Continuity versus split and reconstitution: exploring the molecular developmental corollaries of insect eye primordium evolution

Holometabolous insects like Drosophila proceed through two phases of visual system development. The embryonic phase generates simple eyes of the larva. The postembryonic phase produces the adult specific compound eyes during late larval development and pupation. In primitive insects, by contrast, eye development persists seemingly continuously from embryogenesis through the end of postembryogenesis. Comparative literature suggests that the evolutionary transition from continuous to biphasic eye development occurred via transient developmental arrest. This review investigates how the developmental arrest model relates to the gene networks regulating larval and adult eye development in Drosophila, and embryonic compound eye development in primitive insects. Consistent with the developmental arrest model, the available data suggest that the determination of the anlage of the rudimentary Drosophila larval eye is homologous to the embryonic specification of the juvenile compound eye in directly developing insects while the Drosophila compound eye primordium is evolutionarily related to the yet little studied stem cell based postembryonic eye primordium of primitive insects.     

The effect of manipulating ecdysteroid signaling on embryonic eye development in the locust<Emphasis Type="Italic"> Schistocerca americana</Emphasis>

Adult body plan differentiation in holometabolous insects depends on global induction and control by ecdysteroid hormones during the final phase of postembryogenesis. Studies in Drosophila melanogaster and Manduca sexta have shown that this pertains also to the development of the compound eye retina. It is unclear whether the hormonal control of postembryonic eye development in holometabolous insects represents evolutionary novelty or heritage from hemimetabolous insects, which develop compound eyes during embryogenesis. We therefore investigated the effect of manipulating ecdysteroid signaling in cultured embryonic eye primordia of the American desert locust Schistocerca americana, in which ecdysteroid level changes are known to induce three rounds of embryonic molt. Although at a considerably reduced rate compared to in vivo development, early differentiation and terminal maturation of the embryonic retina was observed in culture even if challenged with the ecdysteroid antagonist cucurbitacin B. Supplementing cultures with 20-hydroxyecdysone (20E) accelerated differentiation and maturation, and enhanced cell proliferation. Considering these results, and the relation between retina differentiation and ecdysteroid level changes during locust embryogenesis, we conclude that ecdysteroids are not an essential but possibly a modulatory component of embryonic retina development in S. americana. We furthermore found evidence that 20E initiated precocious epithelial morphogenesis of the posterior retinal margin indicating a more general role of ecdysteroids in insect embryogenesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by C. Desplan     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

Conservation of the expression of Dll, en, and wg in the eye-antennal imaginal disc of stalk-eyed flies

SUMMARY We studied the developmental basis of exaggerated eye span in two species of stalk-eyed flies ( Cyrtodiopsis dalmanni and Sphyracephala beccarri ). These flies have eyes laterally displaced at the end of eyestalks, and males have greatly exaggerated eye span, which they use as a sexual display. To investigate eye span development we have compared eye-antennal disc morphology and the expression of three key regulator genes of Drosophila head development, Distal-less ( Dll ) , engrailed ( en ), and wingless ( wg ), in the stalk-eyed flies and Drosophila . We found great similarity in the basic division of the disc into anterior-antennal and posterior-eye portions and in the general patterning of Dll, en , and wg . Unexpectedly, our results showed that although the eye and antenna are adjacent in adult stalk-eyed flies, their primordia are physically separated by the presence of an intervening region between the anterior and posterior portions of the disc. This region is absent from Drosophila eye-antennal discs. We chose two stalk-eyed fly species that differed in the degree of eyestalk exaggeration but surprisingly we found no corresponding difference in the size of the en-wg expression domains that mark the boundaries of the dorsal head capsule primordia. In summary, our expression data establish the regional identity of the eye-antennal disc and provide a framework from which to address the developmental genetics of hypercephaly.     

Divergent decapentaplegic expression patterns in compound eye development and the evolution of insect metamorphosis

In the fruit fly Drosophila, the patterning genes decapentaplegic and wingless contribute to the spatial control of retina development in an antagonistic manner. We examined the expression patterns of these genes in the developing visual system of the hemimetabolous grasshopper Schistocerca americana and the primitive holometabolous beetle species Tribolium castaneum. The pattern of wingless expression was strongly conserved as a pair of lateral domains at the anterior margins of both the developing retina and the developing optic lobes. The expression of decapentaplegic, on the other hand, is different. Unlike in Drosophila, no decapentaplegic expression was detected before the onset of photoreceptor differentiation in the retinal precursor tissue of either grasshopper or beetle. Moreover, the subsequent expression of decapentaplegic in the latter species was not concentrated in the moving front of retina differentiation, as in Drosophila, but observed in anterior and posterior regions. Our results indicate that Drosophila eye development contains elements of both ancestral and derived regulatory gene functions. The requirement for decapentaplegic as an antagonist of wingless during the early development of the Drosophila retina might have originated during the evolution of insect metamorphosis.     

Leg development in flies versus grasshoppers: differences in dpp expression do not lead to differences in the expression of downstream components of the leg patterning pathway

All insect legs are structurally similar, characterized by five primary segments. However, this final form is achieved in different ways. Primitively, the legs developed as direct outgrowths of the body wall, a condition retained in most insect species. In some groups, including the lineage containing the genus Drosophila, legs develop indirectly from imaginal discs. Our understanding of the molecular mechanisms regulating leg development is based largely on analysis of this derived mode of leg development in the species D. melanogaster. The current model for Drosophila leg development is divided into two phases, embryonic allocation and imaginal disc patterning, which are distinguished by interactions among the genes wingless (wg), decapentaplegic (dpp) and distalless (dll). In the allocation phase, dll is activated by wg but repressed by dpp. During imaginal disc patterning, dpp and wg cooperatively activate dll and also indirectly inhibit the nuclear localization of Extradenticle (Exd), which divide the leg into distal and proximal domains. In the grasshopper Schistocerca americana, the early expression pattern of dpp differs radically from the Drosophila pattern, suggesting that the genetic interactions that allocate the leg differ between the two species. Despite early differences in dpp expression, wg, Dll and Exd are expressed in similar patterns throughout the development of grasshopper and fly legs, suggesting that some aspects of proximodistal (P/D) patterning are evolutionarily conserved. We also detect differences in later dpp expression, which suggests that dpp likely plays a role in limb segmentation in Schistocerca, but not in Drosophila. The divergence in dpp expression is surprising given that all other comparative data on gene expression during insect leg development indicate that the molecular pathways regulating this process are conserved. However, it is consistent with the early divergence in developmental mode between fly and grasshopper limbs.     

Analysis of the expression pattern of Mysidium columbiae wingless provides evidence for conserved mesodermal and retinal patterning processes among insects and crustaceans

The Wnt family includes a number of genes, such as wingless ( wg), which encode secreted glycoproteins that function in numerous developmental patterning processes. In order to gain a better understanding of crustacean pattern formation, a wg orthologue was cloned from the malacostracan crustacean Mysidium columbiae(mysid), and the expression pattern of this gene was compared with that of Drosophila wg. Although Drosophila wg is expressed in many developing tissues, such as the ventral neuroectoderm, M. columbiae wg (mcowg)expression is detected within only a subset of these tissues. mcowg is expressed in the dorsal part of each developing segment and within the developing eye, but not within the ventral neuroectoderm. Dorsal wg expression in Drosophila is required for heart and muscle development, and conservation of this dorsal wgexpression pattern suggests that mcowgmay function to pattern these tissues in mysids. Consistent with this, expression of Even-skipped (Eve) protein in heart precursor and muscle cells, which is dependent on Wg signaling in Drosophila, is also conserved in mysids. Within the developing mysid eye, mcowg is expressed in a pattern that is similar to the expression pattern of Drosophila wg in the fly eye disc. In Drosophila,Wg inhibits neural differentiation at the anterior margin of the eye disc and patterns the dorsal/ventral axis of the eye. These data indicate that mcowg may function similarly during mysid eye development. Analysis of mcowgexpression provides molecular evidence suggesting that the processes of heart, muscle, and eye patterning are likely to be conserved among insects and crustaceans.     

Expression patterns of dachshund during head development of Gryllus bimaculatus (cricket)

We report that Gryllus bimaculatus dachshund (Gbdac), a cricket homologue of Drosophila dachshund (Dmdac), is expressed in the developing eye and brain. During brain development, Gbdac was first expressed in the medial head region, corresponding to a part of developing protocephalic region, and expressed in the primordial and adult Kenyon cells. During eye development, Gbdac was first expressed in the lateral head region, becoming to the eye primordium and a part of the deutocerebrum. Then, Gbdac was expressed in the posterior region of the eye primordium, prior to the formation of compound eyes. The expression domain shifted to the anterior domain concomitantly with the movement of morphogenetic furrows. Gbdac was also expressed in the developing optic lobes during differentiation of the retina. These expression patterns were compared with those of Dmdac. We found that although developmental processes of the Gryllus eye and brain differ from those of the Drosophila ones, the expression patterns of Gbdac are essentially similar to those of the Dmdac.     

Early development of leg and wing primordia in the Drosophila embryo

The development of the leg and wing primordia in the Drosophila embryo has been traced using molecular markers. Distal-less and disconnected gene expression provide molecular labels for the leg primordia throughout embryonic development, disconnected expression in the developing leg primordia depends on Distal-less activity. The leg primordia arise as discrete clusters of cells that occupy well defined positions in the embryonic ectoderm. At later stages of embryogenesis the primordia become morphologically recognizable and are intimately associated with the development of the Keilin's organs. The presumptive leg disc and the Keilin's organ appear to derive from a common primordium. Similarly the Abnormal leg pattern gene provides a molecular label for the wing and haltere primordia. The dorsal thoracic primordia appear to be of independent origin from the legs.     

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.     

DEAF-1 function is essential for the early embryonic development of Drosophila

The Drosophila protein DEAF-1 is a sequence-specific DNA binding protein that was isolated as a putative cofactor of the Hox protein Deformed (Dfd). In this study, we analyze the effects of loss or gain of DEAF-1 function on Drosophila development. Maternal/zygotic mutations of DEAF-1 largely result in early embryonic arrest prior to the expression of zygotic segmentation genes, although a few embryos develop into larvae with segmentation defects of variable severity. Overexpression of DEAF-1 protein in embryos can induce defects in migration/closure of the dorsal epidermis, and overexpression in adult primordia can strongly disrupt the development of eye or wing. The DEAF-1 protein associates with many discrete sites on polytene chromosomes, suggesting that DEAF-1 is a rather general regulator of gene expression.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.     

Dpp and Hh signaling in the Drosophila embryonic eye field.

We have analyzed the function of the Decapentaplegic (Dpp) and Hedgehog (Hh) signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. As reported previously, Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). We discuss the similarities between vertebrate systems and Drosophila with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling.     

Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.     

Early events in adult eye development of the moth, Manduca sexta

The eye imaginal disc of Manduca sexta is created early in the final larval instar from the adult eye primordium, which is composed of fully differentiated cells of the larval head capsule epidermis. Concomitant with the down-regulation of the larval epidermal program, expression of broad, a marker of pupal commitment, is activated in the primordium. The cells then detach from the cuticle, fold inward, and begin to proliferate at high levels to produce the inverted, eye imaginal disc. These and other events that begin on the first day of the final larval instar appear to mark the initiation of metamorphosis. Little is known about the endocrine control of the initiation of metamorphosis in any insect. The hemolymph titer of juvenile hormone (JH) declines to low levels during this period and the presence of JH is sufficient to repress development in cultured eye primordia. However, maintenance of JH at high levels in vivo by treatment with long-lasting JH mimics has no apparent effect on early steps in eye imaginal disc development. We discuss our findings in the context of the endocrine control of metamorphosis. The initiation of metamorphosis in Manduca, and perhaps a wide range of insect species, appears to involve the overcoming of JH repression by an unidentified, nutrient-dependent, hormonal factor.     

Functional analyses in the milkweed bug Oncopeltus fasciatus (Hemiptera) support a role for Wnt signaling in body segmentation but not appendage development

Specification of the proximal-distal (PD) axis of insect appendages is best understood in Drosophila melanogaster, where conserved signaling molecules encoded by the genes decapentaplegic (dpp) and wingless (wg) play key roles. However, the development of appendages from imaginal discs as in Drosophila is a derived state, while more basal insects produce appendages from embryonic limb buds. Therefore, the universality of the Drosophila limb PD axis specification mechanism has been debated since dpp expression in more basal insect species differs dramatically from Drosophila. Here, we test the function of Wnt signaling in the development of the milkweed bug Oncopeltus fasciatus, a species with the basal state of appendage development from limb buds. RNA interference of wg and pangolin (pan) produce defects in the germband and eyes, but not in the appendages. Distal-less and dachshund, two genes regulated by Wg signaling in Drosophila and expressed in specific PD domains along the limbs of both species, are expressed normally in the limbs of pan-depleted Oncopeltus embryos. Despite these apparently paradoxical results, Armadillo protein, the transducer of Wnt signaling, does not accumulate properly in the nuclei of cells in the legs of pan-depleted embryos. In contrast, engrailed RNAi in Oncopeltus produces cuticular and appendage defects similar to Drosophila. Therefore, our data suggest that Wg signaling is functionally conserved in the development of the germband, while it is not essential in the specification of the limb PD axis in Oncopeltus and perhaps basal insects.