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 mechanism of Drosophila leg development along the proximodistal axis

During development of higher organisms, most patterning events occur in growing tissues. Thus, unraveling the mechanism of how growing tissues are patterned into final morphologies has been an essential subject of developmental biology. Limb or appendage development in both vertebrates and invertebrates has attracted great attention from many researchers for a long time, because they involve almost all developmental processes required for tissue patterning, such as generation of the positional information by morphogen, subdivision of the tissue into distinct parts according to the positional information, localized cell growth and proliferation, and control of adhesivity, movement and shape changes of cells. The Drosophila leg development is a good model system, upon which a substantial amount of knowledge has been accumulated. In this review, the current understanding of the mechanism of Drosophila leg development is described.     

Heads and tails: evolution of antero-posterior patterning in insects

In spite of their varied appearances, insects share a common body plan whose layout is established by patterning genes during embryogenesis. We understand in great molecular detail how the Drosophila embryo patterns its segments. However, Drosophila has a type of embryogenesis that is highly derived and varies extensively as compared to most insects. Therefore, the study of other insects is invaluable for piecing together how the ancestor of all insects established its segmented body plan, and how this process can be plastic during evolution. In this review, we discuss the evolution of Antero-Posterior (A-P) patterning mechanisms in insects. We first describe two distinct modes of insect development - long and short germ development - and how these two modes of patterning are achieved. We then summarize how A-P patterning occurs in the long-germ Drosophila, where most of our knowledge comes from, and in the well-studied short-germ insect, Tribolium. Finally, using examples from other insects, we highlight differences in patterns of expression, which suggest foci of evolutionary change.     

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".     

An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development

The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. We have investigated the in vivo role that such acylation might play in appendage patterning in mouse and Drosophila; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species.     

'De-evolution' of Drosophila toward a more generic mode of axis patterning

The genetics of the establishment of the primary axes of the early embryo have been worked out in great detail Drosophila. However, evidence has accumulated that Drosophila employs a mode of patterning that is not shared with most insects. In particular, the use of the morphogenic gradient of the Bicoid homeoprotein appears to be a novel addition to the fly developmental toolkit. To better understand the ancestral mode of patterning that is probably more widely used by insects, several groups have used Evo-Devo approaches as well as sophisticated genetic manipulations of Drosophila to achieve some form of 'de-evolution' of this derived insect. Genetic manipulations of the beetle Tribolium and the wasp Nasonia have validated most of these results.     

Short and long germ segmentation: unanswered questions in the evolution of a developmental mode

The insect body plan is very well conserved, yet the developmental mechanisms of segmentation are surprisingly varied. Less evolutionarily derived insects undergo short germ segmentation where only the anterior segments are specified before gastrulation whereas the remaining posterior segments are formed during a later secondary growth phase. In contrast, derived long germ insects such as Drosophila specify their entire bodies essentially simultaneously. These fundamental embryological differences imply potentially divergent molecular patterning events. Numerous studies have focused on comparing the expression and function of the homologs of Drosophila segmentation genes between Drosophila and different short and long germ insects. Here we review these comparative data with special emphasis on understanding how short germ insects generate segments and how this ancestral mechanism may have been modified in derived long germ insects such as Drosophila. We break down the larger issue of short versus long germ segmentation into its component developmental problems and structure our discussion in order to highlight the unanswered questions in the evolution of insect segmentation.     

Chinmo and neuroblast temporal identity

Although spatial patterning during embryonic development is well characterized, a corresponding framework for temporal patterning has not been established. In this issue, Zhu et al. (2006) identify the Chinmo protein as conferring temporal identity on the neural progeny of Drosophila neuroblasts, revealing appealing parallels with spatial patterning.     

Establishing positional information through gradient dynamics: A lesson from the Hedgehog signaling pathway

A long standing question in developmental biology is how morphogen gradients establish positional information during development. Although the existence of gradients and their role in developmental patterning is no longer in doubt, the ability of cells to respond to different morphogen concentrations has been controversial. In the Drosophila wing disc, Hedgehog (Hh) forms a concentration gradient along the anterior-posterior axis and establishes at least three different gene expression patterns. In a recent study, we challenged the prevailing idea that Hh establishes positional information in a dose-dependent manner and proposed a model in which dynamics of the gradient, resulting from the Hh gene network architecture, determines pattern formation in the wing disc. In this Extra View, we discuss further the methodology used in this study, highlight differences between this and other models of developmental patterning, and also present some questions that remain to be answered in this system.Key words: Hedgehog, developmental patterning, morphogen, dynamics, mathematical modeling     

Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, we have isolated and analyzed expression of Hremx, the ascidian homologue of ems/emx with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis responded in different ways upon treatment with retinoic acid: the anterior expression domain was unaltered, while the posterior expression domain extended to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. We suggest the possibility that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates.     

Novel modes of localization and function of nanos in the wasp Nasonia

Abdominal patterning in Drosophila requires the function of nanos (nos) to prevent translation of hunchback (hb) mRNA in the posterior of the embryo. nos function is restricted to the posterior by the translational repression of mRNA that is not incorporated into the posteriorly localized germ plasm during oogenesis. The wasp Nasonia vitripennis (Nv) undergoes a long germ mode of development very similar to Drosophila, although the molecular patterning mechanisms employed in these two organisms have diverged significantly, reflecting the independent evolution of this mode of development. Here, we report that although Nv nanos (Nv-nos) has a conserved function in embryonic patterning through translational repression of hb, the timing and mechanisms of this repression are significantly delayed in the wasp compared with the fly. This delay in Nv-nos function appears to be related to the dynamic behavior of the germ plasm in Nasonia, as well as to the maternal provision of Nv-Hb protein during oogenesis. Unlike in flies, there appears to be two functional populations of Nv-nos mRNA: one that is concentrated in the oosome and is taken up into the pole cells before evidence of Nv-hb repression is observed; another that forms a gradient at the posterior and plays a role in Nv-hb translational repression. Altogether, our results show that, although the embryonic patterning function of nos orthologs is broadly conserved, the mechanisms employed to achieve this function are distinct.     

Computational analysis of BMP gradients in dorsal-ventral patterning of the zebrafish embryo

The genetic network controlling early dorsal-ventral (DV) patterning has been extensively studied and modeled in the fruit fly Drosophila. This patterning is driven by signals coming from bone morphogenetic proteins (BMPs), and regulated by interactions of BMPs with secreted factors such as the antagonist short gastrulation (Sog). Experimental studies suggest that the DV patterning of vertebrates is controlled by a similar network of BMPs and antagonists (such as Chordin, a homologue of Sog), but differences exist in how the two systems are organized, and a quantitative comparison of pattern formation in them has not been made. Here, we develop a computational model in three dimensions of the zebrafish embryo and use it to study molecular interactions in the formation of BMP morphogen gradients in early DV patterning. Simulation results are presented on the dynamics BMP gradient formation, the cooperative action of two feedback loops from BMP signaling to BMP and Chordin synthesis, and pattern sensitivity with respect to BMP and Chordin dosage. Computational analysis shows that, unlike the case in Drosophila, synergy of the two feedback loops in the zygotic control of BMP and Chordin expression, along with early initiation of localized Chordin expression, is critical for establishment and maintenance of a stable and appropriate BMP gradient in the zebrafish embryo.     

Morphogens in vertebrate development: How do they work?

The idea that concentration gradients of crucial substances might control the pattern of development, even in the embryos of complex organisms, has been around for a long time, but mostly in obscure forms. Twenty five years ago clear, experimentally testable ideas about how such gradients might work were enunciated, and more recently the morphogen gradient principle was shown to underlie the beginnings of patterning in Drosophila. Is it also central to vertebrate development? Four recent papers raise experimentation to a new level^(1–4), while showing how difficult it might be to pin down the precise form of the mechanism.     

Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae

Embryo segmentation has been studied extensively in the fruit fly, DROSOPHILA: These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt and paired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading to Drosophila at least 520 million years ago. In T. urticae, the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The mite runt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.     

The T-box genes H15 and optomotor-blind in the spiders Cupiennius salei, Tegenaria atrica and Achaearanea tepidariorum and the dorsoventral axis of arthropod appendages

SUMMARY Dorsoventral axis formation in the legs of the fly Drosophila melanogaster requires the T-box genes optomotor-blind ( omb ) and H15 . Evolutionary conservation of the patterning functions of these genes is unclear, because data on H15 expression in the spider Cupiennius salei did not support a general role of H15 in ventral fate specification. However, H15 has a paralogous gene, midline ( mid ) in Drosophila and H15 duplicates are also present in Cupiennius and the millipede Glomeris marginata . H15 therefore seems to have been subject to gene duplication opening the possibility that the previous account on Cupiennius has overlooked one or several paralogs. We have studied omb - and H15 -related genes in two additional spider species, Tegenaria atrica and Achearanea tepidariorum and show that in both species one of the H15 genes belongs to a third group of spider H15 genes that has an expression pattern very similar to the H15 pattern in Drosophila . The expression patterns of all omb -related genes are also very similar to the omb expression pattern in Drosophila . These data suggest that the dorsoventral patterning functions of omb and H15 are conserved in the arthropods and that the previous conclusions were based on an incomplete data set in Cupiennius . Our results emphasize the importance of a broad taxon sampling in comparative studies.     

Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Pattern formation: a new twist to BMP signalling

Dorsal-ventral patterning in Xenopus and Drosophila embryos involves BMP family signalling molecules. Twisted Gastrulation has now been added to the list of proteins that regulate the activity of these molecules, providing new insights into how BMPs are made available to their signalling receptors.     

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.     

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.