Isolation, characterization and expression of limb patterning genes in Euprymna scolopes (Mollusca: Cephalopoda)

Results from genetic, molecular, and developmental studies have shown that the limbs of Drosophila and vertebrates have remarkable similarities in the molecular instructions that convey the positional information that is required for the patterning of their orthogonal axes. The parsimonious explanations for parallels in the genetic programs of two animal lineages that diverged over 500 mya are independent co-option of homologous genes for similar functions or the conservation of an ancestral genetic program. One version of a 'Universal Genetic Program for Limb Development' (UGPLD) contains the following genes: apterous/Lhx2-9, decapentaplegic/Bone Morphogenic Protein, Distal-less/Dllx, fringe/Radical fringe, hedgehog/Sonic hedgehog, and wingless/Wnt1. These genes have never been analyzed as a group in an invertebrate taxon other than Arthropoda.;Cephalopod arms and tentacles have a considerable degree of patterning along their orthogonal axes. These appendages are non-homologous to other structures outside the Mollusca and are a structural novelty within the taxon. This work reports the isolation of cDNA transcripts from the aforementioned genes from the sepiolid Euprymna scolopes, their molecular characterization and corresponding developmental expression patterns. Also included is a general description of the organogenesis of E. scolopes illustrated with photomicrographs of histological serial sections, a report on artificial fertilization trails, embryonic growth rates under different temperature regimes, and the recovery of 17 additional cDNA transcripts, 10 of which are full-length cds from developmentally important genes. This later body of information represents a valuable tool for the future use of this squid as a cephalopod model system for developmental studies.;Esc-ap and Esc-hh were not expressed in the arm crown in patterns consistent with roles in growth or axial organization of arms and tentacles, but with patterning of the brachial nervous system. Other conspicuous expression patterns suggest an Esc-ap role in eye and neural development, particularly for olfaction and vision, and Esc-hh in gut and neural development. Esc-fng was transiently expressed at the tip of the differentiating arms and later in the distal ring of the suckers. Both expression patterns could be associated with the formation of borders; a possible link of the early expression to dorso-ventral patterning is also discussed. Other conspicuous Esc-fng expression patterns could be linked to vasculogenesis.;Esc-Dll expression was assessed by immunolocalization of the anti-Dll most commonly used in comparative studies across taxa. Arm primordia, budding arms and later the tip of the developing arms and tentacles were strongly immunoreactive, in agreement with a possible gene function in outgrowth and proximo-distal patterning of these appendages. The overall results obtained support the proposed Dll roles in the development of body wall outgrowths, appendages, nervous system, and sensory structures.;Esc-BMP2/4 was expressed on the aboral side of the arm buds and later as a wide band over the entire length of each arm and tentacle, suggesting a role in growth and/or dorso-ventral patterning. In general, Esc-BMP2/4 expression was highly dynamic and pleiotropic; gene transcripts were also detected in the developing brain, eyes, buccal mass, gut, gills, branchial hearts, funnel, mantle, fins, and all hard or cartilaginous structures.;Esc-Wnt1 transcripts were detected in arms and tentacles as a mesodermal, hairpin-like expression in mid and late embryonic stages (23-27), suggesting a role in the elongation of the appendages, but might also be related to the organization of their muscular mass. Esc-Wnt4 and Esc-Wnt5 expression was also analyzed in embryos stages 23 to 27; the hybridization signal in their appendages was mesodermal, Esc-Wnt4 at the base and both edges of each limb and always stronger at the posterior side, and Esc-Wnt5 first in the dorsal distal mesenchyme of the limbs and later throughout the appendages.;Gene expression in the developing arms and tentacles of E. scolopes did not conform fully to the UGPLD. The expression patterns of the putative Esc-Dll, Esc-BMP2-4, and the Esc-Wnts suggest that they could play important roles in the growth and patterning of these appendages; this needs to be confirmed by functional analysis.