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.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.     

Wasps, beetles and the beginning of the ends

Recent papers investigating the genes regulating early embryogenesis in the wasp Nasonia vitripennis and the beetle Tribolium castaneum have provided us with important clues as to how early development is controlled in insects other than higher dipterans such as Drosophila melanogaster. The results of these studies demonstrate that in insects that do not have bicoid, anterior patterning is regulated by a combination of maternal orthodenticle and hunchback. Furthermore, during the evolution of long-germ-band development, Nasonia and Drosophila may have evolved different mechanisms to pattern posterior segments, marginalising the important role of the terminal system in short-germ-band embryos.     

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.     

Hypothesis testing in evolutionary developmental biology: a case study from insect wings

Developmental data have the potential to give novel insights into morphological evolution. Because developmental data are time-consuming to obtain, support for hypotheses often rests on data from only a few distantly related species. Similarities between these distantly related species are parsimoniously inferred to represent ancestral aspects of development. However, with limited taxon sampling, ancestral similarities in developmental patterning can be difficult to distinguish from similarities that result from convergent co-option of developmental networks, which appears to be common in developmental evolution. Using a case study from insect wings, we discuss how these competing explanations for similarity can be evaluated. Two kinds of developmental data have recently been used to support the hypothesis that insect wings evolved by modification of limb branches that were present in ancestral arthropods. This support rests on the assumption that aspects of wing development in Drosophila, including similarities to crustacean epipod patterning, are ancestral for winged insects. Testing this assumption requires comparisons of wing development in Drosophila and other winged insects. Here we review data that bear on this assumption, including new data on the functions of wingless and decapentaplegic during appendage allocation in the red flour beetle Tribolium castaneum.     

Pax group III genes and the evolution of insect pair-rule patterning

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.     

hunchback is required for suppression of abdominal identity, and for proper germband growth and segmentation in the intermediate germband insect Oncopeltus fasciatus

Insects such as Drosophila melanogaster undergo a derived form of segmentation termed long germband segmentation. In long germband insects, all of the body regions are specified by the blastoderm stage. Thus, the entire body plan is proportionally represented on the blastoderm. This is in contrast to short and intermediate germband insects where only the most anterior body regions are specified by the blastoderm stage. Posterior segments are specified later in embryogenesis during a period of germband elongation. Although we know much about Drosophila segmentation, we still know very little about how the blastoderm of short and intermediate germband insects is allocated into only the anterior segments, and how the remaining posterior segments are produced. In order to gain insight into this type of embryogenesis, we have investigated the expression and function of the homolog of the Drosophila gap gene hunchback in an intermediate germ insect, the milkweed bug, Oncopeltus fasciatus. We find that Oncopeltus hunchback (Of'hb) is expressed in two phases, first in a gap-like domain in the blastoderm and later in the posterior growth zone during germband elongation. In order to determine the genetic function of Of'hb, we have developed a method of parental RNAi in the milkweed bug. Using this technique, we find that Oncopeltus hunchback has two roles in anterior-posterior axis specification. First, Of'hb is required to suppress abdominal identity in the gnathal and thoracic regions. Subsequently, it is then required for proper germband growth and segmentation. In milkweed bug embryos depleted for hunchback, these two effects result in animals in which a relatively normal head is followed by several segments with abdominal identity. This phenotype is reminiscent to that found in Drosophila hunchback mutants, but in Oncopeltus is generated through the combination of the two separate defects.     

Maternal torso signaling controls body axis elongation in a short germ insect

In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system . Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud . The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood . We functionally analyzed the terminal system in the short germ insect Tribolium. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. We therefore conclude that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

Insights into the urbilaterian brain: conserved genetic patterning mechanisms in insect and vertebrate brain development

Recent molecular genetic analyses of Drosophila melanogaster and mouse central nervous system (CNS) development revealed strikingly similar genetic patterning mechanisms in the formation of the insect and vertebrate brain. Thus, in both insects and vertebrates, the correct regionalization and neuronal identity of the anterior brain anlage is controlled by the cephalic gap genes otd/Otx and ems/Emx, whereas members of the Hox genes are involved in patterning of the posterior brain. A third intermediate domain on the anteroposterior axis of the vertebrate and insect brain is characterized by the expression of the Pax2/5/8 orthologues, suggesting that the tripartite ground plans of the protostome and deuterostome brains share a common evolutionary origin. Furthermore, cross-phylum rescue experiments demonstrate that insect and mammalian members of the otd/Otx and ems/Emx gene families can functionally replace each other in embryonic brain patterning. Homologous genes involved in dorsoventral regionalization of the CNS in vertebrates and insects show remarkably similar patterning and orientation with respect to the neurogenic region (ventral in insects and dorsal in vertebrates). This supports the notion that a dorsoventral body axis inversion occurred after the separation of protostome and deuterostome lineages in evolution. Taken together, these findings demonstrate conserved genetic patterning mechanisms in insect and vertebrate brain development and suggest a monophyletic origin of the brain in protostome and deuterostome bilaterians.     

The origin,patterning and evolution of insect appendages

The appendages of the adult fruit fly and other insects and Arthropods develop from secondary embryonic fields that form after the primary anterior/posterior and dorsal/ventral axes of the embryo have been determined. In Drosophila, the position and fate of the different fields formed within each segment are determined by genes acting along both embryonic axes, within individual segments, and within specific fields. Since the major architectural differences between most Arthropod classes and orders involve variations in the number, type and morphology of body appendages, the elucidation of the embryology and molecular genetics of the origin and patterning of insect limb fields may help to facilitate an understanding of both the mechanism of appendage formation and some of the major steps in the morphological evolution of the Arthropods. In this review, we will discuss recent studies that have advanced our understanding of both the origin and patterning of Drosophila leg and wing secondary fields. These results provide fresh insights into potentially general mechanisms of how body parts develop and evolve.     

Expression patterns of the homeotic genes Scr, Antp, Ubx, and abd-A during embryogenesis of the cricket Gryllus bimaculatus

We have studied embryogenesis of the two-spotted cricket Gryllus bimaculatus as an example of a hemimetabolous, intermediate germ insect, which is a phylogenetically basal insect and may retain primitive features. We observed expression patterns of the orthologs of the Drosophila homeotic genes, Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) during embryogenesis and compared the expression patterns of these genes with the more basal thysanuran insect, Thermobia domestica (the firebrat), and the derived higher dipteran insect, Drosophila melanogaster. Although Scr is expressed commonly in the presumptive posterior maxillary and labial segment in all three insects, the thoracic expression domains vary. Antp is expressed similarly in the three thoracic segments, the limbs, and the anterior abdominal region among these three insects. The early Antp expression in the firebrat and cricket obeys a segmental register in all three thoracic segments, while in Drosophila its initial expression appears in parasegments 4 and 6. Ubx is expressed in the metathoracic (T3) and abdominal segments similarly in the three insects, whereas the expression pattern in the T3 leg differs among them. abd-A is expressed in the posterior compartment of the first abdominal segment and the remaining abdominal segments in all three insects, although its posterior border varies among them.     

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     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

Polyembryony in parasitic wasps: evolution of a novel mode of development

Major developmental innovations have been associated with adaptive radiations that have allowed particular groups of organisms to occupy empty ecospace. Well-known developmental novelties associated with the conquest of new habitats include the evolution of the tetrapode limb, allowing the radiation of vertebrates into a terrestrial habitat, and formation of insect wings that permitted their dispersal into the air. However, an understanding of the evolutionary forces and molecular mechanisms behind developmental novelties still remains tenuous. A little-studied adaptive radiation in insects from the developmental perspective is the evolution of parasitism. The parasitic lifestyle has allowed parasitic insects to occupy a novel ecological niche where they have evolved a plethora of life history strategies and modes of embryogenesis, developing on or within the body of the host. One of the most striking adaptations to development within the body of the host includes polyembryonic development, where certain wasps form clonally up to 2000 embryos from a single egg. Taking advantage of well-established insect phylogeny, techniques developed in a model insect, the fruit fly, and a wealth of knowledge in comparative insect embryology, we are starting to tease apart the evolutionary events that have led to this novel mode of development in insects.     

Expression of pair rule gene orthologs in the blastoderm of a myriapod: evidence for pair rule-like mechanisms?

ABSTRACT: BACKGROUND: A hallmark of Drosophila segmentation is the stepwise subdivision of the body into smaller and smaller units, and finally into the segments. This is achieved by the function of the well-understood segmentation gene cascade. The first molecular sign of a segmented body appears with the action of the pair rule genes, which are expressed as transversal stripes in alternating segments. Drosophila development, however, is derived, and in most other arthropods only the anterior body is patterned (almost) simultaneously from a pre-existing field of cells; posterior segments are added sequentially from a posterior segment addition zone. A long-standing question is to what extent segmentation mechanisms known from Drosophila may be conserved in short-germ arthropods. Despite the derived developmental modes, it appears more likely that conserved mechanisms can be found in anterior patterning. RESULTS: Expression analysis of pair rule gene orthologs in the blastoderm of the pill millipede Glomeris marginata (Myriapoda: Diplopoda) suggests that these genes are generally involved in segmenting the anterior embryo. We find that the Glomeris pairberry-1 (pby-1) gene is expressed in a pair rule pattern that is also found in insects and a chelicerate, the mite Tetraynchus urticae. Other Glomeris pair rule gene orthologs are expressed in double segment wide domains in the blastoderm, which at subsequent stages split into two stripes in adjacent segments. CONCLUSIONS: The expression patterns of the millipede pair rule gene orthologs resemble pair rule patterning in Drosophila and other insects, and thus represent evidence for the presence of an ancestral pair rule-like mechanism in myriapods. We discuss the possibilities that blastoderm patterning may be conserved in long-germ and short-germ arthropods, and that a posterior double segmental mechanism may be present in short-germ arthropods.     

Life history shifts and alterations in the early development of parasitic wasps

Summary

In insects, the regulation of embryonic development has been intensively studied in model species like Drosophila melanogaster. Previous comparative studies have suggested that the developmental processes documented in Drosophila well describe embryogenesis of holometabolous insects generally. However, there have been few attempts to take into account how life history has influenced insect embryogenesis or to characterize early development of species with life histories fundamentally different from flies. Our studies of advanced insects in the order Hymenoptera suggest that punctuated shifts in life history can profoundly influence these events. In particular, alterations associated with the evolution of endoparasitism argue that departures from the fly paradigm may occur commonly among insects that develop under conditions different from typical terrestrial species.