Recent comparative studies have revealed significant differences in the developmental gene networks operating in three holometabolous insects: the beetle Tribolium castaneum, the para- sitic wasp Nasonia vitripennis, and the fruit fly Drosophila melanogaster. In this chapter I discuss these differences in relation to divergent and con- vergent changes in cellular embryology. I speculate on how segmentation gene networks could have evolved to operate in divergent embryological con- texts, and highlight the role that co-option might have played in this process. I argue that insects represent an important example of how diversifi- cation in life-history strategies between lineages can lead to divergence in the genetic and cellular mechanisms controlling the development of hom- ologous adult structures.
16.1 Introduction
Arthropods are defined by a segmented body plan consisting of a series of anteroposteriorly arrayed segmental units with associated jointed append- ages. The insects are traditionally viewed as one of the four major monophyletic arthropod groups, the other three being crustaceans, myriapods, and chelicerates. However, recent molecular phyloge- nies suggest that crustaceans are paraphyletic with respect to the insects; i.e. insects could reasonably be regarded as a monophyletic clade of terrestrial crustaceans (Carapelli et al., 2007). While many insect species have retained the ancestral condi- tion of undergoing metamorphosis from larva to adult through a series of intermediate nymphal
stages (the hemimetabolous insects; Figure 16.1), the holometabolous insects undergo complete metamorphosis from larva to adult via a pupal stage (Brusca and Brusca, 2003). This is consid- ered a derived life-history trait that arose only once during insect evolution (Brusca and Brusca,
2003) (see Figure 16.1). The vast majority of holom- etabolous insects belong to four speciose orders: the Diptera (two-winged flies), the Lepidoptera (butterflies and moths), the Coleoptera (beetles), and the Hymenoptera (wasps, bees, ants, etc.). We currently have a better understanding of the devel- opmental genetic network underlying segmen- tation in a member of the Diptera—particularly the fruitfly D. melanogaster—than for any other insect, or indeed arthropod (Lawrence, 1992) (see Figure 16.2). However, a representative of the Coleoptera, the beetle T. castaneum, and a repre- sentative of the Hymenoptera, the parasitic wasp N. vitripennis, are rapidly being established as powerful insect model systems (Choe et al., 2006; Brent et al., 2007). Recent studies have revealed significant differences in the segmentation gene networks operating in these insects when com- pared with each other and with D. melanogaster (Schröder, 2003; Bucher and Klingler, 2004; Cerny et al., 2005; Choe et al., 2006; Lynch et al., 2006a,b; Olesnicky et al., 2006; Brent et al., 2007; Choe and Brown, 2007). In this chapter, I review and discuss these differences in relation to the modes of cellu- lar embryogenesis exhibited by these insects. Both D. melanogaster and N. vitripennis have evolved a rapid mode of development that required major changes in embryogenesis at the cellular level
171
172 AN I M AL EV O L UTI O N
(Bull, 1982; Lawrence, 1992; Davis and Patel, 2002). In contrast, T. castaneum has retained a more ances- tral mode of cellular embryogenesis (Handel et al.,
2000; Davis and Patel, 2002). I speculate on how insect segmentation gene networks have evolved to operate in these divergent embryological con- texts. A recent molecular phylogeny suggests that the rapid mode of cellular embryogenesis exhib- ited by D. melanogaster and N. vitripennis evolved convergently (Savard et al., 2006). I go on to ask whether convergent changes in gene networks might have underpinned these apparent parallel transitions in cellular embryology. First I review the modes of cellular embryogenesis found within the insects, and discuss the role that life history has played in their evolution.
16.2 The influence of life-history strategy on insect embryogenesis
16.2.1 An evolutionary biologist’s view on development
The principal aim of a developmental biologist is to work towards establishing a more complete picture of how the genetic information contained within an organism’s genome is deployed over developmen- tal time to transform a single cell into a functional multicellular organism. In contrast, the principal aim of many evolutionary developmental biolo- gists is to identify—through comparative analysis of developmental data within a phylogenetic frame- work—the changes in developmental mechanisms
Orthoptera
Hemiptera
Schistocerca sp. Gryllus bimaculatus
Oncopeltus fasciatus
Hemi No
Hemi ?
Seq ?
Seq ?
Hymenoptera
Nasonia vitripennis Yes Long Yes
Bracon hebetor Yes Long ?
Holo
Aphidius ervi No Seq ?
Apis mellifera Yes Long* ?
Coleoptera
Lepidoptera
Diptera
Tribolium castaneum
Callosobruchus maculates
Bombyx mori
Manduca sexta
Drosophila melanogaster
Anopheles gambiae
Holo
Holo
Holo
Yes
No
?
Yes
Seq
Long*
Seq* Long*
Long
Yes
?
? Yes
Figure 16.1 A phylogeny of the insect species discussed in this chapter with embryological features mapped on. The relative relationships of the four holometabolous insect orders follows the study by Savard et al. (2006). Some insects do not fit comfortably into the categories
‘sequential’ or ‘long-germ’ segmentation; for caveats in relation to the categorization of specific species (*) see Davis and Patel (2002). Character states have been left clear where there are uncertainties, i.e. when there is a lack of gene expression data and/or dye injection experiments to ascertain the existence of an extended syncytial blastoderm stage.
E V OL UTION OF DE VE L OP ME NT A L NE T W OR K S 173
Anterior Posterior
1
Hunchback
2
Hunchback
Gaint
Krüppel Knirps Gaint
3
Even-skipped
Fushi-tarazu
4
Engrailed
Wingless
Parasegment boundaries
Figure 16.2 The Drosophila melanogaster segmentation gene cascade. Adapted from Peel et al. (2005).
(1) Maternal genes. Maternal transcripts of the segmentation genes caudal and hunchback are uniformly distributed, whereas maternal bicoid mRNA is tethered to the anterior pole of the egg. Localized at the posterior pole is a complex of maternal proteins and RNAs that includes transcripts of the gene nanos. On fertilization, maternal mRNAs are de-repressed, translated and Bicoid and Nanos proteins form gradients at either end of the egg. Bicoid activates zygotic hunchback expression and represses caudal translation in the anterior, whereas Nanos represses the translation of maternal hunchback in the posterior. As a result Hunchback protein is restricted to the anterior of the egg and protein gra- dients of Bicoid (decreasing posteriorly) and Caudal (decreasing anteriorly) form. In parallel, a maternally encoded terminal patterning system operates during embryogenesis; the product of torso-like—which is expressed within specialized follicle cells situated at both egg poles during oogenesis—catalyses the localized cleavage of a protein encoded by trunk within the perivitelline fluid. The trunk cleavage product acts as a ligand on the receptor tyrosine kinase encoded by torso, triggering a signalling cascade that regulates the zygotic expression of downstream segmentation genes, such as tailless, at either pole of the egg.
(2) Gap genes. The net result of maternal signalling is the activation along the anteroposterior axis of the egg of a series of zygotic gap genes (i.e. giant, Krüppel, tailless), thus named because their mutation leads to gaps in the region of the embryo in which they are normally expressed. The protein products of the gap genes themselves diffuse within the syncytial blastoderm, regulate each other, and thus further refine their expression. Gap genes also play an important role at this stage in regulating the expression of the Hox genes, whose proteins products confer identity to segments.
(3) Pair-rule genes. In the next tier of the Drosophila segmentation cascade are three genes—even-skipped, runt, and hairy—whose expression is driven by the maternal and gap gene transcription factor products. All three genes possess complex regulatory sequences that interpret the aperiodic expression of maternal and gap gene products and drive expression in a periodic pattern of seven stripes. These genes are collect- ively referred to as pair-rule genes since their mutation often leads to abnormalities in alternate segments. The three ‘primary’ pair-rule gene products in turn regulate expression of ‘secondary’ pair-rule genes, such as fushi-tarazu, paired, sloppy-paired, and odd-skipped. Black curve, even-skipped; grey curve, fushi-tarazu.
174 AN I M AL EV O L UTI O N
that underpin divergence in body architecture between lineages. Rather than thinking in terms of developmental time—with the rather arbitrary starting point of zygote or germ cell—evolutionary developmental biologists consider evolutionary timescales, and as such development is not viewed as a linear process but rather as continuous devel- opmental cycles undergoing constant modification in response to selection and drift.
Natural selection can act independently on dis- tinct stages of an organism’s developmental cycle. This is obvious when considering insects. There has clearly been divergence in segment form between insect species, particularly with respect to append- age morphology; compare, for example, the suck- ing mouthparts of the phytophagous milkweed bug Oncopeltus fasciatus (Hughes and Kaufman,
2000) with the mandibles of some carnivorous beetles (Konuma and Chiba, 2007). However, it is clear that, on the whole, the basic insect segmental unit has been conserved. In contrast, oocytes and early eggs exhibit significant morphological differ- ences, a consequence of the numerous and diverse life-history strategies that have evolved within the insects.
16.2.2 All eggs are different, but some eggs are more different than others
Evolutionary shifts in insect life-history strategies often correlate with changes in cellular modes of embryogenesis. This was dramatically illustrated in a study by Grbic and Strand (1998) on two para- sitic wasps belonging to the hymenopteran fam- ily Braconidae. Bracon hebetor is an ectoparasite that lays yolky eggs on the integument of moth larvae. In the lineage leading to Aphidius ervi, however, there has been a transition to an endo- parasitic life history; A. ervi lays a single yolkless egg into the haemocoel of an aphid host. Grbic and
Strand (1998) studied the cellular embryology of these insects and found significant differences. In the eggs of B. hebetor, the cellularization of early cleavage nuclei is delayed until after they form a blastoderm, and all segments develop more or less simultaneously. In contrast, in A. ervi eggs, com- plete cytokinesis (the formation of cell membranes) occurs from the fourth round of nuclear divisions onwards, the early embryo ruptures from the chor- ion within the host haemocoel, and segments form one by one in an anterior to posterior progression. One can only speculate on why the transition to an endoparasitic life history required such dra- matic changes in cellular embryology, but is seems likely they are associated with the transition from receiving nutrients in the form of maternal yolk to the use of nutrients available from the haemo- lymph of the unfortunate host.
Similarly dramatic cellular transitions in embryogenesis have occurred within non-para- sitic insect lineages (for an in-depth review see Davis and Patel, 2002). Although the precise eco- logical reasons remain speculative, it seems likely that in many cases these transitions occurred in response to selection for increases in the speed of embryogenesis. Here I discuss two specific cellular adaptations and how they might have facilitated the faster development of an insect segmented body plan.
The timing of cellularization
In most insect species early nuclear divisions
are superficial; the formation of cell membranes
around early cleavage nuclei is delayed until they
have migrated to the egg surface and formed the
blastoderm. For example, dye injection experi-
ments have demonstrated this to be the case in
the locust Schistocerca gregaria (Ho et al., 1997)
(Figure 16.1). However, this delay is particularly
pronounced in some holometabolous insect lin-
(4) Segment-polarity genes. The pair-rule gene products activate the final tier in the Drosophila segmentation gene cascade, the segment polar- ity genes. These are the genes encoding proteins that actually initiate the formation of segment boundaries, and, as the name suggests, confer polarity to segments. Segment polarity genes are expressed in a series of 14 stripes, with odd and even stripes regulated by a different combin- ation of the pair-rule proteins. The boundary between the expression of two of these genes, engrailed and wingless, becomes the parasegmental boundary, whereas segment boundaries form later, posterior to engrailed expression. Black bar, engrailed; grey bar, wingless.
E V OL UTION OF DE VE L OP ME NT A L NE T W OR K S 175
eages, creating an extended syncytial blastoderm stage: examples of such insects include N. vitripen- nis (Bull, 1982), T. castaneum (Handel et al., 2000), and D. melanogaster (Lawrence, 1992) (Figure 16.1). Within a syncytium, gradients of patterning mol- ecules can form quickly across a field of nuclei, without the need for complex intercellular signal- ling pathways.
The allocation of cells to segments
The temporal dynamics by which cells are allo-
cated to segments varies across insect species. In
insects exhibiting primitive modes of develop-
ment, anterior segments are patterned through the
subdivision of blastoderm nuclei/cells, while pos-
terior segments are patterned sequentially after
the blastoderm stage, within a posteriorly located
cellular zone of extension. Examples of such
insects include the hemimetabolous insects Gryllus
bimaculatus, Schistocerca sp., and O. fasciatus and the
holometabolous insect T. castaneum (see Davis and
Patel, 2002) (Figure 16.1). I shall refer to these as
‘sequentially segmenting’ insects. In many insect
lineages there has been an increase in the number
of anterior segments patterned through subdiv-
ision in the blastoderm (Davis and Patel, 2002); this
has occurred, for example, in some coleopteran lin-
eages (Patel et al., 1994). In many holometabolous
insects this trend has reached its logical extreme,
and all segments form through early subdivision
of embryonic blastoderm nuclei. These insects are
said to exhibit ‘long-germ’ embryogenesis, since
the embryonic germ rudiment typically occupies
almost the entire length of the egg. Examples of
such insects include D. melanogaster (Lawrence,
1992) and N. vitripennis (Bull, 1982) (see below and
Figures 16.1 and 16.2).
16.3 Molecular transitions underlying the evolution of long-germ embryogenesis
During long-germ embryogenesis in D. melanogaster, a cascade of transcription factors acts within a syn- cytium to divide the embryo into progressively smaller domains such that segments develop more or less simultaneously. The D. melanogaster segmentation gene cascade is briefly outlined in
Figure 16.2, but for a more thorough understand-
ing the reader is referred to Lawrence (1992).
In order to identify the changes in gene net-
works that underpinned the evolution of long-germ
embryogenesis, a good understanding of the seg-
mentation mechanisms operating in insects that
have retained sequential segmentation is required.
One such insect is the beetle T. castaneum (Handel
et al., 2005). Recent studies on this holometabolous
insect have revealed significant differences in the
genetic circuitry underlying segmentation when
compared with D. melanogaster (Schröder, 2003;
Bucher and Klingler, 2004; Bucher et al., 2005; Cerny
et al., 2005; Choe et al., 2006; Choe and Brown, 2007;
Schinko et al., 2008). Together, comparative studies
on T. castaneum, N. vitripennis, and D. melanogaster
suggest that the evolution of long-germ embryo-
genesis required distinct molecular transitions to
occur in concert at either pole of the egg (Choe
et al., 2006; Brent et al., 2007).
16.3.1 Molecular transitions at the anterior egg pole: the evolution of maternally encoded anterior patterning gradients
In T. castaneum, head and thoracic segments are patterned through the subdivision of blastoderm nuclei located near the posterior egg pole; the anterior blastoderm forms extra-embryonic tis- sue (Handel et al., 2000) (Figure 16.3a,d). In insects exhibiting long-germ embryogenesis, however, head and thoracic segments are patterned further towards the anterior egg pole (Figure 16.3b,c,e,f). It has been proposed that this spatial shift in anterior patterning required the evolution of an instruct- ive anterior patterning gradient to complement the action of existing posterior determinants (Lynch et al., 2006a). The localization of maternal mRNAs to the anterior pole of the oocyte is observed in T. castaneum, N. vitripennis, and D. melanogaster (Lawrence, 1992; Bucher et al., 2005; Olesnicky and Desplan, 2007) (Figure 16.1). In N. vitripennis and D. melanogaster, mRNA and/or translated protein from anterior and posterior sources of maternal mRNAs form largely non-overlapping, and oppos- ing, instructive patterning gradients (Lawrence,
1992; Lynch et al., 2006a; Olesnicky et al., 2006; Brent
176 AN I M AL EV O L UTI O N
Anterior-Posterior patterning Terminal patterning
(a)
Tribolium castaneum
Hunchback orthodenticle-1
Unknown factor(s)
Nanos
Caudal
(d)
Torso- like
Trunk
Torso ?
Tribolium castaneum
Tailless
Torso- like
Trunk
Torso
Krüppel
Extraembryonic
Anterior head
Gnathal
Thorax Growth zone
Extraembryonic
Anterior head
Gnathal
Thorax Growth zone
(b)
Hunchback
Orthodenticle-1
Giant
Nasonia vitripennis
Nanos
Orthodenticle-1
Caudal
(e)
Orthodenticle-1
Nasonia vitripennis
Orthodenticle-1
Hunchback Krüppel
Other anterior factors
Tailless Tailless
Anterior head
Gnathal
Thorax Abdomen
Anterior Gnathal head
Thorax Abdomen
(c)
Hunchback
Bicoid
Giant
Other anterior factors
Drosophila melanogaster
Krüppel
Caudal
Nanos
(f)
Torso- like
Trunk
Torso
Drosophila melanogaster
Tailless Tailless
Torso- like
Trunk
Torso
Anterior Gnathal Thorax Abdomen Anterior Gnathal Thorax Abdomen
head head
Figure 16.3 A schematic representation of the variation in maternal patterning observed between the holometabolous insects Tribolium castaneum, Nasonia vitripennis, and Drosophila melanogaster (as described in the text and summarized in Table 16.1). Particular focus is placed on variation in the maternal regulation of the zygotically expressed central gap gene Krüppel (a)–(c) and terminal gap gene tailless (d)–(f). Note that in T. castaneum Krüppel is expressed like, but does not function as, a canonical gap gene (see text and Cerny et al., 2005), and tailless is not expressed in the anterior (see text and Schröder et al., 2000, and Schoppmeier and Schröder, 2005). Maternally expressed genes are shown within shaded rectangles, above or beside representations of zygotically expressed genes: gradients of shading within
the rectangles depict expression gradients (not to scale). The mRNA of maternal genes written in bold is known to be anteriorly and/or posteriorly tethered during oogenesis. Genetic interactions depicted for T. castaneum and N. vitripennis are determined from RNA interference experiments and so cannot be assumed to reflect direct regulation (hence the dotted lines). Recent models suggest that the establishment
of the Krüppel gap domain in D. melanogaster can largely be explained by both positive (at low concentrations) and negative (at high concentrations) regulation by maternal hunchback (Papatsenko and Levine, 2008).
E V OL UTION OF DE VE L OP ME NT A L NE T W OR K S 177
et al., 2007) (see Table 16.1 and Figure 16.3b,c,e,f). In T. castaneum, on the contrary, neither of the anteri- orly localized mRNAs identified to date play a significant role in anterior–posterior patterning (Bucher et al., 2005), and the maternal mRNAs of two genes known to be important anterior deter- minants in T. castaneum—hunchback and orthoden- ticle-1—are initially distributed uniformly in the egg (Wolff et al., 1995; Schröder, 2003; Schinko et al.,
2008) (see Table 16.1 and Figure 16.3a,d). It is pos- sible that an anteriorly localized maternal mRNA, whose protein product forms an instructive pat- terning gradient exists in T. castaneum but has been overlooked. However, it is tempting to speculate that there is an association between the retention of sequential segmentation in T. castaneum, and the lack of an instructive anterior patterning gra- dient. It will be interesting to determine whether an instructive anterior patterning gradient has evolved in those beetle lineages in which there has been an increase in the number of segments patterned in the blastoderm prior to gastrulation (Patel et al., 1994).
16.3.2 Molecular transitions at the posterior egg pole: changes in the regulation of pair-rule gene homologues
The primary pair-rule genes (even-skipped, hairy, and runt) are the first genes within the D. mela- nogaster segmentation cascade to be expressed in a periodic pattern of stripes (Jaynes and Fujioka,
2004) (see Figure 16.2). The primary pair-rule genes activate a suite of secondary pair-rule genes, that includes paired, sloppy-paired-1 and -2, and odd-skipped (Jaynes and Fujioka, 2004). They are collectively referred to as pair-rule genes since their mutation often leads to abnormalities in alternate segments. Recent work on T. casta- neum has revealed divergent regulatory interac- tions between the homologues of D. melanogaster pair-rule genes. Choe et al. (2006) showed that it is the homologues of D. melanogaster even-skipped, D. melanogaster runt and D. melanogaster odd- skipped that comprise the primary tier of pair-rule genes in T. castaneum. The authors disrupted the expression of each primary pair-rule gene in turn
using parental RNA interference (RNAi) and then examined the expression of the remaining genes in knockdown embryos. Surprisingly, rather than canonical pair-rule phenotypes, the individual knockdown of each of these genes result in aseg- mental phenotypes in which all but a few anterior segments are deleted. Although direct regulatory interactions were not proven, the results suggested to the authors that T. castaneum even-skipped acti- vates T. castaneum runt, which in turn activates T. castaneum odd-skipped, which completes a regula- tory cycle by repressing T. castaneum even-skipped. On the basis of these data, it was proposed that the genes comprise a regulatory gene circuit, each cycle of which sequentially patterns pairs of seg- ments via the downstream regulation of a second- ary tier of pair-rule gene homologues composed of T. castaneum paired and T. castaneum sloppy- paired (note that T. castaneum hairy does not appear to play a role in trunk segmentation; Choe and Brown, 2007).
This model and the data it is based on raise an
interesting question. How could a regulatory cir-
cuit of transcription factors—that by definition
must operate intracellularly—pattern segments
within a cellularized zone of extension? One pos-
sibility I suggest is that the proposed transcription
factor circuit—or perhaps just some components
of it—constitute an intracellular molecular oscilla-
tor with an analogous role to the molecular oscil-
lators that control sequential segmentation of the
pre-somitic mesoderm during vertebrate develop-
ment (see reviews by Pourquié, 2004, and Gridley,
2006). The vertebrate segmentation clock relies on
the Notch intercellular signalling pathway, both as
a component of the molecular oscillator in some
cases (Pourquié, 2004; Gridley, 2006) and to coord-
inate oscillations amongst neighbouring cells
(Masamizu et al., 2006). Work on myriapods and
chelicerates has shown that some pair-rule gene
homologues and members of the Notch intercellu-
lar signalling pathway are expressed in a dynamic
fashion during sequential segmentation in a man-
ner reminiscent of that seen during vertebrate
sequential segmentation (Stollewerk et al., 2003;
Chipman et al., 2004; Schoppmeier and Damen,
2005), leading to the exciting hypothesis that a
segmentation clock, analogous if not homologous
178 AN I M AL EV O L UTI O N
Table 16.1 A summary of some Tribolium castaneum and Nasonia vitripennis segmentation gene homologues shown by recent studies to exhibit differences in expression and function when compared to Drosophila melanogaster (see also Figures 16.2 and 16.3).
Gene T. castaneum N. vitripennis
bicoid1 bicoid not present in the genomes of insects outside the cychlorrhaphan flies
Evolved from a zerknüllt (zen)–like precursor
Orthodenticle-1 appears to play an analogous role to D. melanogaster Bicoid in these species
orthodenticle-12– 4 mRNA is maternally inherited—unlike in D. melanogaster where expression is purely zygotic—but not localized to the egg poles as in N. vitripennis
Maternal plus zygotic expression becomes anteriorly restricted
Functions with Hunchback to pattern the anterior of the embryo
giant4– 6 No maternal expression as is the case in D. melanogaster
Expressed in two zygotic gap-like domains as in
D. melanogaster, except that the posterior domain is positioned much more to the anterior
Anterior domain controls segment identity via the regulation of Hox genes, but unlike in D. melanogaster, it is not required for segment formation
Required for the formation of all thoracic and abdominal segments, not just the segments in which it is expressed
cauda13, 4,7–10 Maternal mRNA initially uniformly distributed as in
D. melanogaster
Posterior protein gradient forms through translational repression by an unknown factor/factors (i.e. not Bicoid)
Expressed in the cellularized growth zone
Unlike in D. melanogaster, required for the formation of all but the most anterior few segments
Unlike in D. melanogaster, maternal mRNA is localized to the anterior and posterior poles (via distinct mechanisms at either pole)
On fertilization, mRNA is released and forms an opposing anterior and posterior mRNA and, through translation, protein gradients
Anterior gradient functions with maternal Hunchback to activate anterior gap genes: empty spiracles, giant, and (zygotic) hunchback
Posterior gradient functions with Caudal to pattern posterior segments
Largely conserved zygotic head gap gene role
Unlike in D. melanogaster, maternal mRNA is localized to the anterior during oogenesis
On fertilization, mRNA is released and forms an anterior mRNA and, through translation, a protein gradient
Represses central gap gene Krüppel in anterior, preventing repression of anterior gap gene hunchback by Krüppel, and thus plays a permissive role in anterior development
A similar zygotic gap gene role to D. melanogaster
Unlike in D. melanogaster, maternal mRNA localized to posterior during oogenesis
On fertilization, mRNA is released and forms a posterior mRNA and, through translation, a protein gradient
Functions with Orthodenticle-1 to pattern posterior via activation of posterior gap genes
Influence extends further to the anterior than in
D. melanogaster and includes activation of the central gap gene Krüppel
tailless and
the terminal patterning system3,11–13
tailless expressed by small group of cells at the posterior pole of the blastoderm.
In contrast to D. melanogaster, there is no expression at the anterior pole of the blastoderm
Terminal patterning system (torso and torso-like) required for sequential segmentation and formation of anterior extra-
embryonic tissue
tailless expression is activated in anterior and posterior by Orthodenticle-1 (i.e. there is no evidence for a terminal patterning system in N. vitripennis)
Unlike D. melanogaster, the anterior domain is not required for segmentation
Posterior domain has more extensive influence on
posterior patterning than in D. melanogaster
References: 1, Stauber et al. (2002); 2, Schröder (2003); 3, Lynch et al. (2006a); 4, Olesnicky and Desplan (2007); 5, Bucher and Klingler (2004);
6, Brent et al. (2007); 7, Schulz et al. (1998); 8, Wolff et al. (1998); 9, Copf et al. (2004); 10, Olesnicky et al. (2006); 11, Schröder et al. (2000);
12, Schoppmeier and Schröder (2005); 13, Lynch et al. (2006b).
E V OL UTION OF DE VE L OP ME NT A L NE T W OR K S 179
to that operating in vertebrates, controls sequential
segmentation in these arthropods (Peel and Akam,
2003; Stollewerk et al., 2003). However, there is no
evidence, as yet, for the involvement of the Notch
signalling pathway during insect sequential seg-
mentation. Wingless signalling also plays a central
role in the vertebrate segmentation clock (Pourquié,
2004; Gridley, 2006). Perhaps wingless signalling
forms the basis of a possible segmentation clock
in insects (Miyawaki et al., 2004) or alternatively
other signalling pathways might be involved.
In D. melanogaster the periodic expression of
primary pair-rule genes is, somewhat curiously,
activated by an aperiodic series of anteroposteri-
orly restricted domains of gap gene expression
(Figure 16.2). Gap genes play an additional role
in D. melanogaster development; they regulate the
anteroposteriorly restricted domains of Hox gene
expression that confer identity to segments (Irish
et al., 1989). In T. castaneum, most D. melanogaster
gap gene homologues are expressed in restricted
anteroposterior domains, consistent with a gap
gene function, and in a roughly conserved antero-
posterior order, albeit shifted towards the anterior
(Schröder, 2003; Bucher and Klingler, 2004; Cerny
et al., 2005). However, the knockdown by RNAi of
at least two of the D. melanogaster gap gene homo-
logues—Krüppel and giant—does not result in
canonical gap gene phenotypes: i.e. the loss of the
segments in and around their domains of expres-
sion (Bucher and Klingler, 2004; Cerny et al., 2005).
Instead, these segments take on abnormal iden-
tities as a result of the misexpression of Hox genes
(Bucher and Klingler, 2004; Cerny et al., 2005). This
might imply that one of the ancestral roles of gap
gene homologues in insects was to position Hox
gene domains correctly, and that they were only
later recruited to pattern pair-rule genes (Peel and
Akam, 2003). Under this model, gap gene recruit-
ment is correlated with the transition to activating
pair-rule stripes simultaneously in a syncytium,
where control by intercellular signalling becomes
redundant and where a spatial rather than tem-
poral regulatory input is required (Peel and Akam,
2003). Presumably transcription factors expressed
at the right time and place in the posterior blasto-
derm were co-opted to regulate progressively
more posterior pair-rule stripes, thus explaining
the complex nature of the regulatory sequence of D. melanogaster primary pair-rule genes. And per- haps the co-option of D. melanogaster gap gene homologues was favoured, since they had already evolved a spatially and temporally corresponding role in Hox gene regulation.
16.4 Molecular transitions underlying the convergent evolution of long-germ embryogenesis
The four major holometabolous insect orders all contain species exhibiting long-germ embryo- genesis, for example the dipteran D. melanogaster, the lepidopteran Manduca sexta, the coleopteran Callosobruchus maculates, and the hymenopterans Apis mellifera, N. vitripennis, and Bracon hebetor (Grbic and Strand, 1998; see also Davis and Patel,
2002). However, the Lepidoptera, Coleoptera, and Hymenoptera also contain species that have retained, or re-evolved in the case of parasitic hymenopterans (Grbic and Strand, 1998), differing degrees of sequential segmentation; for example, the lepidopteran Bombyx mori, the coleopteran T. castaneum, and the hymenopteran A. ervi (Grbic and Strand, 1998). This has led to the idea that long-germ development evolved multiple times independently during the holometabolous insect radiation (see Davis and Patel, 2002). This scenario now seems more likely due to a recent re-evalua- tion of holometabolous insect phylogeny. The gen- eral consensus surrounding the relationship of the major holometabolous insect orders used to be that the Diptera and Lepidoptera are sister groups, and that the Coleoptera form the basal branch in the tree (Whiting, 2002). However, a recent molecu- lar phylogenetic study supports a reversal in the position of the Hymenoptera and Coleoptera, such that the Hymenoptera now form the basal branch (Savard et al., 2006). Thus, the Diptera and Hymenoptera are now separated by an order that contains many species with clear sequential seg- mentation; the Coleoptera.
Work by the Desplan and Pultz laboratories has begun to reveal the molecular basis to segmen- tation in N. vitripennis, which has an embryonic fate map almost identical to that of D. melanogaster (Bull, 1982; Brent et al., 2007). If N. vitripennis and
180 AN I M AL EV O L UTI O N
D. melanogaster did evolve long-germ embryogen- esis independently, as the latest molecular phyl- ogenies suggest, comparisons between these two species are a first step to determining the extent to which underlying gene network changes were also convergent.
As in D. melanogaster (Figure 16.2), the segmen-
tation cascade operating in N. vitripennis can be
divided into four distinct tiers of maternal, gap,
pair-rule, and segment-polarity genes. The major
genetic differences identified so far between
D. melanogaster and N. vitripennis are found right
at the top of the segmentation cascade and relate to
changes in the maternal contribution to patterning.
These differences are summarized in Table 16.1
and Figure 16.3 and are discussed below.
In D. melanogaster, anterior development is
largely under the control of the Bicoid morphogen
gradient (Lawrence, 1992) (Figures 16.2 and 16.3c).
However, bicoid is known to be an invention of the
higher Diptera (Stauber et al., 2002). In N. vitripennis
anterior patterning is accomplished by two distinct
anterior gradients of patterning molecules (Lynch
et al., 2006a; Brent et al., 2007) (Figure 16.3b). In con-
trast to D. melanogaster, where their expression is
purely zygotic, maternal mRNAs of both orthode-
nticle-1 and giant are tethered to the anterior pole
during oogenesis in N. vitripennis (for details see
Lynch et al., 2006a, Brent et al., 2007, and Olesnicky
and Desplan, 2007). Following fertilization these
mRNAs are translated to form anterior gradients
of Orthodenticle-1 and Giant protein. The mater-
nal Orthodenticle-1 gradient functions to activate
anterior segmentation genes, such as the gap genes
empty spiracles, (zygotic) giant, and hunchback (Lynch
et al., 2006a), while the maternal Giant gradient
functions to set the anterior expression boundary
of the central gap gene Krüppel (Brent et al., 2007)
(Figure 16.3b). The repressive role of maternal Giant
is permissive for anterior development, since, in its
absence, Krüppel expression spreads anteriorly to
repress the anterior gap gene hunchback (Brent et al.,
2007) (Figure 16.3b).
The influence of Orthodenticle-1 and Giant on
embryonic patterning does not extend as far to the
posterior as does Bicoid in D. melanogaster (Lynch
et al., 2006a; Brent et al., 2007). Instead, the influ-
ence of the posterior determinant caudal extends
further towards the anterior; in D. melanogaster hunchback regulates (probably both positively and negatively; Papatsenko and Levine, 2008) (Figure
16.3c) the central gap domain of Krüppel, but in N. vitripennis it is caudal that activates this gap domain (Olesnicky et al., 2006) (Figure 16.3b). Indeed, poster- ior patterning in N. vitripennis exhibits significant differences when compared with D. melanogaster. A posterior gradient of Caudal is established in N. vitripennis—in the absence of translational repression by Bicoid (Figure 16.2 and 16.3c)—via the tethering of caudal mRNA to the posterior pole during oogenesis (Olesnicky et al., 2006; Olesnicky and Desplan, 2007) (Figure 16.3b). The mRNA of orthodenticle-1 is also tethered to the posterior pole during oogenesis, such that, together, gradients of Caudal and Orthodenticle-1 protein control poster- ior patterning (Lynch et al., 2006a; Olesnicky et al.,
2006) (Figure 16.3b).
The large degree of variation between D. mela-
nogaster and N. vitripennis in maternal patterning
suggests that the changes in the gene network
underlying the putative independent evolution of
long-germ embryogenesis within the hymenop-
teran and dipteran lineages might have been very
different. However, many of the observed dif-
ferences could be attributed to the evolution of a
maternal Bicoid gradient within the higher Diptera
(Stauber et al., 2002). Indeed, bicoid and orthodenticle
are both homeobox-containing genes, and Bicoid is
predicted to have usurped the role of Orthodenticle
as an anterior determinant (as exemplified by
T. castaneum; Schröder, 2003; Schinko et al., 2008)
and N. vitripennis (Lynch et al., 2006a) orthodenticle-1
(Table 16.1, Figures 16.3a,b) through gaining affin-
ity for Orthodenticle DNA-binding sites via the
convergent acquisition of a lysine residue at pos-
ition 50 in its homeodomain (Treisman et al., 1989).
Further data from additional long-germ dipterans
(e.g. Anopheles gambiae) and hymenopterans (e.g.
A. mellifera) will be required to map divergent (and
convergent) gene network changes to the dipteran
and/or hymenopteran lineages. For example, it
is interesting that regulation by zygotic giant is
involved in the maintenance and refinement of the
Krüppel gap domain in D. melanogaster (Papatsenko
and Levine, 2008) (Figure 16.3c). Was giant recruited
to play an earlier role in the establishment of the
E V OL UTION OF DE VE L OP ME NT A L NE T W OR K S 181
Krüppel gap domain within the lineage leading to N. vitripennis, or was a role for giant at the mater- nal level a character that has been lost during the dipteran radiation within the lineage leading to D. melanogaster?
There does, however, already appear to be a strong case for differences in the evolution of the terminal patterning system during the putative independent evolution of long-germ development within the hymenopteran and dipteran lineages. In D. melanogaster a terminal patterning system acts to determine the most anterior and posterior regions of the embryo (Lawrence, 1992) (Figure 16.2). The product of the gene torso-like is maternally restricted to the egg poles where it cleaves the protein encoded by the gene trunk (Casali and Casanova, 2001). The Trunk C-terminal then acts as a ligand, binding to the receptor tyrosine kin- ase encoded by torso and triggering a signalling cascade that regulates the zygotic expression of downstream segmentation genes, such as tailless, at either pole of the egg (Casali and Casanova,
2001) (see Table 16.1 and Figure 16.3f). In T. casta- neum, both torso and torso-like are required at the posterior pole for the activation and/or mainten- ance of sequential segmentation (Schoppmeier and Schröder, 2005) (Figure 16.3d). However, in N. vitripennis the expression of tailless has been shown to be dependent on the anterior and pos- terior gradients of maternal Orthodenticle-1 and thus perhaps not a terminal patterning system (Lynch et al., 2006b) (Figure 16.3e). The absence of a D. melanogaster-like terminal patterning system in hymenopterans is further supported by the failure to find homologues of torso or trunk in the A. mellifera genome (Dearden et al., 2006).
The apparent lack of a Drosophila-like ter- minal patterning system in long-germ hymen- opteran insects is intriguing, particularly since one appears to be required for sequential seg- mentation in T. castaneum (Schoppmeier and Schröder, 2005; Lynch et al., 2006b) (Figure 16.3e). Studies on more holometabolous insects, as well as non-holometabolous insects, will be required to determine if a terminal patterning system is a character that has been lost within the hymen- opteran lineage or gained in the lineage leading to the other major holometabolous insect orders.
One possibility is that in some of the insect line- ages that underwent the transition to long-germ embryogenesis (i.e. in dipteran lineages) a ter- minal patterning system that ancestrally played an important role in initiating or maintaining sequential segmentation was co-opted to activate posterior segmentation genes (Schoppmeier and Schröder, 2005), whereas in other lineages under- going parallel transitions (i.e. in hymenopteran lineages) this did not occur.
16.5 General conclusions
16.5.1 The role of co-option in the evolution of segmentation gene networks
A theme emerging from comparative studies on insects is the role that co-option plays in evolution, at different levels of complexity. At the regulatory sequence level, it seems possible, if not likely, that some gap gene homologues—expressed at the right time and in the right place due to an ancestral role in Hox gene regulation—have been co-opted into regulating pair-rule gene homologues, perhaps via the simple acquisition of binding sites (Bucher and Klingler, 2004; Cerny et al., 2005; Choe et al., 2006). At the protein level, Bicoid—or perhaps more accurately Zerknüllt—was co-opted to an anterior patterning role within the higher Diptera via a sim- ple coding mutation that allowed it to recognize the regulatory targets of an existing anterior determin- ant; Orthodenticle (Stauber et al., 2002; Lynch et al.,
2006a). At the intracellular level, existing cytoskel- etal machinery may have been co-opted during the evolution of instructive anterior patterning gradients (Bucher et al., 2005). All these cases are consistent with a long series of simple modifica- tions to developmental gene circuits having, over evolutionary time, underpinned diversification at the cellular level and above. The results of a recent whole-genome study are consistent with the widespread occurrence of gene co-option during insect evolution. Dearden et al. (2006) looked at the presence/absence of homologues of D. melanogaster developmental genes in the A. mellifera genome. They found that, of the developmental genes involved in processes that are known to be diver- gent between these two species (i.e. sex determin-
182 AN I M AL EV O L UTI O N
ation, dosage compensation, meiosis, and germ-cell development), a significant number of those con- served in A. mellifera (c = 19.03; P < 0.001, n = 78) have multiple (i.e. pleiotropic) functions in D. mela- nogaster. This suggests either that the homologues of genes with multiple functions in D. melanogaster have been lost less frequently in the honeybee lin- eage and/or that genes with ancestral conserved functions have been frequently co-opted into new roles in the fruitfly lineage (Dearden et al., 2006).
16.5.2 The evolution of developmental gene networks in relation to adult morphology
The comparative studies reviewed in this chap- ter clearly demonstrate that genetic networks controlling the development of conserved adult structures (i.e. homologous insect segmental units) can diverge significantly over time due to lineage-specific transitions in cellular embryology
associated with changes in life-history strategy. They also suggest that distinct changes in gene networks might underlie convergent transitions in modes of cellular embryogenesis. This implies that over the course of hundreds of millennia, developmental gene networks and the adult morphology they pattern can become ‘decoupled’. Assigning homology, or otherwise, to adult mor- phological features based on comparative devel- opmental genetic data alone is therefore risky. Accurately reconstructing the evolution of animal body plans will require a holistic approach, which includes adequate and intelligent sampling of spe- cies (i.e. perhaps less focus on species exhibiting highly derived modes of embryogenesis), a more thorough understanding of the embryological con- texts in which gene networks operate, and a better appreciation of how evolutionary changes in life- history strategy (i.e. changes in species ecology) can influence the evolution of development.
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