For each lineage, the top row shows schematic representations of a generalized limb bud and an adult limb in two different orientations. Axes are indicated to the left of each limb bud (A, anterior; Po, posterior; Pr, proximal; Di, distal; Do, dorsal; Ve, ventral) Bottom rows show limb buds with gene expression domains pink color). Vertebrate gene expression based on mouse limb development. Arthropod gene expression is a compound reconstruction from chelicerate, myriapod, and hexapod limb development in order to consolidate a complete set of pro-orthologous genes comparable to vertebrate and cephalopod lineages. Cephalopod gene expression is based on findings in this study from the cuttlefish Sepia officinalis and Sepia bandensis. The figure illustrates the conserved and divergent expression patterns of homologous (orthologous) genes, some of which share equivalent roles in patterning the limb axes. The proximodistal axis displays conserved expression of transcription factors at opposite ends; Hth (pro-ortholog of vertebrate Meis genes) and Exd (pro-ortholog of vertebrate Pbx genes) are restricted proximally, whereas Dll (pro-ortholog of vertebrate Dlx genes), Wnt5 (pro-ortholog of Wnt5a) and Sp8/9 (pro-ortholog of vertebrate Sp8 and Sp9 genes, known as Sp6-9 in some arthropods) show distally restricted expression. The typical expression pattern of Dac seen in arthropods (between proximally and distally restricted genes) is not strictly conserved in vertebrates (Dac is the pro-ortholog of vertebrate Dach genes) or cephalopods. However, Dac expression in non-locomotory arthropod appendages (e.g., mandibles) is distally restricted, resembling cephalopod Dac expression (Donoughe and Extavour, 2016). Expression patterns of the diverse family of Wnt genes shows interesting variation. Although, some members of the family show variation in their expression pattern (Wnt1 and Wnt7), there is a general pattern of distal restriction of Wnt expression (represented here by Wnt5, but also seen in many other Wnt ligands) in the three lineages. At the level of individual Wnt members, Wg (pro-ortholog of vertebrate Wnt1) is restricted ventrally in arthropods but not in vertebrates or cephalopods, and Wnt7a (arthropod and cephalopod Wnt7 genes are pro-orthologs of vertebrate Wnt7a) is restricted dorsally in vertebrates but not in arthropods or cephalopods. Restricted expression of Wnt ligands either dorsally or ventrally has not been reported in cephalopods, but the dorsally restricted expression of the Wnt repressor Sfrp1/2/5 suggests a role of polarized Wnt pathway activation in the control of the dorsoventral axis of cephalopod limbs, similar to vertebrates (by dorsal Wnt7a) and arthropods (by ventral Wg). There is a clear restriction of at least one Bmp ligand (vertebrate Bmp7 and cephalopod Bmp2/4; pro-orthologs of arthropod Dpp) and the transcription factor En along the dorsoventral axis in these three lineages. Finally, polarized expression of Hh is conserved in the three lineages (posterior in vertebrates and arthropods, but anterior in cephalopods), which, together with the functional manipulations, indicates conservation of Hh signaling in patterning the anteroposterior limb axis in the three lineages. The asterisk (*) in arthropod Dac indicates that some mouth appendages show a distal expression domain (Donoughe and Extavour, 2016) (Angelini and Kaufman, 2005) more similar to cephalopod Dac limb expression than to Dac expression in arthropod legs. Two asterisks (**) indicate that Wnt5 expression shows variation in arthropods, with a sub-distal expression in chelicerates (Damen, 2002) but distal in hexapods (i.e. flour beetle) (Bolognesi et al., 2008). Three asterisks (***) indicate that Dpp shows variation in its expression domain in arthropods, with some hexapods and chelicerates showing a distal expression domain, whereas in Myriapods and other hexapods it is dorsally restricted, as depicted here (Angelini and Kaufman, 2005). Schematized gene expression domains for vertebrates and arthropods are from the following sources. Mouse gene expression: Meis1 (González-Lázaro et al., 2014), Pbx1 (Capellini et al., 2006), Sp8 (Kawakami et al., 2004), Dlx5 (Vieux-Rochas et al., 2013), Dach1 (Salsi et al., 2008), Wnt1, Wnt5a, Wnt7a, Sfrp2 (Witte et al., 2009), Shh (Riddle et al., 1993), Bmp7 (Choi et al., 2012) and En1 (Loomis et al., 1998). Arthropod expression based on: Chelicerates, Hth, Exd, Dll, Dac (Prpic et al., 2003), Sp8/9 (Königsmann et al., 2017), Wg (Damen, 2002), En (Damen, 2002) and Sfrp1/2/5 (Hogvall et al., 2018); Myriapods, Hth, Exd, Dll, Dac (Prpic and Tautz, 2003), Sp8/9 (Setton and Sharma, 2018), Wg, Dpp (Prpic, 2004), Wnt5, Wnt7, Hh, En (Janssen et al., 2004), Sfrp1/2/5 (Hogvall et al., 2018); Hexapods, flour beetle, Hth, Exd (Prpic et al., 2003), Sp8/9 (Schaeper et al., 2010), Dll (Beermann et al., 2001), Dac (Prpic et al., 2001), Wg, Wnt5 (Bolognesi et al., 2008), Dpp (Sanchez-Salazar et al., 1996), En (Brown et al., 1994); Hexapods, cricket, Hth, Exd, Dll, Dac, Wg, Hh, Dpp, En (Donoughe and Extavour, 2016).