Wnt5 protein and mRNA expression domains in epidermis, muscle and tendon cells during embryonic development.

<p>WNT5 is predominantly expressed in subsets of neurons in the CNS from stage 12 onwards throughout embryonic development (data not shown; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone.0032297-Fradkin2" target="_blank">[28]</a>). However, there is also strong expression from this stage onwards in the epidermis and the musculature. At stage 12, Wnt5 protein (<b>A, B</b>) and <i>Wnt5</i> mRNA (<b>E, F</b>) expression is observed in the epidermis, most prominently in two clusters (<b>arrows</b>), and throughout the somatic mesoderm that will give rise to the body wall musculature. Later in embryonic development at early stage 16 WNT5 protein and <i>Wnt5</i> mRNA are present in the attachment sites (<b>arrows in panels C and G</b>) and at low levels in most muscle fibers including the LTMs 21, 22 and 23 (<b>C, G</b>). At the end of embryonic development at late stage 17, Wnt5 protein (<b>D</b>) and <i>Wnt5</i> mRNA (<b>H</b>) are almost undetectable in the somatic mesoderm. In all panels anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants frequently do not express SR, while the bypassed attachment sites do.

<p>Double labeled stage 16 embryos are shown of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) with anti-Muscle Myosin in green and anti-SR in red (Material and Methods). Asterisks mark the novel attachment sites of the overshooting LTM muscles; white arrowheads mark the locations of the original attachment sites. In <i>Wnt5</i> mutants the novel target sites do not express SR in 65% of the segments containing overshooting muscles, while the bypassed attachment sites usually express SR. The SR positive, original tendon cell is also present in <i>drl^Red2</i> mutants, but is partly masked by the overshooting muscle fiber in panel (<b>C</b>), but clearly visible in panel (F)). These results were confirmed in embryos that express Tau-MYC under the control of a <i>stripe</i> promoter in both <i>Wnt5</i> and <i>drl</i> mutants (data not shown). The following genotypes are shown, the control UAS-Tau-MYC; <i>sr</i>-GAL4 embryos (<b>D</b>), <i>Wnt5⁴⁰⁰</i>; UAS-Tau-MYC/<i>sr</i>-GAL4 (<b>E</b>) and <i>drl^Red2</i>; UAS-Tau-MYC/<i>sr</i>-Gal4 (<b>F</b>). Anti-Muscle Myosin is shown in green and anti-MYC in red. No MYC protein is observed in the ectopic attachment sites. The photographs in Panels (<b>A</b>–<b>C</b>) were taken on a compound microscope and those in Panels (<b>D–F</b>) on a confocal microscope. Anterior is up and ventral is left.</p

LTM muscle fibers 21, 22 and 23 frequently overshoot their attachment sites in <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutant embryos.

<p>Stage 16 embryo body wall muscle preparations stained with anti-Muscle Myosin are shown for the wild type control (<i>w¹¹¹⁸</i>) (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>), <i>drl^Red2</i> (<b>C</b>), <i>Drl-2^E124</i> (<b>D</b>), <i>dnt^42.3</i> (<b>E</b>) and Df(2L)Exel6043 (<b>F</b>). Two hemisegments are displayed for each genotype with one set of muscles 21–23 labelled. In <i>Wnt5</i>, <i>drl</i> and <i>dnt</i> mutants, LTMs frequently bypass their normal attachment at the epidermis at muscle 12 and instead extend ventrally beyond muscle 13 and attach at a novel epidermal site located close to muscle fiber 7. Df(2L)Exel6043 mutant embryos, that lack both DNT and DRL, display this phenotype in all hemisegments of the homozygous animals. The penetrance of these phenotypes is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. The muscle bypass phenotype is schematically shown in panel (<b>G</b>). The * indicates the location of the novel, ectopic epidermal attachment in panels (<b>B</b>), (<b>C</b>), (<b>E</b>), (<b>F</b>) and (<b>G</b>). Anterior is up and ventral is left.</p

The new attachment sites of the bypassed muscle fibers in <i>Wnt5</i> and <i>drl</i> mutants express βPS integrin.

<p>Wild type (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^red2</i> (<b>C</b>) embryos were labelled with anti-βPS Integrin. Muscles 21–23 do exhibit an accumulation of βPS Integrin protein at the tip of the overshooting fibers (white asterix).</p

Muscle attachment defects persist from the embryonic to larval stages in <i>Wnt5</i> and <i>drl</i> mutants.

<p>Third instar larval body walls of <i>w¹¹¹⁸</i> (<b>A</b>), <i>Wnt5⁴⁰⁰</i> (<b>B</b>) and <i>drl^Red2</i> (<b>C</b>) mutant larvae are stained with anti-FAS2 (mAb 1D4). <i>Wnt5⁴⁰⁰</i> larvae and <i>drl^Red2</i> larvae frequently bypass their normal attachment sites and extend ventrally where they form new stable attachments. The original and ectopic tendons cells are indicated by + and *, respectively. FAS2 protein is evident at both sites. The penetrance of the bypass phenotypes is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032297#pone-0032297-t001" target="_blank"><b>Table 1</b></a>. Anterior is up and ventral is left.</p