Morphological transitions and the genetic basis of the evolution of extraembryonic tissues in flies

Changes in the genotype influence changes in morphology during evolution, giving rise to the vast diversity of morphological features that we observe. The ability to describe how genetic change causes morphological transformation is key for a mechanistic understanding of evolutionary change. This thesis examines morphological and genetic aspects of the origin of the amnioserosa, a unique tissue of higher flies (Cyclorrhapha), which combines different aspects of the amnion and the serosa, two protective extraembryonic epithelia commonly found in lower flies and other insects. Typically, extraembryonic epithelia develop from an amnio-serosal fold, which closes about the ventral side of the gastrulating embryo. The outer cell layer of the amnio-serosal fold becomes the serosa. This epithelium disjoins from the amnion and encloses the embryo. The inner cell layer of the amnio-serosal fold becomes the amnion and retains continuity with the dorsal epidermis of the embryo. Higher flies of the taxon Schizophora differ: they develop a single extraembryonic epithelium on the dorsal side called the amnioserosa. Here, a third type is reported in close relatives of Schizophora (lower Cyclorrhapha), in which the amnion is dorsal and the serosa complete. Based on the occurrence of these three extraembryonic tissue organizations in flies two major morphological transitions in the organization of the extraembryonic epithelia of flies are proposed, one that occurred before the cretaceous radiation of flies some 150 million years ago in which the amnion got relocated from the ventral to the dorsal side of the embryo, and another that occurred before the tertiary radiation of flies some 100 million years ago in which the dorsal amnion and the serosa combined into a single epithelium, the amnioserosa. The genetic analysis focuses on the homeobox gene zerknüllt (zen). Most insects express zen in their extraembryonic tissues. In the milkweed bug Oncopeltus, zen controls the fusion of the amnion with the serosa. In the flour beetle Tribolium, zen controls not only the fusion of the amnion with the serosa but also the distinction of serosal from amniotic cells. In the fruitfly Drosophila, zen controls the distinction of amnioserosal from embryonic cells. Here, it is shown that in lower cyclorrhaphan flies, zen controls serosal cell-fate, like in Tribolium. Furthermore, spatial and temporal differences in the extraembryonic expression of zen are reported between Drosophila and lower cyclorrhaphan flies. Extraembryonic zen expression in Drosophila abuts the posterior and lateral germ rudiment, unlike in lower cyclorrhaphans in which the prospective amnion is interspersed. Ectopic expression of zen in Megaselia through injection of capped mRNA had no effect on development, suggesting that spatial expression difference may not explain the morphological reorganization into an amnioserosa-like tissue in evolution. Temporally, extraembryonic zen expression in Drosophila is downregulated during the late phase of germband extension, unlike in lower cyclorrhaphans in which it persists. A new transfection-based technique for delivery of double-stranded RNA to embryonic cells was developed and used to mimic the time course of zen expression of Drosophila in Megaselia. This procedure was sufficient to create an amnioserosa-like tissue in Megaselia. In addition, differences in extraembryonic zen expression were observed between lower cyclorrhaphan species. In one species (Megaselia) it was restricted to dorsal balstoderm, like in Drosophila. In another species (Episyrphus), it extended to the anterior tip of the blastoderm egg. This difference cannot explain the origin of the amnioserosa but may reflect differences in embryonic pattern formation through bicoid, which determines the position of the head by means of localized mRNA. To test this prediction, ca. 60 kb upstream of zen (spanning the expected genomic position of bicoid) were sequenced in Episyrphus. This region did not contain a bicoid homologue. The research described in this thesis demonstrates that temporal change in the expression pattern of a single gene can explain the origin of the amnioserosa. This result shows that even at a macroevolutionary scale, i.e. over large phylogenetic distances, evo-devo studies can provide insight into the causal relationship between gene expression and morphology. In addition, the unique zen expression in Episyrphus and zen locus data from this species point to a possible difference in embryonic patterning in this taxon