Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterised Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity.     

Nanos plays a conserved role in axial patterning outside of the Diptera

Axial patterning is a fundamental event in early development, and molecules involved in determining the body axes provide a coordinate system for subsequent patterning. While orthologs of Drosophila bicoid and nanos play a conserved role in anteroposterior (AP) patterning within at least a subset of Diptera, conservation of this process has not yet been demonstrated outside of the flies. Indeed, it has been argued that bicoid, an instrumental "anterior" factor in Drosophila melanogaster, acquired this role during the evolution of more-derived dipterans. Interestingly, the interaction of Drosophila maternal nanos and maternal hunchback provides a system for patterning the AP axis that is partially redundant to the anterior system. Previous studies in grasshoppers suggest that hunchback may play a conserved role in axial patterning in this insect, but this function may be supplied solely by the zygotic component of hunchback expression. Here we provide evidence that the early pattern of zygotic grasshopper Hunchback expression is achieved through translational repression that may be mediated through the action of grasshopper nanos. This is consistent with the notion that an anterior gradient system is not necessary in all insects and that the posterior pole "probably conveys longitudinal polarity on the ensuing germ anlage".     

A major role for zygotic hunchback in patterning the Nasonia embryo

Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.     

Extreme divergence of a homeotic gene: the bicoid case

Evolutionary developmental genetics (evo-devo) reveals that the plasticity of development is so important that every developmental biology project should carefully take this point into consideration. The example of bicoid, the first discovered morphogen, illustrates how an essential gene can change its function during evolution. The search for bicoid homologues showed that this gene is surprisingly specific to flies (cyclorraphan diptera) and absent in other insects. In fact, recent studies demonstrate that bicoid is a very derived Hox3 homeotic gene. During insect evolution, the ancestral Hox3 gene lost its homeotic function and acquired new roles in oocytes and embryonic annexes. Then, in the lineage leading to modern flies, a duplication of this new gene, followed by functional divergence, led to the formation of bicoid and zerknüllt. Both genes are located within the Drosophila Hox complex; however, they have no homeotic function. Thanks to the power of Drosophila genetics, it is possible to suggest that torso and hunchback may constitute the insect primitive anterior organizer. The bicoid evolutionary history reveals several fundamental mechanisms of the evolution of developmental genes, such as changes of gene regulation, modifications of protein sequences and gene duplication. It also shows the need for studying a wider range of model organisms before generalisations can be made from data obtained with one particular species.     

Localization of bicoid mRNA in late oocytes is maintained by continual active transport

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential to produce the Bicoid protein gradient that patterns the anterior-posterior axis of the embryo. Previous studies have characterized a microtubule-dependent pathway for bicoid mRNA localization during midoogenesis, when bicoid first accumulates at the anterior. We show that the majority of bicoid is actually localized later in oogenesis, when the only known mechanism for mRNA localization is based on passive trapping. Through live imaging of fluorescently tagged endogenous bicoid mRNA, we identify a temporally distinct pathway for bicoid localization in late oocytes that utilizes a specialized subpopulation of anterior microtubules and dynein. The directional movement of bicoid RNA particles within the oocyte observed here is consistent with dynein-mediated transport. Furthermore, our results indicate that association of bicoid with the anterior oocyte cortex is dynamic and support a model for maintenance of bicoid localization by continual active transport on microtubules.     

Maternal Co-ordinate Gene Regulation and Axis Polarity in the Scuttle Fly Megaselia abdita

Axis specification and segment determination in dipteran insects are an excellent model system for comparative analyses of gene network evolution. Antero-posterior polarity of the embryo is established through systems of maternal morphogen gradients. In Drosophila melanogaster, the anterior system acts through opposing gradients of Bicoid (Bcd) and Caudal (Cad), while the posterior system involves Nanos (Nos) and Hunchback (Hb) protein. These systems act redundantly. Both Bcd and Hb need to be eliminated to cause a complete loss of polarity resulting in mirror-duplicated abdomens, so-called bicaudal phenotypes. In contrast, knock-down of bcd alone is sufficient to induce double abdomens in non-drosophilid cyclorrhaphan dipterans such as the hoverfly Episyrphus balteatus or the scuttle fly Megaselia abdita. We investigate conserved and divergent aspects of axis specification in the cyclorrhaphan lineage through a detailed study of the establishment and regulatory effect of maternal gradients in M. abdita. Our results show that the function of the anterior maternal system is highly conserved in this species, despite the loss of maternal cad expression. In contrast, hb does not activate gap genes in this species. The absence of this activatory role provides a precise genetic explanation for the loss of polarity upon bcd knock-down in M. abdita, and suggests a general scenario in which the posterior maternal system is increasingly replaced by the anterior one during the evolution of the cyclorrhaphan dipteran lineage.     

An anterior function for the Drosophila posterior determinant Pumilio

Bicoid is a key determinant of anterior Drosophila development. We demonstrate that the prototypical Puf protein Pumilio temporally regulates bicoid (bcd) mRNA translation via evolutionarily conserved Nanos response elements (NRE) in its 3'UTR. Disruption of Pumilio-bcd mRNA interaction by either Pumilio or bcd NRE mutations caused delayed bcd mRNA deadenylation and stabilization, resulting in protracted Bicoid protein expression during embryogenesis. Phenotypically, embryos from transgenic mothers that harbor bcd NRE mutations exhibited dominant anterior patterning defects and we discovered similar head defects in embryos from pum(-) mothers. Hence, Pumilio is required for normal anterior development. Since bcd mRNA resides outside the posterior gradient of the canonical partner of Pumilio, Nanos, our data suggest that Pumilio can recruit different partners to specifically regulate distinct mRNAs.     

A caudal mRNA gradient controls posterior development in the wasp Nasonia

One of the earliest steps of embryonic development is the establishment of polarity along the anteroposterior axis. Extensive studies of Drosophila embryonic development have elucidated mechanisms for establishing polarity, while studies with other model systems have found that many of these molecular components are conserved through evolution. One exception is Bicoid, the master organizer of anterior development in Drosophila and higher dipterans, which is not conserved. Thus, the study of anteroposterior patterning in insects that lack Bicoid can provide insight into the evolution of the diversity of body plan patterning networks. To this end, we have established the long germ parasitic wasp Nasonia vitripennis as a model for comparative studies with Drosophila. Here we report that, in Nasonia, a gradient of localized caudal mRNA directs posterior patterning, whereas, in Drosophila, the gradient of maternal Caudal protein is established through translational repression by Bicoid of homogeneous caudal mRNA. Loss of caudal function in Nasonia results in severe segmentation defects. We show that Nasonia caudal is an activator of gap gene expression that acts far towards the anterior of the embryo, placing it atop a cascade of early patterning. By contrast, activation of gap genes in flies relies on redundant functions of Bicoid and Caudal, leading to a lack of dramatic action on gap gene expression: caudal instead plays a limited role as an activator of pair-rule gene expression. These studies, together with studies in short germ insects, suggest that caudal is an ancestral master organizer of patterning, and that its role has been reduced in higher dipterans such as Drosophila.     

The formation of the Bicoid morphogen gradient requires protein movement from anteriorly localized mRNA

The Bicoid morphogen gradient directs the patterning of cell fates along the anterior-posterior axis of the syncytial Drosophila embryo and serves as a paradigm of morphogen-mediated patterning. The simplest models of gradient formation rely on constant protein synthesis and diffusion from anteriorly localized source mRNA, coupled with uniform protein degradation. However, currently such models cannot account for all known gradient characteristics. Recent work has proposed that bicoid mRNA spatial distribution is sufficient to produce the observed protein gradient, minimizing the role of protein transport. Here, we adapt a novel method of fluorescent in situ hybridization to quantify the global spatio-temporal dynamics of bicoid mRNA particles. We determine that >90% of all bicoid mRNA is continuously present within the anterior 20% of the embryo. bicoid mRNA distribution along the body axis remains nearly unchanged despite dynamic mRNA translocation from the embryo core to the cortex. To evaluate the impact of mRNA distribution on protein gradient dynamics, we provide detailed quantitative measurements of nuclear Bicoid levels during the formation of the protein gradient. We find that gradient establishment begins 45 minutes after fertilization and that the gradient requires about 50 minutes to reach peak levels. In numerical simulations of gradient formation, we find that incorporating the actual bicoid mRNA distribution yields a closer prediction of the observed protein dynamics compared to modeling protein production from a point source at the anterior pole. We conclude that the spatial distribution of bicoid mRNA contributes to, but cannot account for, protein gradient formation, and therefore that protein movement, either active or passive, is required for gradient formation.     

EVOLUTIONARY ORIGIN OF THE CYCLORRHAPHA (DIPTERA): TEST OF ALTERNATIVE MORPHOLOGICAL HYPOTHESES

Abstract— The higher flies, infraorder Cyclorrhapha [=Muscomorpha (McAlpine, 1989)], have undergone enormous radiation since the Cretaceous (∼100 Myr). Rapid morphological evolution in cyclorrhaphans has made their phylogenetic placement with respect to more primitive clades a long-standing problem in dipteran systematics. Of the two most plausible hypotheses, one treats the Cyclorrhapha as sister group to the orthorrhaphous superfamily Empidoidea [=Empidiformia (Hennig, 1948), Orthogenya (Brauer, 1883)], while the other places them within the empidoids. The debate over cyclorrhaphan origin has heretofore focused on homology interpretations for a few character systems, particularly the male genitalia. We provide the first attempt to assemble and quantify all of the available morphological evidence. By cladistic analysis of these data under alternative codings of genitalic features reflecting opposing homology theories, and then excluding these features altogether, we sought to judge which genitalic theory is better supported by the evidence as a whole, and how much the debate matters to resolving cyclorrhaphan origins. Using the analog of a factorial design, we also measured the effect of alternative transformation series in several other controversial characters, of outgroup choice and of successive weighting. Under all manipulations, including both genitalic codings, the Cyclorrhapha originate within the Empidoidea, near the family Atelestidae. However, trees in which the Empidoidea are constrained to be monophyletic are only 1-6 steps longer (out of ∼150), a fit not significantly worse under a permutation test for monophyly. Adult morphological data may not suffice to settle either the placement of Cyclorrhapha or the debate over genitalic homology. Moreover, the issue of genitalic homology does not appear critical to that of cyclorrhaphan origin.     

Segmentation gene expression in the housefly Musca domestica.

Drosophila and Musca both belong to the group of higher dipteran flies and show morphologically a very similar early development. However, these two species are evolutionary separated by at least 100 million years. This presents the opportunity for a comparative analysis of segmentation gene expression across a large evolutionary distance in a very similar embryonic background. We have analysed in detail the early expression of the maternal gene bicoid, the gap genes hunchback, Krüppel, knirps and tailless, the pair-rule gene hairy, the segment-polarity gene engrailed and the homoeotic gene Ultrabithorax. We show that the primary expression domains of these genes are conserved, while some secondary expression aspects have diverged. Most notable is the finding of hunchback expression in 11-13 stripes shortly before gastrulation, as well as a delayed expression of terminal domains of various genes. We conclude that the early developmental gene hierarchy, as it has been defined in Drosophila, is evolutionary conserved in Musca domestica.     

Establishment of the Deformed expression stripe requires the combinatorial action of coordinate, gap and pair-rule proteins.

In Drosophila embryos, anterior-posterior positional identities are set and maintained by the expression boundaries of homeotic selector genes. The establishment of the initial expression boundaries of the homeotic genes are in turn dependent on earlier acting patterning genes of Drosophila. To define the combinations of early genes that are required to establish a unique blastoderm stripe of expression of the homeotic gene Deformed, we have analysed single and double patterning mutants and heat shock promoter fusion constructs that ectopically express early acting regulators. We find that the activation of Deformed is dependent on combinatorial input from at least three levels of the early hierarchy. The simplest activation code sufficient to establish Deformed expression, given the absence of negative regulators such as fushi-tarazu, consists of a moderate level of expression from the coordinate gene bicoid, in combination with expression from both the gap gene hunchback, and the pair-rule gene even-skipped. In addition, the activation code for Deformed is redundant; other pair-rule genes in addition to even-skipped can apparently act in combination with bicoid and hunchback to activate Deformed.     

An interesting new species of Megaselia from Sulawesi, the ground plan of the Phoridae (Diptera) and phylogenetie implications for the Cyclorrhapha

Megaselia biarticulata sp.n. is described from Sulawesi. Its distinctly two-segmented palp reinforces the view that a two-segmented palp is part of the ground plan of the family. The tibial hair palisades are also postulated as part of the ground plan and serve to link the Phoridae to the Platypezidae. The median furrow on the frons is considered as part of the ground plan. Furthermore it is postulated that it is homologous with the frontal vitta in the Schizophora, and that its invaginated lower end gave rise to the ptilinum. The Empidoidea cannot be ancestral to the Cyclorrhapha and the case for regarding these two taxa as sister groups seems highly tenuous in the light of the inferred ground plan for the Phoridae.     

Phylogenetic relationships and the larval head of the lower Cyclorrhapha (Diptera)

We examined final‐stage larvae of all currently recognized lower cyclorrhaphan (= Aschiza) families, except Ironomyiidae and Sciadoceridae, and those of the higher cyclorrhaphan (= Schizophora) families Calliphoridae, Conopidae, Lonchaeidae, Muscidae, and Ulidiidae, and compared them with larvae of two out‐group families, Rhagionidae and Dolichopodidae, paying particular attention to structures of the head. A set of 86 morphological characters were analysed phylogenetically. The results show that the lower Cyclorrhapha is paraphyletic in relation to the higher Cyclorrhapha. The monophyly of the Cyclorrhapha is strongly supported. The lower Cyclorrhapha is resolved into two clades, based on the Lonchopteridae. Within the Syrphidae the traditional three‐subfamily system is supported, based on the Microdontinae. Within the lower Cyclorrhapha, the larval head is variable in form and arrangement of components. In Lonchopteridae, the mouth lies at the back of an open trough or furrow, comprising ventrally an elongate labium and laterally the maxilla. This arrangement of components appears to facilitate scooping food in water films. In Platypezoidea there is no furrow, and the dorsolateral lobes bearing the antennae are connected by a dorsal extension of the pseudocephalon. The main food‐gathering structure is the hooked apex of the labium, but in Phoridae the mandibles may also be important. In Eumuscomorpha the mandibles are at the apex of the head skeleton. The pseudocephalon is extended and infolded dorsally to form an oral pocket over the mouth. In the Pipunculidae, and the Microdontinae and Syrphinae of the Syrphidae, ventrally it forms a V‐shaped groove or guide along which the mandibles project. The labium is sclerotized apically, and forms a plate or tapered projection. This arrangement of components facilitates holding, piercing and extracting prey tissues. In Eristalinae the pseudocephalon is attached to the mandibles and is formed into a pair of cirri bearing mandibular lobes that lie either side of the mouth. Furthermore, the epipharynx is produced anteriorly in relation to the hypopharynx, and the labium is attached to the anterior part of the epipharynx to form a cavity or atrium. This arrangement is suited to fragmenting and imbibing solid food in Eristalinae with hooked mandibles, and when the mandibles are reduced and the mandibular lobes are inverted and sclerotized, these structures form a filter for separating fluid‐suspended particulate food. In higher Cyclorrhapha an atrium is present as in Eristalinae, but a connection between the pseudocephalon and the mandibles is absent. Instead, the pseudocephalon is bifurcate dorsally and forms a pair of cephalic lobes that ventrally ensheath each mandible. The surface of the sheath may be coated in cirri and other food‐gathering structures. The cephalic lobes, mandibular sheaths and the head skeleton are maneuverable and retractile to a higher degree than in lower Cyclorrhapha. This arrangement of components facilitates feeding on both solid food, in which the mouthooks may extend from the sheath to break the food up, and particulate and suspended food, in which the food‐gathering structures of the sheath scoop up the food. In many higher Cyclorrhapha, maneuverability is enhanced by a break between the labium and the basal sclerite, to which it is fused in all lower Cyclorrhapha. Intermediate characters and states for the structures of the higher cyclorrhaphan larval head are present in out‐groups, and lower Cyclorrhapha and homologies are discussed. Liquidity of the food is an important factor explaining the structure of the larval head in Cyclorrhapha. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 153 , 287–323.