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The conservation of gene networks that specify and differentiate distinct tissues has long been a subject of great interest to evolutionary developmental biologists, but the question of how pre-existing tissue-specific developmental trajectories merge is rarely asked. During the radiation of flies, two extraembryonic epithelia, known as serosa and amnion, evolved into one, called amnioserosa. This unique extraembryonic epithelium is found in fly species of the group Schizophora, including the genetic model organism Drosophila melanogaster , and has been studied in depth. Close relatives of this group develop a serosa and a rudimentary amnion. The scuttle fly Megaselia abdita has emerged as an excellent model organism to study this extraembryonic tissue organization. In this review, development and functions of the extraembryonic tissue complements of Drosophila and Megaselia are compared. It is concluded that the amnioserosa combines cells, genetic pathway components and functions that were previously associated either with serosa development or amnion development. The composite developmental trajectory of the amnioserosa raises the question of whether merging tissue-specific gene networks is a common evolutionary process. This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

The C2H2 zinc finger gene cucoid establishes anterior-posterior (AP) polarity in the early embryo of culicine mosquitoes. This gene is unrelated to genes that establish embryo polarity in other fly species (Diptera), such as the homeobox gene bicoid, which serves this function in the traditional model organism Drosophila melanogaster. The cucoid gene is a conserved single copy gene across lower dipterans but nothing is known about its function in other species, and its evolution in higher dipterans, including Drosophila, is unresolved. We found that cucoid is a member of the ZAD-containing C2H2 zinc finger (ZAD-ZNF) gene family and is orthologous to 27 of the 91 members of this family in D. melanogaster, including M1BP, ranshi, ouib, nom, zaf1, odj, Nnk, trem, Zif, and eighteen uncharacterized genes. Available knowledge of the functions of cucoid orthologs in Drosophila melanogaster suggest that the progenitor of this lineage specific expansion may played a role in regulating chromatin. We also describe many aspects of the gene duplication history of cucoid in the brachyceran lineage of D. melanogaster, thereby providing a framework for predicting potential redundancies among these genes in D. melanogaster.

Mechanisms and evolution of primary axis specification in insects are discussed in the context of the roles of ß-catenin and TCF in polarizing metazoan embryos. Three hypotheses are presented. First, insects with sequential segmentation and posterior growth use cell-autonomous mechanisms for establishing embryo polarity via the nuclear ratio of ß-catenin and TCF. Second, TCF homologs establish competence for anterior specification. Third, the evolution of simultaneous segmentation mechanisms, also known as long-germ development, resulted in primary axis specification mechanisms that are independent of ß-catenin but reliant on TCF, a condition that preceded the frequent replacement of anterior determinants in long germ insects.

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky-Muller incompatibilities (DMI), a way how TEs can contribute to the evolution of endogenous genetic barriers and eventually population divergence. We investigated whether differential TE activity created endogenous selection pressures among conspecific populations of the non-biting midgeChironomus riparius,focussing on aChironomus-specific TE, the minisatellite-likeCla-element, whose activity is associated with speciation in the genus. Using an improved and annotated draft genome for a genomic study with five naturalC. ripariuspopulations, we found highly population-specific TE insertion patterns with many private insertions. A highly significant correlation of pairwise population FSTfrom genome-wide SNPs with the FSTestimated from TEs suggests drift as the major force driving TE population differentiation. However, the significantly higherCla-elementFSTlevel due to a high proportion of differentially fixedCla-elementinsertions indicates that segregating, i.e. heterozygous insertions are selected against. With reciprocal crossing experiments and fluorescent in-situ hybridisation ofCla-elementsto polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings that might be linked to DMI in hybrids. We propose that the inferred negative selection on heterozygousCla-elementinsertions causes endogenous genetic barriers and therefore acts as DMI amongC. ripariuspopulations. The intrinsic genomic turnover exerted by TEs, thus, may have a direct impact on population divergence that is operationally different from drift and local adaptation.

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles., eLife digest With very few exceptions, animals have ‘head’ and ‘tail’ ends that develop when they are an embryo. The genes involved in specifying these ends vary between species and even closely-related animals may use different genes for the same roles. For example, the products of two unrelated genes called bicoid in fruit flies and panish in common midges accumulate at one end of their respective eggs to distinguish head from tail ends. It remained unclear how other fly species, which have neither a bicoid nor a panish gene, distinguish the head from the tail end, or how genes can evolve the specific function of bicoid and panish. Cells express genes by producing gene templates called messenger ribonucleic acids (or mRNAs for short). The central portions of mRNAs, known as protein-coding sequences, are then used to produce the protein. Proteins can play several distinct roles, which they acquire through evolution. This can happen in different ways, for example, genetic mutations in the part of a gene that codes for protein may alter the resulting protein, giving it a new activity. Alternatively, sequences at the beginning and the end of an mRNA molecule that do not code for protein, but regulate when and where proteins are made, can influence a protein’s role by changing its environment. Many genes produce mRNAs with alternative sequences at the beginning or the end, a process known as alternative transcription. Here, Yoon et al. identified three unrelated genes that perform similar roles to bicoid and panish in the embryos of several different moth flies and mosquitoes. These genes appear to have acquired their activity because one of their alternative transcripts accumulated at the future head end, rather than through mutations in the protein-coding sequences. Studying multiple species also made it clear that panish inherited its function from a localized alternative transcript of an old gene that duplicated and diverged. These findings suggest that alternative transcription may provide opportunities for genes to evolve new roles in fundamental processes in flies. Most animal genes use alternative start and stop sites for transcription, but the reasons for this remain largely obscure. This is especially the case in the human brain. The findings of Yoon et al., therefore, raise the question of whether alternative transcription has played an important role in the evolution of the human brain.

The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd’s derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd’s derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd’s transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd’s controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution.

Bone Morphogenetic Proteins (BMPs) pattern the dorsal-ventral axis of bilaterian embryos; however, their roles in the evolution of body plan are largely unknown. We examined their functional evolution in fly embryos. BMP signaling specifies two extraembryonic tissues, the serosa and amnion, in basal-branching flies such as Megaselia abdita, but only one, the amnioserosa, in Drosophila melanogaster. The BMP signaling dynamics are similar in both species until the beginning of gastrulation, when BMP signaling broadens and intensifies at the edge of the germ rudiment in Megaselia, while remaining static in Drosophila. Here we show that the differences in gradient dynamics and tissue specification result from evolutionary changes in the gene regulatory network that controls the activity of a positive feedback circuit on BMP signaling, involving the tumor necrosis factor alpha homolog eiger. These data illustrate an evolutionary mechanism by which spatiotemporal changes in morphogen gradients can guide tissue complexity. DOI: http://dx.doi.org/10.7554/eLife.20894.001

How to generate head-to-tail polarity in a midge Animal genomes do not remain static but gain and lose essential genes over time. One such case is the bicoid gene of flies. Bicoid plays an important role in early Drosophila development during the generation of head-to-tail polarity, but most flies lack bicoid. Which genes then serve the same function in other insects? Now, Klomp et al. report a structurally unrelated gene that serves essentially the same function as bicoid in a mosquito-related midge but does so through a different genetic mechanism. Science , this issue p. 1040

The number of insect species that are amenable to functional genetic studies is growing rapidly and provides many new research opportunities in developmental and evolutionary biology. The holometabolous insects represent a disproportionate percentage of animal diversity and are thus well positioned to provide model species for a wide variety of developmental processes. Here we discuss emerging holometabolous models, and review some recent breakthroughs. For example, flies and midges were found to use structurally unrelated long-range pattern organizers, butterflies and moths revealed extensive pattern formation during oogenesis, new imaging possibilities in the flour beetle Tribolium castaneum showed how embryos break free of their extraembryonic membranes, and the complex genetics governing interspecies difference in head shape were revealed in Nasonia wasps.

Morphogenetic functions of the amnioserosa, the serosa, the amnion, and the yolk sac are reviewed on the basis of recent studies in flies (Drosophila, Megaselia), beetles (Tribolium), and hemipteran bugs (Oncopeltus). Three hypotheses are presented. First, it is suggested that the amnioserosa of Drosophila and the dorsal amnion of other fly species function in a similar manner. Second, it is proposed that in many species with an amniotic cavity, the amnion determines the site of serosa rupture, which, through interactions between the serosa and the amnion, enables the embryo to break free from the amniotic cavity and to close its backside. Finally, it is concluded that the yolk sac is likely an important player in insect morphogenesis.

The purpose of this application, under Articles 23.9.3 and 81.2.3 of the Code, is to conserve the specific name Megaselia abdita Schmitz, 1959 for a widespread Holarctic scuttle fly by giving it precedence over its senior subjective synonym Megaselia griseipennis (Santos Abreu, 1921), which has long been treated as a nomen dubium. A growing literature exists using the name M. abdita. It is proposed that the name Megaselia abdita be given precedence over Megaselia griseipennis (Santos Abreu, 1921) to avoid confusion.

The homeobox gene caudal (cad) regulates posterior development in Drosophila. In early embryos, the cad protein (CAD) is expressed in a posterior-to-anterior concentration gradient, which contributes polarity to the developing embryo. The CAD gradient is complementary to and dependent on the anterior pattern organizer Bicoid (BCD), which represses the translation of ubiquitous maternal cad transcripts in the anterior embryo through a direct interaction with the cad 3' untranslated region (UTR). Here, we show that early embryos of the lower cyclorrhaphan fly Megaselia express the putative cad orthologue Mab-cad throughout the posterior three quarters of the blastoderm but lack maternal transcripts. In transgenic blastoderm embryos of Drosophila, Mab-cad cis-regulatory DNA drives the expression of a reporter gene in a similar pattern, while Mab-cad 3' UTR fails to mediate translational repression of a ubiquitously transcribed reporter. For another lower cyclorrhaphan fly (Lonchoptera) and two related outgroup taxa of Cyclorrhapha (Empis, Haematopota), we report maternal cad expression in ovarian follicles. Together, our results suggest that BCD is not required for the translational repression of Mab-cad, and that maternal cad expression was lost in the Megaselia lineage.

In the common fruit fly Drosophila, head formation is driven by a single gene, bicoid, which generates head-to-tail polarity of the main embryonic axis. Bicoid deficiency results in embryos with tail-to-tail polarity and no head. However, most insects lack bicoid, and the molecular mechanism for establishing head-to-tail polarity is poorly understood. We have identified a gene that establishes head-to-tail polarity of the mosquito-like midge, Chironomus riparius. This gene, named panish, encodes a cysteine-clamp DNA binding domain and operates through a different mechanism than bicoid. This finding, combined with the observation that the phylogenetic distributions of panish and bicoid are limited to specific families of flies, reveals frequent evolutionary changes of body axis determinants and a remarkable opportunity to study gene regulatory network evolution.

The segmentation gene network in insects can produce equivalent phenotypic outputs despite differences in upstream regulatory inputs between species. We investigate the mechanistic basis of this phenomenon through a systems-level analysis of the gap gene network in the scuttle fly Megaselia abdita (Phoridae). It combines quantification of gene expression at high spatio-temporal resolution with systematic knock-downs by RNA interference (RNAi). Initiation and dynamics of gap gene expression differ markedly between M. abdita and Drosophila melanogaster, while the output of the system converges to equivalent patterns at the end of the blastoderm stage. Although the qualitative structure of the gap gene network is conserved, there are differences in the strength of regulatory interactions between species. We term such network rewiring ‘quantitative system drift’. It provides a mechanistic explanation for the developmental hourglass model in the dipteran lineage. Quantitative system drift is likely to be a widespread mechanism for developmental evolution. DOI: http://dx.doi.org/10.7554/eLife.04785.001, eLife digest Similar biological phenomena can result from different processes occurring in different organisms. For example, the early stages of how an insect develops from an egg can vary substantially between different species. Nonetheless, all insects have a body plan that develops in segments. The same outcome occurring as a result of different developmental steps is known as ‘system drift’, but the mechanisms underlying this phenomenon are largely unknown. How the body segments of the fruit fly Drosophila develop has been extensively studied. First, a female fruit fly adds messenger RNA (or mRNA) molecules copied from a number of genes into her egg cells. These mRNA molecules are then used to produce proteins whose concentration varies along the length of each egg. These proteins in turn switch on so-called ‘gap genes’ in differing amounts in different locations throughout the fruit fly embryo. The activity of these genes goes on to define the position and extent of specific segments along the fruit fly's body. Like the fruit fly, the scuttle fly Megaselia abdita has a segmented body. However, mothers of this species deposit somewhat different protein gradients into their eggs. How the regulation of development differs in the scuttle fly to compensate for this change is unknown. Now, Wotton et al. have studied, in detail, how gap genes are regulated in this less well-understood fly species to understand the mechanisms responsible for a specific example of system drift. In the fruit fly, gap genes normally switch-off (or reduce the expression of) other gap genes within the same developing body segment, and Wotton et al. found that the same kind of interactions tended to occur in the scuttle fly. As such, the overall structure of the gap gene network was fairly similar between scuttle and fruit flies. There were, however, differences in the strength of these interactions in the two fly species. These quantitative differences result in a different way of making the same segmental pattern in the embryo. In this way, Wotton et al. show how tinkering with the strength of specific gene interactions can provide an explanation for system drift. DOI: http://dx.doi.org/10.7554/eLife.04785.002

The members of the evolutionarily conserved Hox-gene complex, termed Hox genes, are required for specifying segmental identity during embryogenesis in various animal phyla. The Hox3 genes of winged insects have lost this ancestral function and are required for the development of extraembryonic epithelia, which do not contribute to any larval structure. Higher flies (Cyclorrhapha) such as Drosophila melanogaster contain Hox3 genes of two types, the zerknüllt type and the bicoid type. The zerknüllt gene is expressed zygotically on the dorsal side of the embryo and is required for establishing extraembryonic tissue. Its sister gene bicoid is expressed maternally and the transcripts are localized at the anterior pole of the mature egg. BICOID protein, which emerges from this localized source during early development, is required for embryonic patterning. All known direct bicoid homologues are confined to Cyclorrhaphan flies. Here, we describe Hox3 genes of the non-Cyclorrhaphan flies Empis livida (Empididae), Haematopota pluvialis (Tabanidae), and Clogmia albipunctata (Psychodidae). The gene sequences are more similar to zerknüllt homologues than to bicoid homologues, but they share expression characteristics of both genes. We propose that an ancestral Hox3 gene had been duplicated in the stem lineage of Cyclorrhaphan flies. During evolution, one of the gene copies lost maternal expression and evolved as zerknüllt , whereas the second copy lost zygotic expression and evolved as bicoid . Our finding correlates well with a partial reduction of zerknüllt -dependent extraembryonic tissue during Dipteran evolution.

The Drosophila gene bicoid functions as the anterior body pattern organizer of Drosophila . Embryos lacking maternally expressed bicoid fail to develop anterior segments including head and thorax. In wild-type eggs, bicoid mRNA is localized in the anterior pole region and the bicoid protein forms an anterior-to-posterior concentration gradient. bicoid activity is required for transcriptional activation of zygotic segmentation genes and the translational suppression of uniformly distributed maternal caudal mRNA in the anterior region of the embryo. caudal genes as well as other homeobox genes or members of the Drosophila segmentation gene cascade have been found to be conserved in animal evolution. In contrast, bicoid homologs have been identified only in close relatives of the schizophoran fly Drosophila . This poses the question of how the bicoid gene evolved and adopted its unique function in organizing anterior–posterior polarity. We have cloned bicoid from a basal cyclorrhaphan fly, Megaselia abdita (Phoridae, Aschiza), and show that the gene originated from a recent duplication of the direct homolog of the vertebrate gene Hox3 , termed zerknüllt , which specifies extraembryonic tissues in insects.

The anterior determinant bicoid (bcd) of Drosophila is a homeodomain protein. It forms an anterior-to-posterior gradient in the embryo and activates, in a concentration-dependent manner, several zygotic segmentation genes during blastoderm formation. Its posterior counterpart, the homeodomain transcription factor caudal (cad), forms a concentration gradient in the opposite direction, emanating from evenly distributed messenger RNA in the egg. In embryos lacking bcd activity as a result of mutation, the cad gradient fails to form and cad becomes evenly distributed throughout the embryo. This suggests that bcd may act in the region-specific control of cad mRNA translation. Here we report that bcd binds through its homeodomain to cad mRNA in vitro, and exerts translational control through a bcd-binding region of cad mRNA.

Bone 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 development of the insect head tagma involves massive rearrangements and secondary fusions of segment anlagen during embryogenesis. Due to the lack of reliable morphological markers, the number, identity, and sequence of the head segments, particularly in the pregnathal region, are still a matter of ongoing debates. We examined the complex array of internal structures of the embryonic Drosophila melanogaster head such as the sensory structures and nerves of the peripheral and stomatogastric nervous systems, and we used embryonic head mutations causing a lack of overlapping segment anlagen to unravel the segmental identity and the sequence of the neural elements. Our results provide evidence for seven distinct segments in the Drosophila head, each characterized by a specific set of sensory neurons, consistent with the proposal that insects, myriapods, and crustaceans share a monophyletic evolutionary tree from a common annelid-like ancestor.

Using intracellular horseradish peroxidase injection we traced the developmental fate of early gastrula cells of the procephalic region in the stage 16/17 embryo. Morphogenetic movements in the developing brain are described in three dimensions. The results are related to head segmentation, and an early gastrula fate map of pregnathal head segments is proposed.

The engrailed expression in embryos of a beetle, four midges and a fly has been analysed with special reference to the terminal regions. In all six species the segmental expression pattern is very similar but variability occurs in the clypeolabrum, foregut and hindgut. In some cases, segmental engrailed expression seems to be extended into the hind- and/or foregut. The engrailed expression of these species is compared with published data from other insects.

Organisms exhibit an incredible diversity of form, a fact that makes the evolution of novelty seemingly self-evident. However, despite the "obvious" case for novelty, defining this concept in evolutionary terms is highly problematic, so much so that some have suggested discarding it altogether. Approaches to this problem tend to take either an adaptation- or development-based perspective, but we argue here that an exclusive focus on either of these misses the original intent of the novelty concept and undermines its practical utility. We propose instead that for a feature to be novel, it must have evolved both by a transition between adaptive peaks on the fitness landscape and that this transition must have overcome a previous developmental constraint. This definition focuses novelty on the explanation of apparently difficult or low-probability evolutionary transitions and highlights how the integration of developmental and functional considerations are necessary to evolutionary explanation. It further reinforces that novelty is a central concern not just of evolutionary developmental biology (i.e., "evo-devo") but of evolutionary biology more generally. We explore this definition of novelty in light of four examples that range from the obvious to subtle.

INTRODUCTIONThe insect order Diptera (true flies) has great potential in evolutionary developmental biology, featuring the “fruit fly,” Drosophila melanogaster, one of the best-studied genetic model systems in developmental biology, and many human disease vectors (e.g., mosquitoes, blackflies, sandflies, tsetse flies). However, the embryos of only a few dipteran species are currently amenable to efficient functional analyses of developmental genes. As described here, the phorid fly Megaselia abdita has been used as model in evolutionary developmental biology and has been an important species in forensic studies.

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation—lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)—and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.

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.

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.

Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, we found evidence that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) all of which contribute cells to neuromeres in the central nervous system. Until completion of germ band retraction, the four pregnathal segment remnants and their corresponding neuromeres become arranged in an S-shape. We discuss published evidence for seven head segments and morphogenetic movements during head formation in various insects (and crustaceans).

We discovered extremely small genomes (1C similar to 100 Mb) in the dipteran insects Coboldia fuscipes (Scatopsidae) and Psychoda cinerea (Psychodidae). The small genomes of these species cannot be explained by a fast developmental rate, which has been shown to correlate with small genome sizes in animals and plants but might accommodate the combined effects of other developmental traits, including small egg size, thin blastoderm layer, and long-germ development.

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 zerknüllt ( 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 Krüppel ( 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 .

Establishment of segmental pattern in the Drosophila syncytial blastoderm embryo depends on pair-rule transcriptional regulators. mRNA transcripts of pair-rule genes localise to the apical cytoplasm of the blastoderm via a selective dynein-based transport system and signals within their 3′-untranslated regions. However, the functional and evolutionary significance of this process remains unknown. We have analysed subcellular localisation of mRNAs from multiple dipteran species both in situ and by injection into Drosophila embryos. We find that although localisation of wingless transcripts is conserved in Diptera, localisation of even-skipped and hairy pair-rule transcripts is evolutionarily labile and correlates with taxon-specific changes in positioning of nuclei. We show in Drosophila that localised pair-rule transcripts target their proteins in close proximity to the nuclei and increase the reliability of the segmentation process by augmenting gene activity. Our data suggest that mRNA localisation signals in pair-rule transcripts affect nuclear protein uptake and thereby adjust gene activity to a variety of dipteran blastoderm cytoarchitectures.

We discuss the interplay between evolution and development as reflected in data and concepts since about 1800. Darwin and his “continental apostle” Haeckel put the striking similarity between early vertebrate embryos in an evolutionary context. Haeckel's partly illicit generalizations discredited evolutionary thinking among early experimental embryologists who moreover noted riddles incompatible with contemporary concepts of homology and evolution. Relevant solutions were suggested by the more recent concept of ontogenetic networks that embody complex regulatory properties and genes with partly overlapping functions. Molecular data on development increasingly reveal evolutionary opportunism, for instance when a widespread signaling chain involved in primitive immune defense was apparently recruited later on for dorso-ventral axis determination in some evolutionarily advanced insect groups. Recently, Rickettsia-related bacteria colonizing many arthropod species were found to “manipulate” the first steps of host development to the advantage of their own propagation, but by ways that could also promote host speciation. Molecular genetics can now document evolutionary steps in ontogenetic networks. In the fruit fly for instance, the novel bicoid gene has superseded a crucial patterning function within a pre-existing network—a case of “molecular caenogenesis.” The expression patterns of conserved genes that antagonistically determine dorso-ventral polarity support a literal revolution envisioned almost 200 years ago. This is the dorso-ventral inversion of the body plan in some metazoans—ascribed then to the Articulata, now to the Chordata. The final section posits that the opportunistic character of evolutionary innovations is detrimental to parsimony in development. J. Exp. Zool. (Mol. Dev. Evol.) 302B:69–91, 2004. © 2004 Wiley-Liss, Inc.

In Drosophila, the establishment of the larval head and thorax depends on the transcription factors BICOID and HUNCHBACK, and on signalling mediated by the receptor tyrosine kinase TORSO. Genetic experiments described in two recent papers(1, 2) demonstrate that these factors can, to a large extent, replace each other, revealing a surprising degree of plasticity in establishing larval anterior structures. The commutability of developmental factors might in part reflect the evolutionary history of the system. BioEssays 23:8–11, 2001. © 2001 John Wiley & Sons, Inc.

SJB and RED thank Barbara Van Slyke for expert technical assistance. This work was supported by NIH and NSF (SJB and RED), and by the Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (US-O).

The Drosophila gene bicoid functions at the beginning of a gene cascade that specifies anterior structures in the embryo. Its transcripts are localized at the anterior pole of the oocyte, giving rise to a Bicoid protein gradient, which regulates the spatially restricted expression of target genes along the anterior–posterior axis of the embryo in a concentration-dependent manner. The morphogen function of Bicoid requires the coactivity of the zinc finger transcription factor Hunchback, which is expressed in a Bicoid-dependent fashion in the anterior half of the embryo. Whereas hunchback is conserved throughout insects, bicoid homologs are known only from cyclorrhaphan flies. Thus far, identification of hunchback and bicoid homologs rests only on sequence comparison. In this study, we used double-stranded RNA interference (RNAi) to address the function of bicoid and hunchback homologs in embryos of the lower cyclorrhaphan fly Megaselia abdita (Phoridae). Megaselia-hunchback RNAi causes hunchback -like phenotypes as observed in Drosophila , but Megaselia-bicoid RNAi causes phenotypes different from corresponding RNAi experiments in Drosophila and bicoid mutant embryos. Megaselia-bicoid is required not only for the head and thorax but also for the development of four abdominal segments. This difference between Megaselia and Drosophila suggests that the range of functional bicoid activity has been reduced in higher flies.

In developing insect eggs the cells of the blastoderm adopt either an embryonic or an extraembryonic fate. The extraembryonic tissue consists of epithelia, termed amnion and serosa, which wrap the germ band embryo. The serosa develops directly from part of the blastoderm and surrounds the embryo as well as the yolk. The amnion develops from the margins of the germ band and in most insect species generates a transient ventral cavity for the developing embryo. The amniotic cavity and the serosa have been reduced in the course of dipteran evolution. The insect order of Diptera includes the paraphyletic Nematocera, including gnats and mosquitoes, and the more derived monophyletic Brachycera, the true flies. Nematocera develop within an amniotic cavity and the surrounding serosa, whereas cyclorrhaphan Brachycera do not. This observation implies that the amnion and serosa have been reduced before the radiation of the monophyletic cyclorrhaphan flies. Here I show that an amniotic cavity is formed during embryogenesis of the horsefly Haematopota pluvialis (Tabanidae) and the dancefly Empis livida (Empididae). The results suggest that extraembryonic tissue was reduced in the stem lineage of cyclorrhaphan flies, with consequences for the molecular basis of pattern formation along the anterior-posterior axis of the embryo.

We describe the development of 20 sensory organs in the embryonic Drosophila head, which give rise to 7 sensory nerves of the peripheral nervous system (PNS), and 4 ganglia of the stomatogastric nervous system (SNS). Using these neural elements and the optic lobes as well as expression domains of the segment polarity gene engrailed in the wild-type head of Drosophila embryos as markers we examined the phenotype of different mutants which lack various and distinct portions of the embryonic head. In the mutants, distinct neural elements and engrailed expression domains, serving as segmental markers, are deleted. These mutants also affect the optic lobes to various degrees. Our results suggest that the optic lobes are of segmental origin and that they derive from the ocular segment anteriorly adjacent to the antennal segment of the developing head.

INTRODUCTIONThis protocol describes how to prepare cuticles of Megaselia abdita embryos that have been injected through the chorion and developed under oil.

INTRODUCTIONThis protocol describes the handling of Megaselia abdita embryos from staged egg depositions prior to injection.