Morphological Changes In Crustaceans And Insects

Thus far we have disctissed how changes in pattern determining genes alter morphology in fruit Hies. We now discuss how the three strategies for altering the activities of pattern determining genes can explain examples of natural morphological diversity found among different arthropods. The first two mechanisms, changes in the expression and function of pattern determining genes, can account for changes in limb morphology seen in certain crustaceans and insects. The third mechanism, changes in regulatory sequences, might provide an explanation for the different patterns of wing development in fruit flies and butterflies.

Arthropods Are Remarkably Diverse

Arthropods embrace live groups: trilobites (sadly extinct], hexapods (such as insects}, crustaceans (shrimp, lobsters, crabs, and so on), myr-iapods (centipedes and millipedes), and chelicerates (horseshoe crabs, spiders, and scorpions). The success of the arthropods derives, in part, from their modular architecture. These organisms are composed of a series of repeating body segments that can be modified in seemingly limitless ways. Some segments carry wings, whereas others have antennae, legs, jaws, or specialized mating devices. We know more about the evolutionary processes responsible for the diversification of arthropods than for ¿my other group of animals.

Changes in Ubx Expression Explain Modifications in Limbs among the Crustaceans

Crustaceans include most, but not all, of the arthropods that swim. Some live in the ocean, while others prefer fresh water. They include some of our favorite culinary dishes, such as shrimp, crab, and lobster. One of fhe most popular groups of crustaceans for study is Arlonun, also known as "sea monkeys." Their embryos arrest as tough spores that can be purchased at toy stores. The spores quickly resume development upon addition of .salt water.

The heads of these shrimp contain feeding appendages. The thoracic segment nearest the head, Tl. contains swimming appendages that look like those further back on the thorax (the second through eleventh thoracic segments, T2—Til). Artemia belongs to an order of crustaceans known as branchiopods. Consider a different order of crustaceans, called isopods, Isopods contain swimming limbs on the second through eighth thoracic segments, just like the branchiopods, Hut, the limbs on the first thoracic segment of isopods have been modified, They are smaller than the others and function as feeding limbs (Figure 19-13). These modified limbs are called maxillipeds {otherwise known as jaw feet), and look like appendages found on the head (though these are not shown in the figure).

Slightly different patterns of Ubx expression are observed in branchiopods and isopods. These different expression patterns are correlated with the modification of the swimming limbs on the first thoracic segment of isopods. Perhaps the last shared ancestor of the present branchiopods and isopods contain the arrangement of thoracic limbs seen in Artemia (which is itself a branchiopod): all thoracic segments contain swimming limbs. During the divergence of branchiopods and isopods, the Ubx regulatory sequences changed in isopods, As a result of this change, Ubx expression was eliminated in the first thoracic segment, and restricted to segments T2-T8, It is easy to imagine that Ubx represses one or more "head" patterning genes in the thorax. In Artemia, these head genes are kept off in all 11 thoracic segments, but in isopods the head genes can be expressed in the Tl segment due to the loss of the Ubx repressor. Indeed, expression of the Scr gene is restricted to head regions of branchiopods, but is expressed in T1 of isopods. The expression of Scr in Tl causes maxillipeds to develop in place of normal swimming limbs (see Figure 19-13).

What is the basis for the different patterns of Ubx expression in isopods and branchiopods? There are several possible explanations, but the most likely one is that the Ubx regulatory DNA of isopods acquired mutations. By this model, the Ubx enhancer no longer mediates expression in the first thoracic segment. In fact, there is a tight correlation between the absence of Ubx expression in tire thorax and the development of feedirtg appendages in different crustaceans. For example, lobster embryos lack Ubx expression in the first two thoracic segments and contain two pairs of maxillipeds. Cleaner shrimp lack Ubx expression in the First three thoracic segments rind contain three pairs of maxillipeds.

Why Insects Lack Abdominal Limbs

All insects have six legs, two on each of the three thoracic segments; this applies to every one of the more than one million species of insects. In contrast, other arthropods, such as crustaceans, have a variable number of limbs. Some crustaceans have limbs on every segment in both the thorax and abdomen. This evolutionary change in morphology, the Joss of limbs on the abdomen of insects, is not due to altered expression of pattern determining genes, as seen in the case of maxil-liped formation in isopods. Rather, the Iosh of abdominal limbs in insects is due to functional changes in the Ubx regulatory protein.

In insects, Ubx and abd-A repress the expression of a critical gene that is required for the development of limbs, called Distalless (OH). in developing DrosophiJa embryos, Ubx is expressed at high levels in the metathorax and anterior abdominal segments; abd-A expression extends into more posterior abdominal segments. Together, Ubx and abd-A keep Dl! off in the first seven abdominal segments. Although Ubx is expressed in the metathorax, it does not interfere with the expression of DI] in that segment, because Ubx is not expressed in the de-

FIGURE 19-13 Changing morphologies in two different groups of crustaceans.

in branchiopods Scr expression is restneted to bead regions where it helps promote the devel opment of feeding appendages, while Ubx is expressed in the thorax where it controls the development of swimming limbs. In isopods, Scr expression is detected in both the head and Ifie first thorauc segment (It), and as a result, the swimming limb in 11 is transformed into a feeding appendage (the maxilliped} This posterior expansion of Scr was made possible by the loss of Ubx expression in Tl since Ubx normally represses Scr expression (Source Adaptent from Levine M. 2002. Nature 4 lb: 848-849, fig 2, p 848. Copyright © 2002 Nature Publishing Croup Used with permission.)

head segments thorax branchiopod

head segments thorax branchiopod

632 Comparative Genomics and the Evolution of Animal Diversity veloping T3 legs until after the time when DlS is activated. As a result, Ubx does not interfere with limb development in T3.

In crustaceans, such as the bmnchiopod Artemia already mentioned, there are high levels of both Ubx and DIl in all 11 thoracic segments (Figure 19-14). The expression of Dll promotes the develop-merit of swimming limbs. Why doos Ubx repress Dll expression in the alidorniii.il segments of insects, but not crustaceans? The answer is that the Ubx protein has diverged between insects and crustaceans. This was demonstrated in the following experiment.

The misexpression of Ubx throughout all of the tissues of the presumptive thorax in transgenic Drosophilo embryos suppresses limb development due to the repression of DIL In contrast, the misexpression of the crustacean Ubx protein in transgenic flies does not interfere with Oil gene expression and the formation of thoracic limbs. These observations indicate that the Drosophilo Ubx protein is functionally distinct from Ubx in crustaceans. The fly protein represses Dll gene expression, whereas the crustacean Ubx protein does not,

What is the basis for this Functional difference between the two Ubx proteins? (They share only 32% overall amino acid identity, but their homeodornains are virtually identical — 59/60 matches.) It turns out that the crustacean protein has a short motif containing 29 amino acid residues that block repression activity. When this sequence is deleted, the crustacean Ubx protein is just as effective as the fly protein at repressing DH gene expression [Figure 19-15).

Both the crustacean and fly Ubx proteins contain multiple repression domains. As discussed in Chapter 17, it is likely that these domains interact with one or more transcriptional repression complexes. The "antirepression" peptide present in the crustacean Ubx protein might interfere with the ability of the repression domains to recruit these complexes, When this peptide is attached to the fly protein, the hybrid protein behaves like the crustacean Ubx protein and no longer represses Dll (see Box 19-5, Co-option of Gene Networks for Evolutionary Innovation).

Modification of Flight Limbs Might Arise from the Evolution of Regulatory DNA Sequences

Ubx has dominated our discussion of morphological change in arthropods. Changes in the Ubx expression pattern appear to be responsible for the transformation of swimming limbs into a b c a b c

FIGURE 19-14 Evolutionary changes in Ubx protein function, (a) The Dll enhancer (DIHG 4) is normally activated in three parti ot "spots'1 in Drosophilo embryos These spots go on to form the three pari? of legs in the adult fly (b) The misexpression of the Drosophila Ubx protein (DmUbxHA) strongly suppresses expression from the Dll enhancer, (c) In contrast, the misexpression of the Ubx protein from the bnneshtimp Artemia (AfUbxHA) causes only a slight suppression of the Dll enhancer. (Source: Adapted from Rcnshaugen M et at 2002. Hex protein mutation and macroevolution of the insect body plan Nature 4t5: 914-917, fig 2, part c, p. 915 Copyright © 2002 Nature Publishing Croup Used with permission Images courtesy of William McGinnis and Matt Ronshaugen )

FIGURE 19-14 Evolutionary changes in Ubx protein function, (a) The Dll enhancer (DIHG 4) is normally activated in three parti ot "spots'1 in Drosophilo embryos These spots go on to form the three pari? of legs in the adult fly (b) The misexpression of the Drosophila Ubx protein (DmUbxHA) strongly suppresses expression from the Dll enhancer, (c) In contrast, the misexpression of the Ubx protein from the bnneshtimp Artemia (AfUbxHA) causes only a slight suppression of the Dll enhancer. (Source: Adapted from Rcnshaugen M et at 2002. Hex protein mutation and macroevolution of the insect body plan Nature 4t5: 914-917, fig 2, part c, p. 915 Copyright © 2002 Nature Publishing Croup Used with permission Images courtesy of William McGinnis and Matt Ronshaugen )

maxillipeds in crustaceans, Moreover, the loss of the antirepression motif in the Ubx protein likely accounts for the suppression of abdominal limbs in insects. In this final section on that theme, we review evidence that changes in the regulatory sequences in Ubx target genes might explain the different wing morphologies found in fruit flies and butterflies.

In Drosophila, Ubx is expressed in the developing halteres where it functions as a repressor of wing development. Approximately five to ten target genes are repressed by Ubx. These gones encode proteins that are crucial for the growth and patterning of the wings (Figure 19-1G) and all are expressed in the developing wing. In Ubx mutants, these genes are no longer repressed in the halteres, and as a result, the halteres develop into a second set of wings.

Fruit flies are dipterans, and all of the members of this order contain a single pair of wings and a set of halteres. Tt is likely that Ubx functions as a repressor of wing development in all dipterans. Butterflies belong to a different order of insects, the lepidopterans. All of the members of this order (which also includes moths) contain two pairs of wings rather than a single pair of wings and a set of halteres. What is the basis for these different wing morphologies in dipterans and lepidopterans?

The two orders diverged from a common ancestor more than 250 million years ago. This is about the time of divergence that separates humans and nonmamalian vertebrates such as frogs. It would seem to be a sufficient period of time to alter Ubx gene function through any or all of the three strategies that we have discussed. The simplest mechanism would be to change the Ubx expression pattern so that it is lost in the progenitors of the hindwings in lepidoptera. Such a loss would permit the developing hindwings to express all of the genes that are normally repressed by Ubx. The transformation of swimming limbs into maxillipeds in isopods provides a clear precedent for such a mechanism. However, there is no obvious change in the Ubx expression pattern in flies and butterflies; Ubx is expressed at high levels throughout the developing hindwings of butterflies-

That leaves us with two possibilities. First, the Ubx protein is functionally distinct in flies and butterflies. The second is that each of the approximately five to ten target genes that are repressed by Ubx in Drosophila have evolved changes in their regulatory DNAs so that crustacean insect crustacean insect

FIGURE 19-15 Comparison of Ubx in crustaceans and in insects, (a) ubx in crustaceans. The C-termtnal antirecession peptide blocks the activity of the N-terminal repression domain (b) Ubx in insects. The C-terminal antirepressior peptide was lost throught mutatier* (Source: Adapted from Ronshaugen M. el at. 2002 Ho* protein mtrtation and man revolution of the insect body plan Nature 415:914-917, fig 4, part b, p. 916 Copyright © 2002 Mature Publishing Group. Used with perrnissforf.il c

FIGURE 19-15 Comparison of Ubx in crustaceans and in insects, (a) ubx in crustaceans. The C-termtnal antirecession peptide blocks the activity of the N-terminal repression domain (b) Ubx in insects. The C-terminal antirepressior peptide was lost throught mutatier* (Source: Adapted from Ronshaugen M. el at. 2002 Ho* protein mtrtation and man revolution of the insect body plan Nature 415:914-917, fig 4, part b, p. 916 Copyright © 2002 Mature Publishing Group. Used with perrnissforf.il

Box 19-5 Co-option of Gene Networks for Evolutionary Innovation

The regulatory gene Distal-less ([Dll) has been implicated in the development of most or all animal (imbs, including the antennae and legs of Drosophila, the swimming limbs and maxillipeds of crustaceans, the fins of fish, and the limbs of mice (Box 19-5 Figure 1). In all of these esses, Dll is required for the extension of limbs away from the body. The extensive conservation of Distal-leas expression in virtually all animals has led to the proposal that the ancestral animals, perhaps the pre-Cambnan flattish round worm, contained small protuberances or "placodes" with sites of Dll expression- These rudimentary placodes in the ancestor might have led to the evolution of limbs in the higher animals.

Dll is not dedicated to the elongation of animal limbs since it is also expressed in other types of tissues. One interesting example is seen in the wings of butterflies. Many consider the eyespots of butterfly wings to be among the most beautiful patterns encountered in nature, tt is thought that these eye-spots are used as decoys that allow butterflies to evade predators. Dll is expressed in the progenitors of the eyespots, called foci (Box 19-5 Figure 2) It is difficult to argue that the eyespot is a degenerate limb. Rather, it would appear that Dll regulates a distinct set of target genes in the foci to help control the pigmentation pattern of the eyespot. Presumably, Dll regulates a different set of target genes in the developing limbs of butterflies, just as it does in other animals The distinct use of Dll in the eyespots represents an example of "co-option" A pre-existing regulatory gene is redeployed for a new purpose.

6M4 Comparative Genomics and the Evolution of Animal Diversity Box 19-5 (Continued)

6M4 Comparative Genomics and the Evolution of Animal Diversity Box 19-5 (Continued)

BOX 19-5 FIGURE 1 Distalless expression in various animal embryos. The embryos shown a/e stained with Dll antibody- Top row: arthropod (fruit fly in left pane) and butterfly in center panel) and crustacean (right panel). Bottom row from the left: echinoderm (sea urchin), annelid, and vertebrate (chicken and zebralish). (Source: Photos provided courtesy of Steve Paddock and Sean Carroll.)

BOX 19-5 FIGURE 2 The expression of DU and other pattern determining genes in the eyespot of B. anynana. Dll (red) is expressed in the eyespot1: of the developing butterfly wir>gs. (Source: Courtesy of Craig Brunetti and Sean CarroB. Brünett! et al. 2001. Current Biology 11:1578, fig 2, parte b and d.)

they are no longer repressed by LTbx in butterflies (see Figure 19-16), An individual predisposed to gambling would lay odds on the former mechanism: a change in Ubx protein function. It seems easier to modify repression activity than to change the regulatory sequences of five to ten different IJbx target genes. We have seen that this type of mech-

dipterans dipterans b wg

FIGURE 19-16 Changes in the regulatory DNA of Ubx target genes.

(a) The Ubx repressor ts expressed in the halteres of dipterans and hindwings of lepidopterans (orange), (b) Different target genes contain Ubx repressor sites in dipterans. These have been lost in lepidopterans.

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