Cooption of an appendage-patterning gene cassette in the head segmentation of arachnids.

Abstract

The jointed appendages of arthropods have facilitated the spectacular diversity and success of this phylum. Key to the regulation of appendage outgrowth is the Krüppel-like factor (KLF)/specificity protein (Sp) family of zinc finger transcription factors. In the fruit fly, <i>Drosophila melanogaster</i>, the <i>Sp6-9</i> homolog is activated by <i>Wnt-1/wingless</i> (<i>wg</i>) and establishes ventral appendage (leg) fate. Subsequently, <i>Sp6-9</i> maintains expression of the axial patterning gene <i>Distal-less</i> (<i>Dll</i>), which promotes limb outgrowth. Intriguingly, in spiders, <i>Dll</i> has been reported to have a derived role as a segmentation gap gene, but the evolutionary origin and regulation of this function are not understood because functional investigations of the appendage-patterning regulatory network are restricted to insects. We tested the evolutionary conservation of the ancestral appendage-patterning network of arthropods with a functional approach in the spider. RNAi-mediated knockdown of the spider <i>Sp6-9</i> ortholog resulted in diminution or loss of <i>Dll</i> expression and truncation of appendages, as well as loss of the two body segments specified by the early <i>Dll</i> function. In reciprocal experiments, <i>Dll</i> is shown not to be required for <i>Sp6-9</i> expression. Knockdown of <i>arrow</i> (Wnt-1 coreceptor) disrupted segmentation and appendage development but did not affect the early <i>Sp6-9</i> expression domain. Ectopic appendages generated in the spider "abdomen" by knockdown of the Hox gene <i>Antennapedia-1</i> (<i>Antp-1</i>) expressed <i>Sp6-9</i> comparably to wild-type walking legs. Our results support (<i>i</i>) the evolutionary conservation of an appendage-patterning regulatory network that includes canonical Wnt signaling, <i>Sp6-9</i>, and <i>Dll</i> and (<i>ii</i>) the cooption of the <i>Sp6-9/Dll</i> regulatory cassette in arachnid head segmentation.