericson1" target="_blank">[15]</a>. Areas boxed in red correspond to the 5E1 epitope <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Bosanac1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Maun1" target="_blank">[27]</a>. (B) Western blot with <i>B. anynana</i> proteins extracted from wing discs showing three potential Hh fragments with the predicted sizes of 19 kD, 25 kD, and 37 kD (arrows) previously characterized from <i>D. melanogaster</i> Hh <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051087#pone.0051087-Lee1" target="_blank">[21]</a>. (C) No bands were detected with the control NS1 medium. The left lane of each photo is the protein standard.</p

Time course of PI-uptake under the DCF conditions.

<p>(<b>A</b>) Representative confocal images of PI-uptake for HeLa cells transfected with wild-type Cx40 or Cx40 mutants (V85I or L221I). Time points of 0, 4 and 20 minutes of incubation with PI are displayed. During 20 minutes of incubation with PI, only cells expressing the V85I and L221I mutants showed PI uptake. Scale bar = 50 µm. (<b>B</b>) Ratio of current PI fluorescence intensity over the initial baseline fluorescence over a 20 minute incubation. L221I showed the fastest rate of PI-uptake, with V85I having a slightly slower rate of uptake, however, both L221I and V85I had similar levels of PI-uptake near the end of 20 minute incubation. The addition of 100 µM CBX blocked PI-uptake. Cx40 expressing cells failed to show any PI-uptake.</p

Propidium iodide-uptake under divalent cation-free conditions.

<p>(<b>A</b>) Representative images of propidium iodide (PI)-uptake under divalent cation-free (DCF) conditions for isolated, individual, transfected HeLa cells. Successful transfection can be identified by their tagged green/yellow fluorescent proteins (green colour in the first column images). PI-uptake (red colour in the second column images) can be seen in cells expressing Cx26-GFP, V85I-YFP and L221I-YFP, but no uptake was seen in cells expressing YFP alone or Cx40-YFP. Scale bar = 20 µm. (<b>B</b>) Quantification of PI-uptake under DCF conditions. V85I-YFP (67.6%, n = 10) and L221I-YFP (83.2%, n = 10) showed a significant increase in PI-uptake compared to Cx40-YFP (4.6%, n = 14, ***indicates P<0.001). Other AF-linked Cx40 mutants, Q49X, L229M and I75F, were also studied and did not show any PI-uptake.</p