Higher cyclorrhaphan flies including Drosophila develop a single extraembryonic epithelium (amnioserosa), which closes the germband dorsally. In most other insects two extraembryonic epithelia, serosa and amnion, line the inner eggshell and the ventral germband, respectively. How the two extraembryonic epithelia evolved into one is unclear. Recent studies have shown that, in the flour beetle Tribolium and in the milkweed bug Oncopeltus, the homeobox gene zerknü llt (zen) controls the fusion of the amnion with the serosa before dorsal closure. To understand the origin of the amnioserosa in evolution, we examined the expression and function of zen in the extraembryonic tissue of lower Cyclorrhapha. We show that Megaselia abdita (Phoridae) and Episyrphus balteatus (Syrphidae) develop a serosa and a dorsal amnion, suggesting that a dorsal amnion preceded the origin of the amnioserosa in evolution. Using Krü ppel (Kr) and pannier (pnr) homologues of Megaselia as markers for serosal and amniotic tissue, respectively, we show that after zen RNAi all extraembryonic tissue becomes indistinguishable from amniotic cells, like in Tribolium but unlike in Drosophila, in which zen controls all aspects of extraembryonic development. Compared with Megaselia and Episyrphus, zen expression in Drosophila is extended to cells that form the amnion in lower Cyclorrhapha and is down-regulated at the developmental stage, when serosa cells in lower Cyclorrhapha begin to expand. These expression differences between species with distinct extraembryonic tissue organizations and the conserved requirement of zen for serosa development suggest that the origin of an amnioserosa-like epithelium was accompanied by expression changes of zen.Megaselia ͉ Episyrphus ͉ Drosophila ͉ EvoDevo ͉ homology

The major goal of ecological evolutionary developmental biology, also known as "eco-evo-devo," is to uncover the rules that underlie the interactions between an organism's environment, genes, and development and to incorporate these rules into evolutionary theory. In this chapter, we discuss some key and emerging concepts within eco-evo-devo. These concepts show that the environment is a source and inducer of genotypic and phenotypic variation at multiple levels of biological organization, while development acts as a regulator that can mask, release, or create new combinations of variation. Natural selection can subsequently fix this variation, giving rise to novel phenotypes. Combining the approaches of eco-evo-devo and ecological genomics will mutually enrich these fields in a way that will not only enhance our understanding of evolution, but also of the genetic mechanisms underlying the responses of organisms to their natural environments.

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SUMMARYBone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.

The homeobox gene bicoid functions as an anterior pattern organizer of the Drosophila embryo, but other than in higher flies (Cyclorrhapha), bicoid orthologues appear to be absent from insect genomes. In Drosophila, bicoid is expressed in an anterior-to-posterior protein gradient and regulates spatially restricted expression domains of segmentation genes in a concentration-dependent manner. hunchback, a direct transcriptional target of Bicoid, complements the "morphogen" activity of Bicoid. hunchback is activated by Bicoid throughout the anterior half of the blastoderm and a Bicoid-binding cis-regulatory element has been identified immediately upstream of the proximal hunchback promoter P2 of Drosophila and other higher Cyclorrhapha (Schizophora). bicoid and Bicoid-dependent hunchback regulation are thought to have originated during or before the radiation of extant Cyclorrhapha, although the precise occurrence of these traits in lower Cyclorrhapha remains unknown. Previously, we have described a bicoid orthologue in Megaselia, a species of the lower cyclorrhaphan family Phoridae. Here, we report the occurrence of bicoid in two additional lower cyclorrhaphan families, Lonchopteridae and Platypezidae. We show that Megaselia Bicoid is required for anterior expression of Megaselia hunchback, and binds upstream of its P2 promoter. Furthermore, we report the expression of lacZ reporter constructs under the control of hunchback regulatory sequences from a range of lower cyclorrhaphan and non-cyclorrhaphan flies in transgenic Drosophila embryos. Our results are consistent with a cyclorrhaphan origin of bicoid and suggest that a Bicoid-binding enhancer upstream of the hunchback P2 promoter evolved at the latest in the last common ancestor of Megaselia and Schizophora.

Insects have undergone dramatic evolutionary changes in extraembryonic development, which correlate with changes in the expression of the class-3 Hox gene zen. Here, we review the evolution of this gene in insects and point out how changes in zen expression may have affected extraembryonic development at the morphological and the genetic level.

INTRODUCTIONThis protocol describes a combined heat/formaldehyde fixation procedure, as well as a manual devitellinization procedure, for pregastrular Megaselia abdita embryos.