Interactive Fly, Drosophila

engrailed


EVOLUTIONARY HOMOLOGS


Table of contents

Engrailed proteins

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for 'Homeobox' engrailed-type protein signature.

Engrailed in other insects

The median neuroblast lineage of grasshopper has provided a model for the development of differing neuronal types within the insect central nervous system. According to the prevailing model, neurons of different types are produced in sequence. Contrary to this, each ganglion mother cell from the median neuroblast produces two neurons of asymmetric type: one is Engrailed positive (of interneuronal fate); and one is Engrailed negative (of efferent fate). The mature neuronal population, however, results from differential neuronal death. This yields many interneurons and relatively few efferent neurons. Also contrary to previous reports, no evidence is found for glial production by the median neuroblast (Jia, 2002).

In adult grasshopper Engrailed is expressed in the interneurons but not the efferent neurons within the dorsal unpaired median (DUM) groups of neurons, which arise embryonically from the median neuroblasts (MNBs). The interneurons and the efferents of the DUM group have somata in one tight cluster, but their primary neurites and axonal branches follow very different pathways within the CNS. Together, these observations prompted a hypothesis that En regulates aspects of neuronal pathfinding and neuronal morphology. Indeed, in Drosophila embryos, the En-positive CNS neurons are interneurons, whereas efferent neurons are En negative. Moreover, genetic manipulation of engrailed expression alters the expression of neuronal cell adhesion molecules, and at the same time results in profound disruptions of normal neuronal morphology in the CNS. Studies in cockroach also implicate En in pathfinding choices made by sensory neurons (Jia, 2002).

The MNB lineage of grasshopper has provided a longstanding model for the development of large NB lineages containing differing neuronal types within the insect CNS. Perhaps more so than within any other NB lineage, individual neurons and small neuronal groups of like type have been broadly investigated, not only from a developmental perspective, but also in morphological, biochemical and behavioral studies of the adult. The present study focuses on the mechanisms by which the MNB gives rise to different neuronal types across the lineage. The prevailing lineage model is a 'sequential' one, whereby the ganglion mother cell (GMC) divisions produce sibling pairs first of efferent neurons, then pairs of interneurons, either local interneurons or intersegmental interneurons. The morphology of neurons in the MNB (or DUM) groups during embryogenesis and in adulthood is consistent with this model. However, En-positive neurons are produced from the MNB early in the lineage. Engrailed expression is specific to interneurons, and the validity of the sequential model became suspect. MNB and midline precursor (MP) lineages were traced across embryogenesis in grasshopper, with the aim of discovering the order in which neurons of different types were produced, and what role En might have in this process (Jia, 2002).

The results confirmed some earlier findings, but at the same time led to a significant revision in understanding of the MNB lineage and its progeny. As in Drosophila, the MNB in grasshopper does not produce midline glia, contrary to previous reports. More importantly, the MNB lineage is asymmetric in its production of neuronal progeny. Throughout the lineage, each GMC generates two nonidentical siblings: one En-positive and one En-negative neuron. A proportion of each neuronal type dies during the course of embryogenesis, but death is most pronounced among the En-negative efferent neurons. Thus, differential cell death, rather than a timed lineage sequence that produces different neuronal types, accounts for the significantly smaller number of efferent neurons versus interneurons in the final neuronal populations (Jia, 2002).

It is argued that the continued production of neurons of two essentially different types within a lineage, followed by selective death of some neurons, is normal across insect neuronal lineages. This provides a flexible and responsive mechanism whereby neuronal populations can be tailored to match segmental diversity across the insect body plan, without reconfiguring individual neuroblast lineages. A similar mechanism would likewise provide a ready means of matching neuronal populations in the CNS with changes in peripheral body form across the course of insect evolution, while not altering the central mechanism of neuronal production. This view is concordant with numerous findings across a diversity of organisms, where divisions of precursors yield progeny of asymmetric type, followed by selective cell death (Jia, 2002).

Evidence has long been available from grasshopper, and then from Drosophila, that sibling neurons of apparently unlike, or asymmetric type are produced from MPs1-3 and from the first GMCs of some NB lineages. And, at least one postembryonic lineage in Manduca produces siblings of two types. However, these findings have not been incorporated into a more general model of neuronal lineages, possibly for two reasons: in contrast to other MPs, MPs4-6 were thought each to produce progeny of like type, and in the few NB lineages with early progeny of apparently asymmetric type, the range of neuronal types produced across the lineage is unknown. Without this larger framework, it is hard to judge the significance of any phenotypic difference. Differences between siblings might be ascribed to a particular variation between neurons of the same basic type or they might, instead, typify an essential asymmetry that is maintained across a lineage. The neuronal progeny of embryonic NB lineages in Drosophila are now known in some detail, and it is striking that two-thirds of lineages contain interneurons as well as either motoneurons, or neurosecretory cells of an efferent type. Interneurons predominate in the extant lineages, but this is not surprising in light of the extensive cell death found among neurons of efferent fate, and it is suspected that the same is true of Drosophila. Of the remaining lineages in Drosophila, many generate interneurons having two distinctly different axonal projections, either anterior or posterior, or ipsilateral or contralateral. Both of these dichotomies could represent an essential asymmetry in lineages that do not produce efferent neurons; the MP2 siblings, for example, are asymmetric in that they have either an anterior or a posterior interneuronal projection (Jia, 2002).

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. The mechanism by which the leech primary blast cell clones acquire segment polarity has been examined -- i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the leech embryo, segment polarity is first evident when the primary blast cells initiate their subsidiary divisions. For example, a primary p blast cell undergoes two rounds of division parallel to the AP axis to generate grand-daughter cells p.aa, p.ap, p.pa and p.pp, thereafter switching to transverse divisions. A single p blast cell clone will eventually give rise to about 70 differentiated descendants, and the relative AP positions of the four grand-daughter cells in the germinal band predicts the overall AP disposition of their descendants within the differentiated blast cell clone. It is not currently known how the anterior and posterior blast cell progeny acquire their differing fates, but the specification of those differences is a crucial step in establishing the segment polarity of each individual primary blast cell clone. The primary o blast cell clone develops in much the same way as the p blast cell clone, although it differs with respect to the details of cleavage pattern and the exact set of descendants produced. The earliest o blast cell divisions are also parallel to the AP axis, and its progeny cells o.aa, o.apa, and o.app are arrayed within the germinal band in an order that predicts the AP disposition of their differentiated descendants (Seaver, 2001).

Using a laser microbeam, single cells were ablated from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which those blast cell progeny that are known to express the engrailed gene, or their lineal precursors, were specifically ablated. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and these findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms (Seaver, 2001).

The expression of the en gene in the leech, H. triserialis, is similar in several regards to that seen in arthropods. Embryonic expression begins at a stage when the segmental repeat is only a few cells in length, and forms a transverse stripe across the dorsoventral width of each nascent segment. But there are also some noteworthy differences. In Helobdella the onset of en expression follows the appearance of segmentally iterated cell divisions, which may indicate that it is simply a downstream marker of segmental periodicity. Furthermore, the en stripes of the leech embryo do not define developmental compartments of cell lineage restriction, nor are these stripes restricted to the posterior of the segment, e.g. the en-expressing cell p.ap generates neurons and epidermal cells that lie in the anterior half of the anatomical segment (Seaver, 2001 and references therein).

The present findings reveal other, even more fundamental differences in the mechanism by which leeches and fruit flies establish segment polarity. In the fly embryo, the patterning of AP positional values within the segmental repeat is primarily dependent upon cell interactions occurring along the AP axis. Moreover, the en transcription factor plays a pivotal role in initiating and maintaining those cell interactions. In the leech embryo, no evidence is found that cell interactions oriented along the AP axis are required to specify anterior and posterior cell fates within the segmental repeat, and ablation of blast progeny that normally express en (cell p.ap) or their lineal precursors (cell p.a; cell o.aa) does not significantly alter the fate of other blast cell progeny within that clone or in more anterior or posterior clones. It may be that the en protein is only involved in the specification of cell-autonomous fates during leech segmentation (Seaver, 2001 and references therein).

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis has dismissed the Articulata and thus challenged the segmentation homology hypothesis. In Platynereis, engrailed and wingless orthologs are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, these results are interpreted as molecular evidence of a segmented ancestor of protostomes (Prud'homme, 2003).

During posterior growth, both during normal juvenile segment formation and after caudal regeneration, Pdu-en is expressed in ectodermal circular stripes in developing segments. This segmental expression appears in continuous rings of cells immediately after the growth zone has produced them (in younger, posterior-most segments) and persists in differentiating (more anterior) segments. The pattern is more complicated on the ventral face, since, in addition to the continuous segmental expression, Pdu-en is expressed in mesodermal groups of cells and in forming ganglia of the ventral nerve cord. A longitudinal section shows that the segmental stripes of expression occur long before segmental coelomic cavities or segmental boundaries are visible. As segments mature, it becomes apparent that continuous segmental stripes of Pdu-en expression are always restricted to the anterior-most row of epidermal cells within a segment immediately posterior to the forming segmental groove corresponding to the actual segmental boundary. These segmental grooves are the only ones to form and do not seem to shift during segment differentiation, as indicated by the relative position of an appendage marker, distal-less. Hence, this expression pattern suggests that during postlarval growth in Platynereis, engrailed is involved both in the establishment of the segmental boundaries in the ectoderm and in the specification of particular cell types in the mesoderm and the central nervous system (Prud'homme, 2003).

Pdu-wnt1 is also expressed early in ectodermal stripes in each developing segment both during normal juvenile segment formation and after caudal regeneration, although the signal level is much weaker compared to that in Pdu-en. Pdu-wnt1 is expressed in the posterior-most ectodermal cells of each developing trunk segment, immediately anterior to the segmental boundary. In contrast with Pdu-en, the thickness of Pdu-wnt1 stripes increases in proportion with the segment length. Pdu-wnt1 is also expressed in the posterior part and in an anterior-proximal spot of the parapodia, as well as in the proctodaeum (Prud'homme, 2003).

Based on morphological landmarks (i.e., segmental grooves), these results suggest that Pdu-en and Pdu-wnt1 are expressed in adjacent domains on either side of the segmental boundary and play a role in the formation and maintenance of this boundary. According to these observations, Pdu-en and Pdu-wnt1 are most likely expressed in directly neighboring cells. However, due to technical difficulties with double in situ stainings, it has not been been possible to ascertain this point (Prud'homme, 2003).

Spiders belong to the chelicerates, which is a basal arthropod group. To shed more light on the evolution of the segmentation process, orthologs of the Drosophila segment polarity genes engrailed, wingless/Wnt and cubitus interruptus have been recovered from the spider Cupiennius salei. The spider has two engrailed genes. The expression of Cs-engrailed-1 is reminiscent of engrailed expression in insects and crustaceans, suggesting that this gene is regulated in a similar way. This is different for the second spider engrailed gene, Cs-engrailed-2, which is expressed at the posterior cap of the embryo from which stripes split off, suggesting a different mode of regulation. Nevertheless, the Cs-engrailed-2 stripes eventually define the same border as the Cs-engrailed-1 stripes. The spider wingless/Wnt genes are expressed in different patterns from their orthologs in insects and crustaceans. The Cs-wingless gene is expressed in iterated stripes just anterior to the engrailed stripes, but is not expressed in the most ventral region of the germ band. However, Cs-Wnt5-1 appears to act in this ventral region. Cs-wingless and Cs-Wnt5-1 together seem to perform the role of insect wingless. Although there are differences, the wingless/Wnt-expressing cells and en-expressing cells seem to define an important boundary that is conserved among arthropods. This boundary may match the parasegmental compartment boundary and is even visible morphologically in the spider embryo. An additional piece of evidence for a parasegmental organization comes from the expression domains of the Hox genes that are confined to the boundaries, as molecularly defined by the engrailed and wingless/Wnt genes. Parasegments, therefore, are presumably important functional units and conserved entities in arthropod development and form an ancestral character of arthropods. The lack of engrailed and wingless/Wnt-defined boundaries in other segmented phyla does not support a common origin of segmentation (Damen, 2002).

The structure of the insect head as revealed by the distribution of engrailed related protein (Engrailed) was examined in the insect orders Diptera (Drosophila), Siphonaptera (the flea), Orthoptera (the cricket) and Hemiptera (milkweed bug). The results of this comparative embryology in conjunction with genetic experiments on Drosophila lead to the following conclusions: (1) The insect head is composed of six Engrailed accumulating segments, four postoral (intercalary, mandibular, maxillary and labial) and two preoral (ocular and antennal). The potential seventh and eighth segments (clypeus or labrum) do not accumulate Engrailed (The exception is Drosophila. Among the six insect orders studied, the Drosophila is the only one showing expression in the clypeolabrum). (2) The structure known as the dorsal ridge (associated with the eye-antennal disc) is not specific to the Diptera but is homologous to structures found in other insect orders. (3) A part of this structure is a single segment-like entity composed of labial and maxillary segment derivatives which produce the most anterior cuticle capable of taking a dorsal fate. The segments anterior to the maxillary segment produce only ventral structures. (4) As in Drosophila, the process of segmentation of the insect head is fundamentally different from the process of segmentation in the trunk. Whereas segmentation in the trunk is driven by gap and pair rule genes, in the head it is driven by head gap genes as well as engrailed, and wingless. (5) The pattern of Engrailed accumulation and its presumed role in the specification and development of head segments appears to be highly conserved while its role in other pattern formation events and tissue-specific expression is variable. An overview of the pattern of Engrailed accumulation in developing insect embryos provides a basis for discussion of the generality of the parasegment and the evolution of Engrailed patterns. It is concluded that the parasegment may not be the fundamental unit of pattern. The parasegment is clearly not the primary unit of homeotic gene expression (Rogers, 1996).

The mosquito Anopheles gambiae En protein shows significant divergence from the Drosophila protein. The overall sequence identity is only 35% and is confined to 7 domains. Four of these domains, the En/Inv domains are found in both the Drosophila En and Inv proteins and in all En-class proteins, including those of mouse. These include the homeodomain, a region surrounding the first intron of Drosophila En, and a C-terminal region. Two other domains are En specific, including murine En, and are not found in Invected. There is another region, the Dipteran-specific En domain, found in Drosophila and mosquito, but not in Inv or mouse En. An engrailed cDNA from mosquito was expressed from a Drosophila engrailed minimal promoter. The promoter fragment used includes 2.6 kb of regulatory DNA that causes transposons to home to the endogenous Drosophila engrailed gene at high frequencies. This transposon was inserted onto a Drosophila chromosome that produces no functional Engrailed proteins. When this transposon integrates near the engrailed promoter, adult viability is restored to engrailed mutant flies showing that the highly divergent mosquito Engrailed protein can replace the Drosophila Engrailed protein at all stages of development. Insertion of this transposon into the adjacent invected gene, which is transcribed in a pattern similar to engrailed, leads to only embryonic rescue, suggesting an important difference in the regulation of these two genes (Whitley, 1997).

Developmental processes have been traditionally viewed to be invariant within higher taxa. However, examples are known whereby closely related species exhibit alterations in early embryogenesis yet appear very similar as adults. Such developmental changes are thought to occur in response to shifts in life history. In insects, the regulation of embryonic development has been intensively studied in model species, such as Drosophila melanogaster. Previous comparative studies suggest that the developmental processes documented in Drosophila adequately describe embryogenesis of advanced, holometabolous, insects generally. There have been few attempts, however, to take into account how life history has influenced early development of insects or to characterize early development of species with life histories fundamentally different from flies. A comparison was carried out of early development in two species from the same family of parasitic wasps that exhibit very different life histories. Bracon hebetor is an ectoparasite that lays large, yolky eggs on the integument of its host that develop much like the free-living honeybee and Drosophila. In contrast, Aphidius ervi is an endoparasite that lays small and apparently yolk-free eggs that develop in the hemocoel of the host. This wasp exhibits a radically different mode of early development at both the cellular and molecular level from B. hebetor. The developmental changes in A. ervi reflect functional adaptations to its derived endoparasitic life history and argue that departures from the fly paradigm may occur commonly among insects whose eggs develop under conditions different from typical terrestrial species (Grbic, 1998).

To compare patterning events at the molecular level, B. hebetor and A. ervi embryos were stained with antibodies that recognize conserved epitopes of Eve, En, and Ubx/Abd-A in different insect species. Eve, a primary pair-rule gene is expressed in the Drosophila syncytium, forms a characteristic seven-stripe pattern with double segment periodicity. En, which is regulated by Eve, is a segment polarity gene that specifies the posterior segmental compartments. Ubx and Abd-A are Drosophila homeotic proteins that specify the posterior thorax and abdomen. In B. hebetor, Eve is expressed in a largely conserved fashion when compared to Drosophila and other long germband insects. Initially, a broad domain of Eve expression splits into broad pair-rule stripes, followed by a split of the individual pair-rule stripes in rapid anteroposterior progression to form segmentally iterated stripes. After germband retraction, Eve localizes in the cells of the dorsolateral mesoderm and neurons; a pattern conserved in all examined insects. In B. hebetor, En is expressed in a rapid anteroposterior progression, forming a mature pattern of segmentally iterated stripes that localize to the posterior segmental compartments. The antibody against Ubx/Abd-A stains the region from the posterior thorax to the penultimate abdominal segment. When A. ervi embryos are stained with anti-Eve, neither a pair-rule nor a segmental pattern is detected. In the extended germband, however, an Eve antigen is detected in dorsolateral mesoderm and neurons. En stripes appear when embryos initiate germband extension. These stripes form sequentially as the germband extends, resulting in a mature pattern of segmentally iterated stripes that localize to the posterior segmental compartments. Ubx/Abd-A is expressed in the posterior thorax and abdomen in the retracted germband stage. Thus, despite the divergence of early patterning, late patterning in B. hebetor and A. ervi includes conserved expression of En and Ubx/Abd-A. This expression pattern in the germband, the phylotypic stage in insects, suggests conservation of this stage irrespective of how development begins (Grbic, 1998).

The transcription factor Engrailed (En) controls the topography of axonal projections by regulating the expression of cell-adhesion molecules but it is not known whether it also controls the choice of individual synaptic target cells. In the cercal sensory system of the larval cockroach (Periplaneta americana), small numbers of identified wind-sensitive sensory neurons form highly specific synaptic connections with 14 identified giant interneurons, and target-cell choice is independent of the pattern of axonal projections. En is a putative positional determinant in the array of cercal sensory neurons. In the present study, double-stranded RNA (dsRNA) interference was used to abolish En expression. This treatment changes the axonal arborization and synaptic outputs of an identified En-positive sensory neuron so that it comes to resemble a nearby En-negative cell, which is itself unaffected. Thus, it has been demonstrated directly that En controls synaptic choice, as well as axon projections (Marie, 2000a).

In the first larval stage (or instar) of the cockroach, P. americana, each cercus bears two wind-sensitive filiform hairs; after moulting several days later, the second instar cercus bears an array of 39 hairs, and their associated sensory neurons. The axonal arborizations and patterns of synaptic connections of the distal-most 25 of the second-instar sensory neurons to 10 giant interneurons in the central nervous system (CNS) have been examined. En is expressed only in the medially-born sensory neurons of the cercus, suggesting that it could apprise them of their position. Two engrailed genes from the cockroach, Pa-en1 and Pa-en2, have been cloned and sequenced. This has allowed use of dsRNA interference to abolish En expression, thereby testing the hypothesis that En controls axonal guidance and choice of synaptic partners in this circuit of identified neurons (Marie, 2000a).

A 1:1 mixture of Pa-en1 and Pa-en2 dsRNA was injected into the cercus of first-instar cockroaches immediately after hatching, the period when most of the second-instar sensory neurons are developing. Because hemocytes take up much of the first dose, a second booster injection was added a few hours after the first. Six days later, when the full array of second-instar neurons is present, staining with the 4D9 antibody shows a complete abolition of En expression, both in the nuclei of medial sensory neurons and in the surrounding medial epidermis. Significantly, no reduction in En staining in the CNS was seen, suggesting that the dsRNA does not penetrate the perineurium and glial sheath (Marie, 2000a).

To examine the neuronal consequences of knocking out En, attention was focussed on two sensory neurons: 6m, which expresses En strongly, and 6d, which is En-negative in normal cerci. Their morphology was examined when the animals moulted into the second instar. Neuron 6m has a distinctive anatomy exclusive to medial neurons. This contrasts with the typical anatomy of a lateral neuron, exemplified by 6d. After dsRNA treatment, the axonal projection of 6m was reproducibly altered, rendering it indistinguishable from arbors of lateral-type neurons such as 6d. The most noticeable changes include an alteration in the trajectory of the ventral axon, and the loss of dorsal branches. In contrast, the axonal arborization of 6d is unperturbed by the application of the dsRNA. Thus, knockout of En appears to cause a complete switch in the anatomy of 6m, from medial-type to lateral-type, rather than a partial transformation (Marie, 2000a).

In the cockroach cercal system, axonal projections are not the primary determinant of synaptic specificity, as they are in the cricket. Instead, electron microscopy has shown that synaptic specificity is produced by local cell-cell recognition events. Neurons with lateral-type morphology can form medial-type synapses, so it was important to determine whether inhibition of En also alters the specificity of synapse formation in neuron 6m. Intracellular recordings show that dsRNA treatment causes 6m to make significantly weaker monosynaptic connections to giant interneuron 2. This could indicate a change in the selection of synaptic partners by 6m, but could equally be caused by a reduction in the ability of the neuron to form synapses. Therefore, a more significant indicator of a change in synaptic specificity would be the establishment of novel synapses with inappropriate giant interneurons. In fact, after dsRNA application, 6m does form synaptic connections to giant interneurons 3 and 6; these connections are never seen in control animals. These connections are normally characteristic of lateral sensory neurons such as 6d, the synaptic outputs of which remain unperturbed after dsRNA application. Thus, En expression in 6m normally prevents it from forming (lateral-type) synapses with giant interneurons 3 and 6, and promotes the formation of (medial-type) synapses with giant interneuron 2 (Marie, 2000a).

It is well established that differential expression of En can control retinal axon projections in the vertebrated tectum through its regulation of ephrins, and, in Drosophila, En downregulates the cell-adhesion molecules Connectin and Neuroglian. En expression can control the projection pattern of an identified neuron. The magnitude of the changes produced by removing En suggests that it affects the pattern of expression of several adhesion molecules, rather than a single one. En directs the choice of individual target cells by the presynaptic neuron, suggesting that it also regulates the expression of neuronal recognition molecules, a function that is likely to be conserved throughout the animal kingdom (Marie, 2000a).

engrailed-related genes have been isolated in numerous taxa. Within the insects, some species have a single engrailed-related gene while others have two copies, raising the question of when and how often gene duplications have occurred. The cloning of two engrailed- related genes, Pa-en1 and Pa-en2, is reported in the cockroach Periplaneta americana. By comparing conserved domains and by carrying out a phylogenetic analysis, it is concluded that these two genes are likely to be the product of a recent duplication in the cockroach lineage. Pa-en1 and Pa-en2 are co-expressed during early embryogenesis and their segmental pattern of expression appears in an anterior-posterior progression. Potential splice variants of Pa-en2, which lack some regulatory domains, were also isolated. The roles these splice variants may play in regulating developmental processes are discussed (Marie, 2000b).

Both Pa-En1 and Pa-En2 possess the four conserved Engrailed-related domains. Pa-En2 also possesses a domain and a short motif which appear to be specific to orthologous insect Invected proteins. These specific features are: (1) the Invected-specific domain, located N terminal to domain I, which is also found in Drosophila Invected and Bombyx Invected orthologs (this domain has also been found in the Engrailed-related protein of Tribolium); (2) the RS dipeptide motif, located between domain II and the homeodomain, which is also found in Drosophila and Bombyx Invected proteins, in the single Tribolium Engrailed-related protein as well as in the partial sequences of Schistocerca, Thermobia and Oncopeltus. Two Engrailed-specific domains have been identified based on analysis of fly and moth sequences. Neither Pa-En1 nor Pa-En2 contains these domains (Marie, 2000b).

Phylogenetic analysis shows Pa-en1 and Pa-en2 to be closely related. The recent finding of two partial engrailed-related sequences in the primitive insect Thermobia and the finding of two full-length engrailed-related genes in (the relatively primitive) cockroach casts doubt on the original idea that a duplication of a single engrailed ancestral gene occurred in the ancestor of higher insects, Lepidoptera and Diptera. A phylogenetic analysis was performed based on all the available insect full length Engrailed-related proteins, to ask whether the Pa-en1 and Pa-en2 genes are the products of a recent duplication in the cockroach lineage or reflect an early duplication event in some ancestral insect (Marie, 2000b).

A tree was generated by maximum parsimony from the sequences made up of domains I, II, III and the homeodomain of several insect Engrailed-related proteins. A straightforward interpretation of this analysis is that Pa-en1 and Pa-en2 result from a recent duplication in the cock-roach lineage. In addition, both trees fail to group the Bombyx engrailed and invected genes with their presumed respective orthologs in the fly; this is in agreement with an analysis using the neighbor-joining method (Marie, 2000b).

A striking feature of engrailed-related genes is the high level of conservation in domains I, II, III and the homeodomain, in contrast to the rest of the sequence, which is highly divergent. Assuming paralogs retain redundant original functions, necessitating the formation of complexes with the same co-factors prior to binding the same DNA targets, high selection pressure would maintain sequences of the conserved domains. Duplicated paralogs would be available for co-option to new functions, which would stabilise the duplications within the genome. For example, this could be achieved by the duplicated gene acquiring a new expression pattern; it has been shown that Drosophila engrailed and invected genes have subtly different expression patterns in the CNS. In addition even though mouse engrailed-2 can functionally substitute for engrailed-1, mutations in these genes produce different phenotypes because of their divergent expression patterns (Marie, 2000b).

It is concluded, by comparison of conserved domains and by phylogenetic analyses, that Pa-en1 and Pa-en2 are likely to be the product of a recent duplication in the cockroach lineage. Since the maximum parsimony method and the arguably more rigorous maximum likelihood method both give high bootstrap values to group Pa-en1 and Pa-en2, this conclusion seems robust. In the bee, the fact that two engrailed-related fragments share the unusual feature of lacking an intron in the homeobox, also suggests an independent duplication, in this case after the separation of Hymenoptera and Diptera. In crustacea, independent duplication of engrailed-related genes has also been proposed. Despite these obvious examples of duplication, the nature of the ancestral arthropod engrailed-related gene remains unclear. A key observation may turn out to be that the engrailed-related gene of the primitive crustacean Artemia, encodes a protein with Engrailed and Invected-specific domains (Marie, 2000b).

The pattern of expression of engrailed-related genes is similar in all arthropods once they have reached the fully-developed germ-band stage, at which they are expressed in a reiterated pattern, in the posterior part of each segment. In Drosophila, engrailed determines the identity of the posterior segmental compartment and it is assumed that this role is conserved in arthropods. However in insects, the generation of the familiar reiterated expression pattern is determined by the type of embryogenesis adopted by a particular insect. In long germ-band insects such as Drosophila, engrailed is expressed almost simultaneously in all the segments of the cellular blastoderm. In contrast, Tribolium is an intermediate germ-band insect in which the segmental pattern of engrailed expression first appears in the gnathal segments and then follows an anterior-posterior progression as the germ band develops. In the short germ-band grasshopper Schistocerca, the first segments to express engrailed are the thoracic segments, followed by the gnathal and then the abdominal segments in an anterior-posterior sequence. In the short germ-band cockroach, Pa-en1 and Pa-en2 seem to be co-expressed and follow an anterior-posterior progression of segmental appearance. However, Pa-en1 and Pa-en2 are expressed in the intercalary segment before any abdominal expression, whereas in grasshopper, beetle, cricket and milkweed bug, some abdominal expression precedes or coincides with intercalary segment expression. In both cockroach and grasshopper, ocular spot expression precedes abdominal expression. The pattern of Pa-en1 and Pa-en2 expression also differs from that of the primitive firebrat, in which en-r1 is expressed in the intercalary segment and en-r2 in the ocular spots before any thoracic expression. The functional significance, if any, of these subtle differences in patterns of engrailed-related expression in different intermediate and short-germ band insects remains to be determined (Marie, 2000b).

The transcription factor Engrailed (En) directs, in the cockroach (Periplaneta americana) cercal system, the shape of the axonal arborization and the choice of postsynaptic partners of an identified sensory neuron (6m). The second larval stage (or instar) of the cockroach has an array of 39 filiform hairs on each cercus, each with an associated sensory neuron. These neurons project to the terminal ganglion and form synapses with defined subsets of 'giant' interneurons), thus mediating the animal's escape response to wind. En is normally expressed only by neurons born in the medial part of the cercal epidermis. Knock-out of En using double-stranded RNA interference transforms 6m so that it resembles a neighboring neuron that normally does not express the en gene, has a different arbor anatomy, and makes different connections. The development of 6m has been characterized and en expression was perturbed at different stages. The results show that En is not required before neuron birth in order for 6m to become a neuron, but that it is required in the postmitotic neuron to control axonal arborization and synaptic specificity. Knock-out of En subsequent to 6m entering the CNS does not change the axonal trajectory and has minor effects on axonal branches but causes the formation of synaptic connections typical of an En-negative cell. This suggests that En controls target recognition molecules independently from those guiding the axon. In contrast, double-stranded RNA injection 1 day later does not have any effects on the phenotype of 6m, suggesting that the period of synapse formation is over by the time En levels have fallen or, if synapse turnover occurs, that En is not required to maintain the specificity of synaptic connections. It is concluded that persistent en expression is required to determine successive stages in the differentiation of the neuron, suggesting that it is not far upstream from those genes encoding axon guidance and synaptic recognition molecules (Marie, 2002).

Homologs of the Drosophila segment polarity gene engrailed have been cloned from many insect species, as well as other arthropods and non-arthropods. Partial cDNAs of two engrailed homologs, which are called engrailed-related genes, have been cloned from the phylogenetically basal insect, the firebrat or Thermobia domestica (Order Thysanura), and possibly as many as four engrailed-related genes from the phylogenetically intermediate insect, the milkweed bug or Oncopeltus fasciatus (Order Hemiptera). Previous to these findings, only single engrailed-related homologs had been found in phylogenetically intermediate insect species (Tribolium and Schistocerca) and in the crustacean Artemia, while two engrailed -related homologs have been found in more derived orders (Hymenoptera and the engrailed and invected genes of lepidopterans and dipterans). Consequently, a phylogenetic analysis of insect engrailed-related genes was performed to determine whether insects ancestrally had one or two engrailed-related genes. Evidence has been found of concerted evolution among engrailed-related paralogs, however, that masks the true phylogenetic history of these genes. Concerted evolution would result in sequence homogenization, such that the en and inv paralogs of a given species appear more similar in sequence to one another than they do to their true orthologs in a different species. Concerted evolution would have its basis in selection for sequence covariation, because not only might two proteins bind to identical DNA target sequences, but they would presumably interact with a common cofactor, Extradenticle, for proper function. The phylogeny may only be decipherable, therefore, by examining the presence or absence of engrailed-specific and invected-specific motifs, which will require cloning the full length cDNAs from more species. A primary difference between the two firebrat genes is the presence (Td-en-r2) or absence (Td-en-r1) of an arginine-serine (RS) dipeptide. The Bombyx and Drosophila en genes also lack the RS dipeptide sequences, whereas it is present in Bombyx and Drosophila invected. The RS dipeptide motif lies within domain II, which is both necessary and sufficient for interaction of Engrailed proteins with Pbx proteins. The RS dipeptide is not present in chordates or other non-arthopod engrailed-related genes. Its conservation among arthropods implies that it has an important function, but this has not been tested. Since the murine En proteins lack the RS motif in domain II, it is not necessary for the interaction of the Pbx proteins and En-2. In addition, the embryonic expression pattern of the two Thermobia engrailed-related genes was examined; like Drosophila engrailed and invected, they are expressed in very similar patterns, but show one temporal difference in pregnathal segments that correlates with the tentative phylogenetic placement of the genes. In Drosophila, intercalary expression is not established until after the abdominal stripes begin to appear. Furthermore, ocular expression precedes intercalary expression. In contrast, the firebat en-r genes differ from this pattern in that en-r2 ocular spots form before the thoracic stripes, whereas en-r1 ocular expression is not present until after the abdominal stripes begin to appear. Conversely, the en-r1 intercalary stripe forms before the en-r2 intercalary stripe. It is interesting that en-r2 expression is more similar to en-r in pterygote insects on this basis, while en-r1 expression resembles that in malacostracan crustaceans, a similarity that mirrors the sister grouping of en-r2 with pterygote en-r genes and the outgroup placement of en-r1 in the phylogenetic analysis. Thermobia engrailed-related expression also confirms that the dorsal ridge is an ancient structure in insects. As germ band elongation is completed, this en-r expression patch of cells lies dorsal to the maxillary and labial stripes, midway between them. This dorsal anterior labial en-r patch is present in other insects as well. The dorsal ridge of insects is composed of two parts, one that expresses en and is formed from the dorsal portions of the labial and maxillary segments and the other that expresses the labial gene and is formed from the dorsalmost cells of the pregnathal and mandibular segments (Peterson, 1998).

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. The expression patterns of the genes even-skipped, engrailed, and wingless in a centipede, Lithobius atkinsoni, were examined. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods (Hughes, 2002).

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite Archegozetes longisetosus, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost. engrailed (en) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the Drosophila genes Antennapedia (Antp),Sex combs reduced (Scr), Deformed (Dfd), proboscipedia (pb), and orthodenticle (otd) confirm the direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost (Telford, 1998).

Engrailed and the generation and diversification of butterfly eyespot color patterns

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene, all of which break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties. hh is expressed in all cells of the posterior compartment of the butterfly wing disc, as it is in Drosophila, but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Higher levels of hh transcripts accumulate specifically in cells that flank the developing foci. In the presence of high levels of Hh, Patched function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci, cells that receive and transduce the Hh signal have increased levels of ptc transcription. Activation of ptc transcription, accompanied by the accumulation of Ci protein occurs in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. these results indicate that the Hh signal is received and transduced by cells that will differentiate as foci. These expression patterns break the A/P compartmental restrictions on gene expression known in Drosophila. During the course of eyespot evolution, there is a relaxation of the strict En-mediated repression of ci that occurs in the posterior compartment of Drosophila. During focal establishment, en and invected are targets, rather than inducers of Hh signaling. In most species of butterflies, eyespots are found only in the posterior compartment of the wing. But in those species in which eyespots are found in the anterior compartment, both En/Inv and Ci are coexpressed in eyespot foci, including the one in the anterior compartment. Thus the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions. It is suggested that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci is recruited from the circuit that acts along the A/P boundary of the wing. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures (Keys, 1999).

A fundamental challenge of evolutionary and developmental biology is understanding how new characters arise and change. The recently derived eyespots on butterfly wings vary extensively in number and pattern between species and play important roles in predator avoidance. Eyespots form through the activity of inductive organizers (foci) at the center of developing eyespot fields. Foci are the proposed source of a morphogen, the levels of which determine the color of surrounding wing scale cells. However, it is unknown how reception of the focal signal translates into rings of different-colored scales, nor how different color schemes arise in different species. Several transcription factors, including butterfly homologs of the Drosophila Engrailed/Invected and Spalt proteins have been identified. These are deployed in concentric territories corresponding to the future rings of pigmented scales that compose the adult eyespot. A new Bicyclus anynana wing pattern mutant, Goldeneye, has been isolated in which the scales of one inner color ring become the color of a different ring. These changes correlate with shifts in transcription factor expression, suggesting that Goldeneye affects an early regulatory step in eyespot color patterning. In different butterfly species, the same transcription factors are expressed in eyespot fields, but in different relative spatial domains that correlate with divergent eyespot color schemes. These results suggest that signaling from the focus induces nested rings of regulatory gene expression that subsequently control the final color pattern. Furthermore, the remarkably plastic regulatory interactions downstream of focal signaling have facilitated the evolution of eyespot diversity (Brunetti, 2001).

To distinguish between different potential mechanisms of eyespot development and evolution, candidate genes involved in eyespot color pattern formation were sought. A screen was performed for gene products that are expressed during the period of scale cell differentiation (12 to 36 hours after pupation) and that have patterns that are correlated with the concentric rings of Bicyclus anynana eyespots. Among the various proteins and transcripts surveyed (these included Cubitus interruptus, Schnurri, SMAD, Brinker, aristaless, dachshund, and teashirt), only the Engrailed/Invected (Engrailed and/or Invected, hereafter denoted by En/Inv) and Spalt (Sal) transcription factors are expressed in patterns of scale-forming cells that correlate with eyespot formation. All identified proteins are expressed in cells in the region of the focus at the center of each eyespot field. Remarkably, a second domain of En/Inv expression arises in the 16 hour pupal wing in a distinct ring of cells outside of the focal region and at the periphery of each eyespot field. In addition, Sal is expressed in rings of cells between the focal region and the ring of En/Inv-expressing cells. Based upon physical landmarks of the developing wing and by comparison of the relative size and position of the concentric rings of gene expression patterns with the colored rings of the adult eyespot, correlations between protein expression patterns and the three colored rings of B. anynana eyespots were found. The En/Inv, Sal, and Dll expression in the focus corresponds to the white center in the adult eyespot. The territory marked by Sal and Dll expression, but not En/Inv expression, appears to correspond to the domain of the black ring of scales in the adult eyespot. Additionally, the outer ring of En/Inv expression correlates with the position of the gold ring of scales in the adult wing. A gene product for which the pattern of expression correlates with the outermost dark-brown ring of scales has not been identified (Brunetti, 2001).

From observations of the temporal and spatial relationships between En/Inv, Sal, and Dll expression, two important inferences can be made: (1) the switch from synchronous coincident expression of these three proteins in the center of the eyespot field to their asynchronous, nonoverlapping expression in the outer rings of the field suggests that they are under different regulatory controls when the foci are first established than when the eyespot field is elaborated; (2) the sequential appearance of the rings, in particular the expression of En/Inv in cells just outside of the Sal domain, suggests that one mechanism for generating concentric patterns of gene expression may be to exclude the expression of one gene from another's domain (Brunetti, 2001).

The transplantation of eyespot foci between species or of selected lines of B. anynana differing in eyespot color composition induces eyespot patterns characteristic of the host animal, suggesting that the response to the focal signal (not the signal itself) is different between species. It is possible that the differences in cells' responses to focal signaling could arise as a result of changes in the expression patterns of regulators. Alternatively, direct responses to focal signaling may be similar between species, but the regulators may interact with different downstream genes involved in scale pigmentation and structure. To determine when during development differences arise between the eyespot color schemes of various species, the expression patterns were compared of En/Inv, Sal, and Dll in B. anynana (Nymphalidae, Satyrinae), Precis coenia (Nymphalidae, Nymphalinae), Vanessa cardui (Nymphalidae, Nymphalinae), and Lycaeides melissa (Lycaenidae, Lycaeninae). In each of the examined species, which represent two different families of butterflies and three different genera within the Nymphalidae, the expression patterns of En/Inv, Sal, and Dll are different, yet they mark territories in the pupal wing that often correlate with color pattern schemes on the adult wing. For example, in P. coenia, the Sal territory in the pupal wing marks the entire area encompassed by the adult eyespot. In addition, the coexpression of En/Inv, Sal, and Dll in P. coenia forewings in an asymmetric patch of scales at the center of the pupal eyespot corresponds to the white/blue scales at the center of the adult eyespot. The coexpression of the same genes in scale-building cells outside of this central spot correlates with the black ring of scales on the adult. In V. cardui, a species closely related to P. coenia, En/Inv is expressed in an outer ring of scale-building cells that correlates with the black ring of scales in the adult eyespot. However, in L. melissa, a crescent of En/Inv expression correlates with the future position of orange scales on the adult, and En/Inv and Sal coexpression correlates with the metallic-looking patch of scales at the center of the eyespot field (Brunetti, 2001).

From comparative data, it is concluded that eyespot color pattern diversity is generated by regulatory differences at two distinct stages of eyespot development that evolve independently of each other: (1) during the focal signaling stage, through the generation of different combinations and patterns of expression of regulatory genes such as en/inv, sal, and Dll; and (2) during the scale differentiation stage, through differences in the response of pigmentation genes to the upstream regulators (Brunetti, 2001).

These results indicate that at least one tier of spatially regulated transcription factors is interposed between focal signaling and scale color differentiation. How the graded distribution of a focal signal is translated into the concentric territories of En/Inv, Sal, and Dll expression is therefore of special interest. In B. anynana, it is suggested that this occurs through response thresholds of, and negative cross-regulation among, genes regulated by the signal. For example, one of the simplest explanations for the exclusion of En/Inv and Sal expression from each other's territories outside of the focus could be the repression of one gene by the product of the other. The reciprocal effects of the Goldeneye mutation on En/Inv and Sal expression are strongly suggestive of negative crossregulation. The establishment, through negative crossregulation, of distinct spatial domains of downstream genes in response to a single activator is a common theme illustrated by the subdivision of the Drosophila embryonic mesoderm and neuroectoderm and of the proximodistal axis of Drosophila limb fields. In P. coenia, however, the nested nonexclusive expression of Sal and En/Inv suggests that here these genes do not crossregulate. Rather, the nested expression pattern outside of the focus is most simply explained by different threshold responses of these two genes to the focal signal; these responses are analogous to the threshold responses of genes to long-range signals in the Xenopus mesoderm and the Drosophila imaginal wing field (Brunetti, 2001).

The deployment of En/Inv, Sal, and Dll in all of the species examined also raises some interesting possible scenarios regarding the origin and diversification of eyespots and the evolution of the underlying genetic regulatory system that controls eyespot pattern formation. It has been proposed that eyespots have a single origin and are derived from simpler spot patterns of uniform color that evolved into organizing centers. Because all three proteins are deployed in color-correlated patterns in this well-diverged group of butterflies, it is likely that these genes were recruited into the developmental program early during the evolution of eyespots. Furthermore, it is intriguing that while the three proteins have distinct expression patterns during scale differentiation, they are coexpressed during focus formation. It is tempting to speculate, on the basis of the data presented here, that the evolution of eyespots in response to diverse selective environments involved the modification of the deployment of genes that were originally expressed in simpler spot patterns into additional concentric patterns organized around and by cells in the center of the eyespot field (Brunetti, 2001).

Engrailed in other invertebrates

engrailed is a homeobox gene essential for developmental functions such as differentiation of cell populations and the onset of compartment boundaries in arthropods and vertebrates. Functional study on engrailed in an unsegmented animal, the nematode Caenorhabditis elegans, has been performed. In the developing worm embryo, ceh-16/engrailed is predominantly expressed in one bilateral row of epidermal cells (the seam cells). ceh-16/engrailed primes a specification cascade through three mechanisms: (1) it suppresses fusion between seam cells and other epidermal cells by repressing eff-1/fusogen expression; (2) it triggers the differentiation of the seam cells through different factors, including the GATA factor elt-5, and (3) it segregates the seam cells into a distinct lateral cellular compartment, repressing cell migration toward dorsal and ventral compartments (Cassata, 2005).

The two Engrailed pan-specific antibodies, Mab4D9 and Mab4F11, reveal strikingly different Engrailed accumulation patterns in both of the malacostracan crustaceans Porcellio scaber (Isopoda) and Procambarus clarkii (Decapoda), compared with insects. The signal detected with Mab4D9 resides in the posterior part of each segment, including the appendages, the ventral and lateral sides of the trunk and the CNS. However, Mab4F11 reveals a signal only in small groups of neurons in the CNS and PNS, primarily localized in the pereon. Similar Mab4D9 and Mab4F11 patterns are observed in the crayfish P. clarkii, except that no Mab4F11 signal is detected in the pleon. To address the possibility of multiple engrailed paralogs, partial cDNAs of two engrailed genes, Ps-en1 and Ps-en2, were cloned from P. scaber, and their expression patterns were studied using whole-mount in situ hybridization. Although the Ps-en1 and Ps-en2 patterns are comparable in early development, they become distinct in late embryogenesis. Ps-en1 is expressed in the CNS, where Mab4F11 stains, but also accumulates in the epidermis. In contrast, Ps-en2 is expressed in the lateral aspect and limbs of all segments. Phylogenetic analysis of en sequences from crustaceans and insects suggests that the two en genes from the apterygote insect Thermobia domestica (Thysanura) may be related to en1 and en2 of higher crustaceans (Abzhanov, 2000).

One possible implication of these results is that there was an ancient duplication of en genes, which resulted in two distinct paralog groups in higher crustaceans and apterygote insects. This ancient duplication event appears limited to the insects and malacostracan crustaceans. Since only a single en ortholog has been reported from onychophorans, spiders, myriapods and branchiopod crustaceans, the en1 and en2 group genes appear to be a shared and derived feature of the Insecta and Malacostraca. This is in accord with some recent arthropod phylogenies based on molecular and/or developmental data. However, higher crustacean en genes, unlike their apterygote homologs, have highly dissimilar expression patterns. It has been proposed that after a duplication event, the duplicate sister genes could be preserved by complementary mutations leading to division of the ancestral gene’s expression patterns/functions rather than gaining novel functions. As well as the multiple vertebrate engrailed genes used to demonstrate this new hypothesis, the data presented on the malacostracan genes seem to provide an additional support for the 'subfunctionalization' model (Abzhanov, 2000).

An engrailed-class gene has been identified in a member of the Onychophora, and the distribution pattern of its protein determined during embryogenesis. This work adds to the understanding of the evolution of development; in addition, it contributes toward the clarification of the phylogenetic position of the Onychophora. Transient expression is observed in a portion of each developing segment. By the time limbs have formed, segmental expression of en-class protein is restricted to the mesoderm. This pattern shares important spatio-temporal characteristics with those of Annelida and Arthropoda, both of which have members that express en-class genes segmentally in mesoderm and ectoderm. A parsimonious interpretation of these results showing en expression only in the mesoderm, is that a segmented ancestor common to Annelida, Arthropoda and Onychophora had iterated expression of an en gene in both mesoderm and ectoderm, as has been observed in the leech (Annelida) and the honeybee (Arthropodia). Expression in the mesoderm may have been lost in the fruit fly and similarly, expression in the ectoderm may have been lost in the onychophoran. However, given that sequental expression of an en-class gene restricted to mesoderm has been observed in Amphioxus, an intriguing alternative possibliity is that en-class gene expression in Onychophora represents a more primitive segmental pattern (Wedeen, 1997).

There is an ongoing discussion of whether segmentation in different phyla has a common origin. The presumably conserved segment-polarity network and the organization into parasegments can be seen as an ancestral character for arthropods. In the closely related onychophorans, engrailed expression points to a comparable organization. However, segment polarity gene orthologs are apparently not involved in body segmentation in other segmented phyla. In annelids, engrailed is expressed in segmentally iterated spots in the CNS and in mesodermal cells, but is probably not involved in body segmentation as in arthropods. The establishment of segment polarity in leeches is independent of cell interactions along the anteroposterior axis; this is in contrast to the situation in arthropods, where anterior and posterior fates of the segments are specified by intercellular signaling between wg- and en-expressing cells. Furthermore, there are no indications that the annelid embryo is constructed from units like the parasegment. In the leech, progeny of particular teloblasts overlap with respect to segmental boundaries and do not form genealogical units as in crustaceans. Some key aspects of arthropod segmentation are thus not present in annelids. The segmentation of annelids and arthropods, therefore, seems to be brought about by different mechanisms. This is an important argument against a common origin of segmentation in annelids and arthropods. In chordates it is also doubtful whether engrailed plays a role in somitogenesis. engrailed but not wingless is expressed in reiterated pattern in the somites of the cephalochordate amphioxus, which suggests that the segment polarity gene network as present in arthropods is not conserved. Furthermore, vertebrate engrailed orthologs do not play a role in somite formation or maintenance of the somite boundaries. This points to a different mode of segmentation in vertebrates and arthropods, and does not support a common origin of segmentation. However additional evidence is required to prove this (Damen, 2002).

Echinoderms possess one of the most highly derived body architectures of all metazoan phyla, with radial symmetry, a calcitic endoskeleton, and a water vascular system. How these dramatic morphological changes evolved has been the subject of extensive speculation and debate, but remains unresolved. Because echinoderms are closely related to chordates and postdate the protostome/deuterostome divergence, they must have evolved from bilaterally symmetrical ancestors. The expression domains in echinoderms are reported for three important developmental regulatory genes (distal-less, engrailed and orthodenticle), all of which encode transcription factors that contain a homeodomain. The reorganization of body architecture involves extensive changes in the deployment and roles of homeobox genes. These include modifications in the symmetry of expression domains and the evolution of several new developmental roles, as well as the loss of roles conserved between arthropods and chordates. New developmental roles include roles for en, in skeletogenesis, otd and dll in podia (tube-feet that function in locomotion, feeding and sensory perception and are extensions of the water vascular system), dll in larvaL brachiolar arms and subtrochal cells, engrailed in rudiment invagination, and otd in the ciliated band. Some of these modifications seem to have evolved very early in the history of echinoderms, whereas others probably evolved during the subsequent diversification of adult and larval morphology. These results demonstrate the evolutionary lability of regulatory genes that are widely viewed as conservative (Lowe, 1997).

Vertebrate segmentation has been proposed as an evolutionary inheritance either from some metameric protostome or from a more closely related deuterostome. In an attempt to clarify these evolutionary possibilities, the developmental expression of AmphiEn was studied. AmphiEn is the engrailed homolog in amphioxus, an invertebrate (Phylum Chordata, Subphylum Urochordata) that is the closest living invertebrate relative to the vertebrate line. The protein includes a homeodomain (EH4) of the engrailed class as well as four additional conserved regions EH1, EH2, EH3 and EH5. In Drosophila, EH1 and EH5 are required for full repression activity, and EH2 and EH3, just preceding the homeodomain, influence the targeting of Engrailed. In neurula embryos of amphioxus, AmphiEn is expressed along the anteroposterior axis in metameric stripes, each located in the posterior part of a nascent or newly formed segment. This pattern resembles the expression stripes of the segment-polarity gene engrailed, which has a key role in establishing and maintaining the metameres in embryos of Drosophila and other metameric protostomes. Later in development, amphioxus embryos express AmphiEn in non-metameric patterns -- transiently in the embryonic ectoderm and dorsal nerve cord. Nerve cord expression occurs in a few cells approximately midway along the rostrocaudal axis and also in a conspicuous group of anterior cells in the cerebral vesicle at a level previously identified as corresponding to the vertebrate diencephalon. Compared to vertebrate engrailed expression at the midbrain/hindbrain boundary, AmphiEn expression in the cerebral vesicle is relatively late. Thus, it is uncertain whether the cerebral vesicle expression marks the rostral end of the amphioxus hindbrain; if it does, then amphioxus may possess little or no homology to the vertebrate midbrain. The segmental expression of AmphiEn in forming somites suggests that the functions of engrailed homologs in establishing and maintaining a metameric body plan may have arisen only once during animal evolution. If so, the protostomes and deuterostomes probably shared a common segmented ancestor (Holland, 1997).

Morphological studies suggest that insects and crustaceans of the Class Malacostraca (such as crayfish) share a set of homologous neurons. However, expression of molecular markers in these neurons has not been investigated, and the homology of insect and malacostracan neuroblasts, the neural stem cells that produce these neurons, has been questioned. Furthermore, it is not known whether crustaceans of the Class Branchiopoda (such as brine shrimp) or arthropods of the Order Collembola (springtails) possess neurons that are homologous to those of other arthropods. Assaying expression of molecular markers in the developing nervous systems of various arthropods could resolve some of these issues. Expression of Even-skipped and Engrailed, two transcription factors that serve as insect embryonic CNS markers, was examined across a number of arthropod species. This molecular analysis allows verification the homology of previously identified malacostracan neurons and identification of additional homologous neurons in malacostracans, collembolans and branchiopods. Engrailed expression in the neural stem cells of a number of crustaceans was also found to be conserved. It is concluded that despite their distant phylogenetic relationships and divergent mechanisms of neurogenesis, insects, malacostracans, branchiopods and collembolans share many common CNS components (Duman-Scheel, 1999).

The arrangement of serotonin- and engrailed-expressing cells was examined in the embryonic ventral nerve cord of the American lobster Homarus americanus Milne Edwards, 1873 (Malacostraca, Pleocyemata, Homarida), and the spatial relationship of these two cell classes was explored by a double-labelling approach. The goal of this study was to determine whether the lobster serotonergic neurons are homologous to similar cells present in representatives of the Hexapoda and other Arthropoda. The results indicate that, in fact, these neurons in the lobster ventral nerve cord have corresponding counterparts in many other mandibulate taxa. Based on the finding of these homologies, the arrangement of serotonergic neurons in a model trunk ganglion of the mandibulate ground pattern was reconstructed as comprising an anterior and a posterior pair of serotonergic neurons per hemiganglion, each cell with both an ipsilateral and a contralateral neurite. Starting from this ground pattern, the evolutionary diversification of this class of neurons within the Mandibulata is discussed (Harzsch, 2003).

Based on the homologies suggested above, the arrangement of serotonergic neurons in a model trunk ganglion of the mandibulate ground pattern can be reconstructed as comprising an anterior and a posterior pair of serotonergic neurons per hemiganglion, each cell with both an ipsilateral and a contralateral neurite. Starting from this ground pattern, the Hexapoda retained both cell pairs but abandoned the ipsilateral neurites. The Pterygota (winged insects) then reduced the anterior cell pair in the thoracic and abdominal ganglia but retained it in the ganglia of the mandible and maxilla one and two. In some hexapod species, an additional third serotonergic cell accompanies the posterior cell pair in the ganglia of the mandible, maxilla one and two, and thorax one, which may be an apomorphic (derived or specialized) character in the ground pattern of the Pterygota. In the grasshopper Schistocerca americana, this cell is also part of the lineage generated by neuroblast 7-3 as is the pair of posterior serotonergic neurons. These authors confirmed by dye injection that the third cell is also present as progeny of 7-3 in other thoracic and abdominal ganglia, but in these segments it does not express serotonin. Although similar supernumerary serotonergic neurons are also present in D. melanogaster, it is not know if they are clonally related since segment-specific differences in the lineage generated by neuroblast 7-3 have not yet been examined in this species (Harzsch, 2003).

The engrailed gene is well known from its role in segmentation and central nervous system development in a variety of species. In molluscs, however, engrailed is involved in shell formation. So far, it seemed that engrailed had been co-opted uniquely for this particular process in molluscs. In the gastropod mollusc Patella vulgata, an engrailed ortholog is expressed in the edge of the embryonic shell and in the anlage of the apical sensory organ. Surprisingly, a dpp-BMP2/4 ortholog is expressed in cells of the ectoderm surrounding, but not overlapping, the engrailed-expressing shell-forming cells. It is also expressed in the anlage of the eyes. A compartment boundary exists between the cells of the embryonic shell and the adjacent ectoderm. It is concluded that engrailed and dpp are most likely involved in setting up a compartment boundary between these cells, very similar to the situation in, for example, the developing wing imaginal disc in Drosophila. It is suggested that engrailed became involved in shell formation because of its ancestral role, which is to set up compartment boundaries between embryonic domains (Nederbragt, 2002).

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Ten genes expressed in midlevel neural domains were examined, namely tailless (tll), paired box homeobox 6 (pax6), emptyspiracles-like (emx), barH, orthopedia (otp), developing brain homeobox (dbx), lim domain homeobox 1/5 (lim1/5), iroquois (irx), orthodenticle-like (otx), and engrailed (en). These genes are all expressed in chordates at least in the midbrain of the central nervous system, and thus, as a group, their domains are more posteriorly located than the anterior set. Some have the anterior border of the domain in the forebrain (tll, pax6, emx, lim1/5, and otx), and some have their anterior border in the midbrain (otp, barH, dbx, irx, and en). Most have posterior borders in the midbrain, but two (en and irx) have posterior borders in the anterior hindbrain. Thus, while all are expressed in the midbrain, each differs in its anterior and posterior extent. Several of the chordate genes (pax6, dbx, en, and irx) have separate posterior expression domains running the length of the chordate hindbrain and spinal cord at different dorsoventral levels of the neural tube (Lowe, 2003).

In S. kowalevskii, these ten orthologs are expressed in circumferential bands in the ectoderm at least of the mesosome (collar) or anterior metasome, that is, more posteriorly than the anterior group. Each gene differs in the exact anteroposterior extent of its domain -- some are expressed in part or all of the prosome. The most broadly expressed orthologs of this group are pax6, otp, lim1/5, irx, and otx. All are expressed in the prosome (relatively weakly for otx), mesosome (weakly in the case of otp and lim1/5), and anterior metasome, all ceasing by the level of the first gill slit. pax6 is strongest at the base of the proboscis, and lim1/5 is expressed most strongly in a dorsal patch at the base of the proboscis. The most narrowly expressed orthologs are barH, tll, emx, and en. tll is detected in early stages in the anterior prosome, posterior prosome, and anterior mesosome and in later stages restricted to the anterior mesosome. The emx domain is a single ring in the anterior mesosome plus an additional domain in the ciliated band in the posterior metasome, the only gene of the 25 to be expressed in the band cells. barH and en are both expressed in narrow ectodermal bands; barH in the anterior mesosome and en in the anterior metasome. A dorsal view of both en and barH reveals a dorsal narrow gap in expression in the midline. Ventrally, no such gap is observed. Two additional spots of en expression are detected in the ectoderm on either side of the dorsal midline in the proboscis. In the most posterior ring of otx expression in the metasome, a similar gap in expression is observed. otp is expressed predominantly in a punctate pattern in the apical layer of prosome ectoderm and in a diffuse pattern in the basal layer of prosome ectoderm, similar to dlx. It is also expressed in a circumferential ring of intermittant ectodermal cells in the posterior mesosome and then in two parallel lines of cells bilateral to the dorsal axon tract of the anterior metasome. Early dbx expression is most strongly detected in an ectodermal ring in the developing mesosome overlapping the posterior domain of tll. dbx is also expressed in the prosome at low levels throughout the ectoderm and at high levels in scattered individual cells or groups of cells. Later expression is restricted to two ectodermal bands marking the anterior and posterior limits of the mesosome. An additional endodermal domain of expression is observed predominantly in the ventral anterior pharyngeal endoderm (Lowe, 2003).

otx, en, and irx deserve description in more detail because in chordates, especially vertebrates, the products of these regionally expressed genes are thought to interact in setting up the midbrain-hindbrain boundary and the isthmic organizer. Furthermore, the otx domain at the midbrain level is the site from which neural crest cells migrate ventrally to the first branchial arch. In S. kowalevskii, otx is expressed at low but readily detectable levels in the prosome ectoderm and at high levels in four closely spaced ectodermal rings: one at the base of the prosome, two in the mesosome, and one in the anterior metasome. This fourth stripe of otx expression crosses the site where the first gill slit perforates the ectoderm. As evidence, beyond morphology, that the hemichordate gill slit is homologous to the chordate gill slit/branchial arch, the pax1/9 ortholog, known to be expressed in chordate gill slits, is expressed in the endoderm of the developing S. kowalevskii gill slit. Gill slit expression of pax1/9 is observed in the adult of P. flava. Thus, chordates and hemichordates have in common the association of the posterior limit of the otx domain with the position of the first gill slit or branchial arch (Lowe, 2003).

In hemichordates, the en domain overlaps the posterior part of the otx domain, and the irx domain runs through both of these, as is also the case in chordates. However, otx expression in S. kowalevskii extends slightly more posteriorly than does en, whereas in chordates the en domain extends slightly more posteriorly (Lowe, 2003).

In summary, for this midlevel group of genes, the S. kowalevskii orthologs are expressed in the mesosome and anterior metasome (with some domains extending anteriorly into the prosome), that is, more posteriorly than those genes of the anterior group. In general, expression domains that end posteriorly near the midbrain-hindbrain boundary in chordates, end in the anterior metasome in hemichordates. Although the anterior metasome is not the site of an obvious morphological boundary, it is the site of the first gill slit. The first gill slit/branchial arch in chordates is at the same body level as the midbrain-hindbrain boundary (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

To gain insights into the relation between evolution of cis-regulatory DNA and evolution of gene function, tissue-specific enhancers of the engrailed gene of the basal chordate amphioxus (Branchiostoma floridae) were identified and their ability to direct expression in both amphioxus and its nearest chordate relative, the tunicate Ciona intestinalis, was examined. In amphioxus embryos, the native engrailed gene is expressed in three domains -- the eight most anterior somites, a few cells in the central nervous system (CNS) and a few ectodermal cells. In contrast, in C. intestinalis, in which muscle development is highly divergent, engrailed expression is limited to the CNS. To characterize the tissue-specific enhancers of amphioxus engrailed, it was first showed that 7.8 kb of upstream DNA of amphioxus engrailed directs expression to all three domains in amphioxus that express the native gene. The amphioxus engrailed muscle-specific enhancer was identified as the 1.2 kb region of upstream DNA with the highest sequence identity to the mouse en-2 jaw muscle enhancer. This amphioxus enhancer directed expression to both the somites in amphioxus and to the larval muscles in C. intestinalis. These results show that even though expression of the native engrailed has apparently been lost in developing C. intestinalis muscles, they express the transcription factors necessary to activate transcription from the amphioxus engrailed enhancer, suggesting that gene networks may not be completely disrupted if an individual component is lost (Beaster-Jones, 2007).

On the origin and evolution of the tripartite brain

The many different nervous systems found in bilaterally symmetric animals may indicate that the tripartite brain appeared several times during the course of bilaterian evolution (see Bilaterian evolutionary tree). However, comparative developmental genetic evidence in arthropods, annelids, urochordates, and vertebrates suggests that the development of a tripartite brain is orchestrated by conserved molecular mechanisms. Similarities in the underlying genetic programs do not necessarily reflect a common origin of structures. Nevertheless, 3 lines of evidence support a monophyletic origin of the tripartite brain and possibly also an elongated central nervous system (CNS): structural homology, character identity networks, and the functional equivalence of character identity genes. Monophyly of the brain also implies that the brain was secondarily reduced and lost multiple times during the course of evolution, leading to extant brainless bilaterians. The likelihood of secondary loss can be estimated by metazoan divergence times and through reconstructed cases such as limb loss in tetrapods or eye loss in fish. When scaled to molecular clock dates, monophyly of the tripartite brain indicates that existing brainless Bilateria had several hundred million years’ time for the secondary modification and eventual loss of a primitive/ancestral brain and CNS. To corroborate this conjecture, ancestral character identity genes of living brainless Bilateria can be tested for their potential to substitute Drosophila or Mus homologs in tripartite brain development (Hirth, 2010).

Comparative developmental genetic analyses in arthropods, annelids, urochordates, and vertebrates provide experimental evidence suggesting that brain and CNS development in these taxa is orchestrated by conserved molecular genetic mechanisms. Similarities in the underlying developmental genetic programs do not necessarily reflect a common origin of structures. However, monophyly of the tripartite brain is supported by 3 lines of evidence, namely (1) structural homology, (2) character identity networks (ChINs), and (3) functional equivalence of character identity genes. (Hirth, 2010).

Homology signifies common descent and can be defined as a relationship between traits of organisms that are shared as a result of common ancestry. Structural homology refers to a morphological character that is (a) derived from a common ancestor possessing this character, (b) built on the same basic plan, and (c) consists of comparable elements. The latter was already exemplified by Darwin, who referred to 'the relative position or connection in homologous parts; they may differ to almost any extent in form and size, and yet remain connected together in the same invariable order'. Textbook examples are the different forelimbs of tetrapods where similar bones are connected in the 'same invariable order', irrespective of the different functions they serve. The same principle of 'relative position or connection in homologous parts' applies to 'midbrain' structures in Drosophila and Mus, although function seems to be conserved as well; in both cases, GABAergic, serotonergic, and dopaminergic neural circuit elements involved in the control of locomotor behavior are located in the same relative position to other neural processing centers, namely posteriorly to light-sensing organs and anteriorly to gustatory and 'facial' innervation (Hirth, 2010).

The principle 'connected together in the same invariable order' of comparable elements may also apply to ventral/spinal cord motor neurons in Drosophila, Platynereis (Annelida), and Mus; they are located ventrally to nonneural tissue and dorsally to the midline. As a stand-alone criterion, however, 'relative position or connection in homologous parts' would be insufficient to signify homology because similar structures with similar positions can have multiple causes, including parallel or convergent evolution. However, related to brain evolution, this criterion is supported by developmental genetic evidence suggesting that insect and mammalian brains are built on the same basic plan, which is executed by the spatiotemporal activities of pleiotropic genes that constitute character identity networks (ChINs) (Hirth, 2010).

ChINs refer to genetic regulatory networks that control the developmental program and, hence, the specification of character identities, such as forelimbs. ChINs for brain and CNS development include Otx-Pax-Hox modules acting along the anterior-posterior axis, BMP-Msx-Nkx modules acting along a DV axis, and bHLH-Par-Numb modules acting along an apico-basal axis. Together, these modules are necessary for the correct formation of an orthogonal, species-specific CNS. Character identity genes are central to ChINs; their knockdown/mutational inactivation does obstruct the formation of a character identity. The majority of character identity genes are transcription factors, with textbook examples such as Pax6 genes, which are essential for the formation of light-sensing organs, or otd/Otx genes, which are essential for anterior brain formation. As stated earlier, similarities in developmental genetic programs do not necessarily reflect a common origin of character identities, which is exemplified, for example, by the role of distal-less(Dll/Dlx) genes in the formation of non-homologous appendages. However, the situation is different if taxa-specific ChINs, which comprise homologous genes, specify taxa-specific character identities to which structural homology applies, namely their construction depends on a conserved genetic program (basic plan) and consists of comparable elements that are arranged 'in the same invariable order'. This principle applies to the above-mentioned midbrain-derived neural circuit elements that are required for the control of locomotor behavior: in both flies and mice, FGF8, Engrailed, and Pax2/5/8 genes are essential for the formation and specification of these structures (Hirth, 2010).

ChINs of different taxa can comprise homologous genes that control the developmental program and, hence, the specification of character identities sharing a common descent. It has therefore been predicted that the phenotype caused by knockdown of a character identity gene (i.e. transcription factor) 'can be reversed with a gene from the clade that shares this character, but not by genes from species that diverged before the origin of this character'. Experimental evidence conforming to this prediction is available. Human Otx2 can rescue defective 'forebrain' formation in Drosophila orthodenticle (otd) mutants, and otd can replace mouse Otx2 in fore- and midbrain formation, but only if otd is accompanied by the Otx2 regulatory sequences required for epiblast-specific translational control. Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain formation, but not for limb development. Mouse Emx1 can rescue brain defects in Drosophila empty spiracles(ems) mutants, but Acropora [Anthozoa, Cnidaria (radially symmetric organisms including corals, anemones and jelly fish)] Emx is not able to replace ems in fly brain development (Hirth, 2010).

Based on these results, and in line with Wagner's prediction of the existence of equivalent functions of character identity genes (Wagner, 2007), it can be concluded that: (1) The character of a 'fore-, mid-, and hindbrain' develops under the control of ChINs comprising otd/Otx, ems/Emx, and en/EN1 genes that are shared in a clade including flies and mice. (2) The failure of Drosophilaengrailed to rescue limb formation in MusEN1 mutants indicates that the appendages of mice and flies are not homologous. (3) Most likely, the epiblast did not exist in the last common ancestor of Drosophila and Mus and is an evolutionary novelty of the lineage leading to Mus (Boyl, 2001). (4) Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain'. The last conclusion has major implications for the evolutionary origin of the tripartite brain and depends on the position of anthozoans/cnidarians relative to bilaterally symmetric animals in a phylogenetic tree (Hirth, 2010).

A phylogenetic tree is based on evolutionary relationships and can be reconstructed using cladistics. The cladistic concept is relative; it scores characters for their presence and absence and, if present, for their state in each of the taxa of interest. Current cladistics scores morphological as well as molecular characters (i.e. genes, genomes, and developmental pathways) against each other, and the resulting phylogenetic tree is based on sister and out groups in order to be rooted. For example sister groups like arthropods and onychophorans share a common panarthropod ancestor, and together they are a sister group to cycloneuralians (including nematodes), which together belong to the ecdysozoans, which are a sister group to lophotrochozoans. Out groups provide necessary additional information about the origin of a character in sister groups; they are used for character polarity which enables the application of the parsimony criterion in order to infer whether the character is primitive/ancestral or derived (Hirth, 2010).

Monophyly of bilaterally symmetric animals and subsequent interpretations about the origin and evolution of the brain and CNS hinge on the identification of genuine sister and out groups and, thus, on how deep a phylogenetic tree is rooted. Current cladistics is work in progress which is exemplified by the allocation of Cnidaria (but also Acoela) as either a sister or out group to bilaterians. The ambiguity is caused by mounting evidence suggesting that cnidarians possess genetic toolkits similar to those active in bilaterian axis and cell type specification, including neurogenesis (Gaillot, 2009). Depending on additional characters and genuine out groups to Cnidaria, this can be interpreted to mean that: (i) cnidarians are de facto bilaterians and a genuine sister group to the rest of bilaterally symmetric animals, namely P+D, or (ii) cnidarians are a genuine out group to P+D. It follows that the positioning of Cnidaria has an impact on the positioning of Urbilateria and the origin of a brain and CNS. Cnidarians possess nerve nets and nerve rings but, so far, no evidence of a centralized nervous system has been found [Gaillot, 2009]. Several extant Protostomia and Deuterostomia (P+D) also possess a net-like nervous system. In the first scenario, Urbilateria would be the last common ancestor of both Cnidaria and P+D, suggesting that a nerve net is a primitive/ancestral character. In the second scenario, Urbilateria would be the last common ancestor of P+D, and unrelated to Cnidaria, suggesting that the cnidarian nerve net is unrelated to the nerve net of extant bilaterally symmetric animals (the position of Acoela would require further considerations) (Hirth, 2010 and references therein).

As mentioned, the presence of genetic toolkits does not necessarily reflect a common origin of characters. The existence of equivalent functions of character identity genes, however, allows inferences as to whether genes diverged before the origin of a character or not (Wagner, 2007). Murine, but not the cnidarian AcroporaEmx gene, can rescue brain defects in Drosophila ems mutants, suggesting that Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain', which is a primitive/ancestral character to mice and Drosophila. These data suggest that Acropora and the last common ancestor of mice and Drosophila did not share the character 'tripartite brain'; it may also indicate that the last common ancestor of Cnidaria and P+D possessed a nerve net-like nervous system. The latter notion is supported by the functional equivalence of another character identity gene. The cnidarian Hydra achaete scute homolog has proneural activity in Drosophila, can heterodimerize with daughterless, and is able to form ectopic sensory organs in the peripheral nervous system of Drosophila; in addition, it is also able to partially rescue adult external sensory organ formation in viable achaete scutecomplex mutations. Unfortunately, whether Hydra achaete scute is able to rescue defects in Drosophila achaete scute mutant brain and CNS development was not tested (Hirth, 2010).

Together, these data suggest that the morphological character 'tripartite brain' evolved after the Cnidaria-P+D split. It depends on the placement of cnidarians in- or outside the clade Bilateria, whether it is reasonable to propose that Urbilateria already possessed a tripartite brain and probably also a complex CNS, or whether Urbilateria was likely to be 'brainless'. If Cnidaria and P+D are true sister groups of a clade Bilateria, it follows that Urbilateria was brainless. Independent of the position of Urbilateria, the above-mentioned data corroborate monophyly of the tripartite brain, as well as its evolutionary origin after the cnidarian-P+D split but before the Protostomia-Deuterostomia (P/D) split. This concept, though, is challenged by the fact that several proto- and deuterostomians do not possess a brain and complex CNS. The conundrum is most obvious when the complex CNS of arthropods is compared to that of cycloneuralians, which are supposed to be sister groups. Comparative data for cycloneuralians are scant, except for Caenorhabditis elegans. The nematode does not possess a complex brain and CNS, yet monophyly of the brain is corroborated by functional equivalence of a character identity gene: the ems homolog ceh-2 of C. elegans is able to rescue ems mutant brain defects in Drosophila. These data suggest that C. elegans secondarily lost the character 'tripartite brain' and most likely also a complex CNS, as should be postulated for other brainless proto- and deuterostomians that are monophyletic to Drosophila and mice. (Hirth, 2010).

Monophyly of the tripartite brain and its evolutionary origin after the cnidarian-P+D split and before the P/D split implies that the brain and CNS were secondarily reduced and eventually lost multiple times and independently during the course of protostomian and deuterostomian evolution. For the quality and consistency of this conjecture, it is necessary to consider (1) metazoan divergence times, (2) the likelihood of secondary loss, as illustrated by reconstructed cases, and (3) the proposal of an experimental paradigm that can test ancestral character identity genes of brainless bilaterians for their potential to control brain development in Drosophila or mice. (Hirth, 2010).

Molecular clock dates suggest that the cnidarian-P+D split occurred somewhere around 630 million years ago (Mya), the P/D split around 555 Mya, and the Arthropoda-Priapulida split around 540 Mya. These estimates suggest that a primitive/ancestral tripartite brain likely evolved within 75 million years between 630 and 555 Mya and then continued to evolve into taxon- and species-specific characters such as the extant Drosophila and mouse brain. Monophyly implies that ancestral priapulids shared with arthropods the character of a tripartite brain and possibly a segmented CNS. Fossil evidence supports ancestral segmentation in priapulids (e.g. Markuelia) even though extant priapulids are nonsegmented and their CNS is 'only' composed of a nerve ring and a single ventral cord running the length of the body. Scaled against molecular clock dates, monophyly of the tripartite brain suggests that the derived character of the priapulid brain and CNS would have had several hundred million years' time for its secondary modification. Such a scale of divergence time can be extrapolated to other taxa as well, and implies that also other extant brainless P+D would have had several hundred million years' time for the secondary, independent modification and eventual loss of a tripartite brain and CNS (Hirth, 2010).

Comparative developmental genetics and phylogenomics reveal that morphological evolution is most likely driven by gene duplication and gene loss, together with changes in differential gene regulation, including mutations in cis-regulatory elements of pleiotropic developmental regulatory genes. These genetic modifications can account not only for the acquisition of novel morphological characters but also for the modification and eventual loss of a morphological character. The latter is illustrated by limbless tetrapods, such as whales, snakes, and flightless birds. Limbless tetrapods are descended from limbed ancestors, and limblessness has been shown to be polygenic, involving pleiotropic regulatory genes that act as modifiers to suppress limb development. In snakes, for example, differential regulation of HoxC genes accounts for the failure to activate the signaling pathways required for proper limb development, eventually leading to limbless snakes. Independent reduction and limb loss in tetrapods occurred repeatedly over several millions of years for lizards, and over 10-12 or up to 20 million years for whales (Hirth, 2010).

The secondary loss of morphological characters is also exemplified in fish. In different natural populations of threespined stickleback fish, the secondary loss of the pelvis occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The selective pressures causing secondary loss can be manifold, including energy limitation and environmental constraints which are most obvious for nervous system structures that are characterized by high energy consumption. For example, populations of cave fish have undergone convergent eye loss at least 3 times within the last 1 million years, whereas populations that continuously lived on the surface retained their eyes. These examples illustrate that the secondary loss of a morphological character can occur repeatedly during the course of evolution within a time frame of million years. In comparison, monophyly of the tripartite brain calibrated by metazoan divergence times suggests that extant brainless P+D would have had several hundred million years’ time, possibly from 555 Mya onwards, for the secondary modification and eventual loss of an ancestral/primitive tripartite brain and CNS, the mechanisms of which remain unknown (Hirth, 2010).

The secondary, independent loss of the brain and CNS multiple times during the course of protostomian and deuterostomian evolution is a conjecture that can be tested experimentally. Wagner's prediction states that the phenotype caused by knockdown of a character identity gene can be rescued with a gene from the clade that shares a particular character, but not by genes from species that diverged before the origin of this character (Wagner, 2007). Monophyly of the tripartite brain implies that extant brainless P+D species were once able to develop a primitive/ancestral tripartite brain; therefore, these brainless species should possess ChINs for the development and specification of a tripartite brain, unless they have secondarily lost the necessary genes during the course of evolution. The potential functional equivalence of character identity genes in brain development can be tested in those cases where brainless P+D species have retained the ChINs or at least a character identity gene. Thus, genes from species that have secondarily lost the character tripartite brain but have retained, for example, otd/Otx genes with an archetypical 'brain function' might be able to rescue, at least in part, brain phenotypes in Drosophila otd or Mus Otx2 mutants. These experiments are feasible and can be tested as homologs of character identity genes controlling tripartite brain development have been identified in brainless P+D species. It will be interesting to see whether Otx or Engrailed genes from brainless brachiopods or echinoderms like sea cucumber are able to substitute their Drosophila or murine homologs in the development and specification of a tripartite brain (Hirth, 2010).


Table of contents


engrailed: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of mutation | References

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