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Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer Associates; 2000.

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Developmental Biology. 6th edition.

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Metamorphosis: The Hormonal Reactivation of Development

In most species of animals, embryonic development leads to a larval stage with characteristics very different from those of the adult organism. Very often, larval forms are specialized for some function, such as growth or dispersal. The pluteus larva of the sea urchin, for instance, can travel on ocean currents, whereas the adult urchin leads a sedentary existence. The caterpillar larvae of butterflies and moths are specialized for feeding, whereas their adult forms are specialized for flight and reproduction, often lacking the mouthparts necessary for eating. The division of functions between larva and adult is often remarkably distinct (Wald 1981). Cecropia moths, for example, hatch from eggs and develop as wingless juveniles (caterpillars) for several months. All this development enables them to spend a day or so as fully developed winged insects, mating quickly before they die. The adults never eat, and in fact have no mouthparts during this short reproductive phase of the life cycle. As might be expected, the juvenile and adult forms often live in different environments.

During metamorphosis, developmental processes are reactivated by specific hormones, and the entire organism changes to prepare itself for its new mode of existence. These changes are not solely ones of form. In amphibian tadpoles, metamorphosis causes the developmental maturation of liver enzymes, hemoglobin, and eye pigments, as well as the remodeling of the nervous, digestive, and reproductive systems. Thus, metamorphosis is often a time of dramatic developmental change affecting the entire organism.

Amphibian Metamorphosis

Morphological changes associated with metamorphosis

In amphibians, metamorphosis is generally associated with the changes that prepare an aquatic organism for a primarily terrestrial existence. In urodeles (salamanders), these changes include the resorption of the tail fin, the destruction of the external gills, and a change in skin structure. In anurans (frogs and toads), the metamorphic changes are more dramatic, and almost every organ is subject to modification (see Figure 2.4; Table 18.1). Regressive changes include the loss of the tadpole's horny teeth and internal gills, as well as the destruction of the tail. At the same time, constructive processes such as limb development and dermoid gland morphogenesis are also evident. The means of locomotion changes as the paddle tail recedes while the hindlimbs and forelimbs develop. The tadpole's cartilaginous skull is replaced by the predominantly bony skull of the frog. The horny teeth used for tearing pond plants disappear as the mouth and jaw take a new shape, and the tongue muscle develops. Meanwhile, the large intestine characteristic of herbivores shortens to suit the more carnivorous diet of the adult frog. The gills regress, and the gill arches degenerate. The lungs enlarge, and muscles and cartilage develop for pumping air in and out of the lungs. The sensory apparatus changes, too, as the lateral line system of the tadpole degenerates, and the eye and ear undergo further differentiation (see Fritzsch et al. 1988). The middle ear develops, as does the tympanic membrane characteristic of frog and toad outer ears. In the eye, both nictitating membranes and eyelids emerge.

Table 18.1. Summary of some metamorphic changes in anurans.

Table 18.1

Summary of some metamorphic changes in anurans.

When an animal changes its habitat and mode of nutrition, one would expect the nervous system to undergo dramatic changes, and it certainly does. One readily observed consequence of anuran metamorphosis is the movement of the eyes forward from their originally lateral position (Figure 18.1).* The lateral eyes of the tadpole are typical of preyed-upon herbivores, whereas the frontally located eyes of the frog befit its more predatory lifestyle. To catch its prey, the frog needs to see in three dimensions. That is, it has to acquire a binocular field of vision wherein input from both eyes converges in the brain (see Chapter 13). In the tadpole, the right eye innervates the left side of the brain, and vice versa. There are no ipsilateral (same-side) projections of the retinal neurons. During metamorphosis, however, these additional ipsilateral pathways emerge, enabling input from both eyes to reach the same area of the brain (Currie and Cowan 1974; Hoskins and Grobstein 1985a). In Xenopus, these new neuronal pathways result not from the remodeling of existing neurons, but from the formation of new neurons that differentiate in response to thyroid hormones (Hoskins and Grobstein 1985a,b). Some larval neurons, such as certain motor neurons in the tadpole jaw, switch their allegiances from larval muscle to the newly formed adult muscle (Alley and Barnes 1983). Still other neurons, such as those innervating the tongue (a newly formed muscle not present in the larva), have lain dormant during the tadpole stage and first form synapses during metamorphosis (Grobstein 1987). Thus, the anuran nervous system undergoes enormous restructuring during metamorphosis. Some neurons die, others are born, and others change their specificity.

Figure 18.1. Eye migration and associated neuronal changes during metamorphosis of the Xenopus laevis tadpole.

Figure 18.1

Eye migration and associated neuronal changes during metamorphosis of the Xenopus laevis tadpole. The eyes of the tadpole are laterally placed, so there is relatively little binocular field of vision. The eyes migrate dorsally and rostrally during metamorphosis, (more...)

Biochemical changes associated with metamorphosis

In addition to the obvious morphological changes, important biochemical transformations occur during metamorphosis. In tadpoles (as in freshwater fishes), the major retinal photopigment is porphyropsin. During metamorphosis, the pigment changes to rhodopsin, the characteristic photopigment of terrestrial and marine vertebrates (Wald 1945, 1981; Smith-Gill and Carver 1981; Hanken and Hall 1988). Tadpole hemoglobin is changed into an adult hemoglobin that binds oxygen more slowly and releases it more rapidly than does tadpole hemoglobin (McCutcheon 1936; Riggs 1951). The liver enzymes change also, reflecting the change in habitat. Tadpoles, like most freshwater fishes, are ammonotelic; that is, they excrete ammonia. Many adult frogs (such as the genus Rana, but not the more aquatic Xenopus) are ureotelic, excreting urea, like most terrestrial vertebrates, which requires less water than excreting ammonia. During metamorphosis, the liver begins to synthesize the urea cycle enzymes necessary to create urea from carbon dioxide and ammonia (Figure 18.2).

Figure 18.2. Development of the urea cycle during anuran metamorphosis.

Figure 18.2

Development of the urea cycle during anuran metamorphosis. (A) The major features of the urea cycle, by which nitrogenous wastes can be detoxified and excreted with minimal water loss. (B) Emergence of urea cycle enzyme activities correlated with metamorphic (more...)

Hormonal control of amphibian metamorphosis

The control of metamorphosis by thyroid hormones was demonstrated by Guder-natsch (1912), who discovered that tadpoles metamorphosed prematurely when fed powdered sheep thyroid gland. In a complementary study, Allen (1916) found that when he removed or destroyed the thyroid rudiment from early tadpoles (thus performing a thyroidectomy), the larvae never metamorphosed, instead becoming giant tadpoles.

The metamorphic changes of frog development are all brought about by the secretion of the hormones thyroxine (T4) and triiodothyronine (T3) from the thyroid during metamorphosis (Figure 18.3). It is thought that T3 is the more important hormone, as it will cause metamorphic changes in thyroidectomized tadpoles in much lower concentrations than will T4 (Kistler et al. 1977; Robinson et al. 1977).

Figure 18.3. Formulae of thyroxine (T4) and triiodothyronine (T3).

Figure 18.3

Formulae of thyroxine (T4) and triiodothyronine (T3).

Regionally specific changes

The various organs of the body respond differently to hormonal stimulation. The same stimulus causes some tissues to degenerate while causing others to develop and differentiate. For instance, tail degeneration is clearly associated with increasing levels of thyroid hormones. The degeneration of tail structures is relatively rapid, as the bony skeleton does not extend to the tail, which is supported only by the notochord (Wassersug 1989). The regression of the tail is brought about by apoptosis, and it occurs in four stages. First, protein synthesis decreases in the striated muscle cells of the tail (Little et al. 1973). Next, there is an increase in concentrations of digestive enzymes within the cells. Concentrations of lysosomal proteases, RNase, DNase, collagenase, phosphatase, and glycosidases all rise in the epidermis, notochord, and nerve cord cells (Fox 1973). Cell death is probably caused by the release of these enzymes into the cytoplasm. After cell death occurs, macrophages collect in the tail region, digesting the debris with their own proteolytic enzymes (Kaltenbach et al. 1979). The result is that the tail becomes a large sac of proteolytic enzymes (Figure 18.4). The major proteolytic enzymes involved appear to be collagenases and other metalloproteinases whose synthesis depends on thyroid hormones. If a metalloproteinase inhibitor (TIMP) is added to the tail, it prevents tail regression (Oofusa and Yoshizato 1991; Patterson et al. 1995).

Figure 18.4. Increase in lysosomal protease activity during tail regression in Xenopus laevis.

Figure 18.4

Increase in lysosomal protease activity during tail regression in Xenopus laevis. The lysosomal enzymes are thought to be responsible for digesting the tail cells. (After Karp and Berrill 1981.)

The response to thyroid hormones is specific to the region of the body. Tadpole head and body epidermis differentiate a new set of glands when exposed to T3. In the tail, however, T3 causes the death of the epidermal cells and a tail-specific suppression of stem cell divisions that could give rise to more epidermal cells. The result is the death of the tail epidermal cells, while the head and body epidermis continues to function (Nishikawa et al. 1989). These regional epidermal responses appear to be controlled by the regional specificity of the dermal mesoderm. If tail dermatome cells (mesodermal cells that generate the tail dermis) are transplanted into the trunk, the epidermis they contact will degenerate upon metamorphosis. Conversely, when trunk dermatome is transplanted into the tail, those regions of skin persist. Changing the ectoderm does not alter the regional response to thyroid hormones (Kinoshita et al. 1989).

Organ-specific response to thyroid hormones is dramatically demonstrated by transplanting a tail tip to the trunk region or by placing an eye cup in the tail (Schwind 1933; Geigy 1941). The tail tip placed in the trunk is not protected from degeneration, but the eye retains its integrity despite the fact that it lies within the degenerating tail (Figure 18.5). Thus, the degeneration of the tail represents an organ-specific programmed cell death. Only specific tissues die when a signal is given. Such programmed cell deaths are important in molding the body. The degeneration of the human tail during week 4 of development resembles the regression of the tadpole tail (Fallon and Simandl 1978).

Figure 18.5. Regional specificity during frog metamorphosis.

Figure 18.5

Regional specificity during frog metamorphosis. Tail tips regress even when transplanted to the trunk. Eye cups, however, remain intact even when transplanted into the regressing tail. (After Schwind 1933.)

Coordination of developmental changes

One of the major problems of metamorphosis is the coordination of developmental events. For instance, the tail should not degenerate until some other means of locomotion—the limbs—has developed, and the gills should not regress until the animal can utilize its newly developed lung muscles. The means of coordinating metamorphic events appears to be a difference among tissues and organs in their responsiveness to different amounts of hormone (Saxén et al. 1957; Kollros 1961). This model is called the threshold concept. As the concentration of thyroid hormones gradually builds up, different events occur at different concentrations of the hormones. If tadpoles are deprived of their thyroids and are placed in a dilute solution of thyroid hormones, the only morphological effects are the shortening of the intestines and accelerated hindlimb growth. However, at higher concentrations of thyroid hormones, tail regression is seen before the hindlimbs are formed. These experiments suggest that as thyroid hormone levels gradually rise, the hindlimbs develop first and then the tail regresses. Similarly, when T3 is given to tadpoles, it induces the earliest-forming bones at the lowest dosages and the last bones at higher dosages, mimicking the natural situation (Hanken and Hall 1988). Thus, the timing of metamorphosis appears to be regulated by the sensitivity of different tissues to thyroid hormones.

To ensure that this timing system works, two of the organs most sensitive to thyroxine are the thyroid itself and the pituitary gland, which regulates thyroid hormone production. Thyroid hormones initially create positive feedback to the pituitary gland, causing the anterior pituitary to induce the thyroid to produce more T3 and T4 (Saxén et al. 1957; White and Nicoll 1981). Later, as an effect of metamorphosis, the thyroid partially degenerates, and inhibitors of thyroid hormone functions are made (Goos 1978).


Amphibian metamorphosis and frog calls. For photographs of amphibian metamorphosis (and for the sounds of the adult frogs), check out the metamorphosis and frog call sections of the CD-ROM. [Click on Amphibian]

Molecular responses to thyroid hormones during metamorphosis

Thyroid hormones appear to work largely at the level of transcription, activating the transcription of some genes and repressing the transcription of others (Lyman and White 1987; Mathison and Miller 1987). The transcription of the genes for albumin, carbamoylphosphate synthase, adult globin, adult skin keratin, and the Xenopus homologue of sonic hedgehog is activated by thyroid hormones. The transcription of the sonic hedgehog gene in the intestine is particularly interesting, since it suggests that the regional patterning of the organs formed during metamorphosis might be generated by the reappearance of some of the same molecules that structured the embryo (Stolow and Shi 1995; Stolow et al. 1997).

But these are relatively late responses to thyroid hormones. The earliest response to T3 is the transcriptional activation of the thyroid hormone receptor (TR) genes (Yaoita and Brown 1990; Kawahara et al. 1991). Thyroid hormone receptors are members of the steroid hormone receptor superfamily of transcription factors. There are two major types of T3 receptors, TRα and TRβ. Interestingly, the mRNAs and proteins of both TRs are present at relatively low levels in the premetamorphosis tadpole and then increase before thyroid hormone is released or metamorphosis begins (Table 18.2; Kawahara et al. 1991; Baker and Tata 1992). The thyroid hormone receptors may bind to their specific sites on the chromatin even before thyroid hormones are present, and they are thought to repress gene transcription. When T3 or T4 enters the cell and binds to the chromatin-bound receptors, the hormone-receptor complex is converted from a repressor to a strong transcriptional activator (Wolffe and Shi 1999). At this time, the synthesis of TRs accelerates dramatically, coinciding with the onset of metamorphosis.

Table 18.2. Relative accumulation of TRα and TRβ mRNA in Xenopus tadpoles following treatment with T3 and prolactin.

Table 18.2

Relative accumulation of TRα and TRβ mRNA in Xenopus tadpoles following treatment with T3 and prolactin.

The injection of exogenous T3 causes a twofold to fivefold increase in TRα message and a 20- to 50-fold increase in the mRNA for TRβ. Thus, T3 binds to its TR and transcribes the TR gene. This “autoinduction” of T3 receptor message by T3 may play a significant role in the acceleration of metamorphosis (Figure 18.7). The more T3 receptors a tissue has, the more competent it should be to respond to small amounts of T3. Thus, metamorphic climax, that time when the visible changes of metamorphosis occur rapidly, may be brought about by the enhanced production and induction of more T3 receptors. The TR does not work alone, however, but forms a dimer with the retinoid receptor, RXR. This dimer binds thyroid hormones and can enter the nucleus to effect transcription (Wong and Shi 1995; Wolffe and Shi 1999).

Figure 18.7. Hypothetical model for the acceleration of metamorphosis in Xenopus by the autoinduction of T3 receptors by T3.

Figure 18.7

Hypothetical model for the acceleration of metamorphosis in Xenopus by the autoinduction of T3 receptors by T3. (A) The premetamorphosis tadpole is characterized by low levels of thyrotropin (thyroid hormone-releasing factor), thyroid hormones, and T (more...)

The hormone prolactin has been found to inhibit the up-regulation of TRα and TRβ mRNAs. Moreover, if the up-regulation of the TR is experimentally blocked by prolactin, the tail is not resorbed, and the adult-specific keratin gene is not activated (Tata et al. 1991; Baker and Tata 1992). Injections of prolactin stimulate larval growth and inhibit metamorphosis (Bern et al. 1967; Etkin and Gona 1967), but there is dispute as to whether this finding reflects the natural role of prolactin (Takahashi et al. 1990; Buckbinder and Brown 1993). We still do not know the mechanisms by which levels of thyroid hormone are regulated in the tadpole, nor do we know how the reception of thyroid hormone elicits different responses (proliferation, differentiation, cell death) in different tissues.

Metamorphosis in Insects

Types of insect metamorphosis

Whereas amphibian metamorphosis is characterized by the remodeling of existing tissues, insect metamorphosis often involves the destruction of larval tissues and their replacement by an entirely different population of cells. Insects grow by molting—shedding their cuticle—and growing new cuticle as their size increases. There are three major patterns of insect development. A few insects, such as springtails and mayflies, have no larval stage and undergo direct development. These are called the ametabolous insects (Figure 18.11A). These insects have a pronymph stage immediately after hatching, bearing the structures that have enabled it to get out of the egg. But after this transitory stage, the insect begins to look like a small adult; after each molt, they are bigger, but unchanged in form (Truman and Riddiford 1999). Other insects, notably grasshoppers and bugs, undergo a gradual, hemimetabolous metamorphosis (Figure 18.11B). After spending a very brief period of time as a pronymph (whose cuticle is often shed as the insect hatches), the insect looks like an immature adult. This immature stage is called a nymph. The rudiments of the wings, genital organs, and other adult structures are present, and these structures become more mature with each molt. At the last molt, the emerging insect is a winged and sexually mature adult.

Figure 18.11. Modes of insect development.

Figure 18.11

Modes of insect development. Molts are represented as arrows. (A) Ametabolous (direct) development in a silverfish. After a brief pronymph stage, the insect looks like a small adult. (B) Hemimetabolous (gradual) metamorphosis in a cockroach. After a very (more...)

In the holometabolous insects (Figure 18.11C: flies, beetles, moths, and butterflies), there is no pronymph stage. The juvenile form that hatches from the egg is called a larva. The larva (caterpillar, grub, maggot) undergoes a series of molts as it becomes larger. The stages between these larval molts are called instars. The number of molts before becoming an adult is characteristic for the species, although environmental factors can increase or decrease the number. The instar stages grow in a stepwise fashion, each being qualitatively larger than the previous one. Finally, there is a dramatic and sudden transformation between the larval and adult stages. After the last instar stage, the larva undergoes a metamorphic molt to become a pupa. The pupa does not feed, and its energy must come from those foods it ingested while a larva. During pupation, the adult structures are formed and replace the larval structures. Eventually, an imaginal molt enables the adult (“imago”) to shed the pupal case and emerge. While the larva is said to hatch from an egg, adults are said to eclose from the pupa.

Eversion and differentiation of the imaginal discs

In holometabolous insects, the transformation from juvenile into adult occurs within the pupal cuticle. Most of the old body of the larva is systematically destroyed by apoptosis, while new adult organs develop from undifferentiated nests of cells, the imaginal discs . Thus, within any larva, there are two distinct populations of cells: the larval cells, which are used for the functions of the juvenile insect, and the thousands of imaginal cells, which lie within the larva in clusters, awaiting the signal to differentiate.

In Drosophila, there are ten major pairs of imaginal discs, which construct many of the adult organs, and an unpaired genital disc, which forms the reproductive structures (Figure 18.12). The abdominal epidermis forms from a small group of imaginal cells called histoblasts, which lie in the region of the larval gut. Other nests of histoblasts located throughout the larva form the internal organs of the adult. The imaginal discs can be seen in the newly hatched larva as local thickenings of the epidermis. Whereas most of the larval cells have a very limited mitotic capacity, the imaginal discs divide rapidly at specific characteristic times. As the cells proliferate, they form a tubular epithelium that folds in upon itself in a compact spiral (Figure 18.13A). The largest disc, that of the wing, contains some 60,000 cells, whereas the leg and haltere discs contain around 10,000 (Fristrom 1972). At metamorphosis, these cells proliferate, differentiate, and elongate (Figure 18.13B).

Figure 18.12. The locations and developmental fates of the imaginal discs in Drosophila melanogaster.

Figure 18.12

The locations and developmental fates of the imaginal discs in Drosophila melanogaster. (After Fristrom et al. 1969.)

Figure 18.13. Imaginal disc elongation.

Figure 18.13

Imaginal disc elongation. Scanning electron micrograph of Drosophila last-instar leg disc (A) before and (B) after elongation. (From Fristrom et al. 1977; photograph courtesy of D. Fristrom.)


Development of a holometabolous insect. To watch the stages of fruit fly development, see the QuickTime movies of the growing larva. This segment also shows methods for “peeling” a pupa. Photographs of each of the imaginal discs are also shown, along with instructions for dissecting out these discs. [Click on Fruit Fly]

The fate map and elongation sequence of the leg disc are shown in Figure 18.14. At the end of the third instar, just before pupation, the leg disc is an epithelial sac connected by a thin stalk to the larval epidermis. On one side of the sac, the epithelium is coiled into a series of concentric folds “reminiscent of a Danish pastry” (Kalm et al. 1995). As pupation begins, the cells at the center of the disc telescope out to become the most distal portions of the leg—the claws and the tarsus. The outer cells become the proximal structures—the coxa and the adjoining epidermis (Schubiger 1968). After differentiating, the cells of the appendages and epidermis secrete a cuticle appropriate for the specific region. Although the disc is composed primarily of epidermal cells, a small number of adepithelial cells migrate into the disc early in development. During the pupal period, these cells give rise to the muscles and nerves that serve that structure.

Figure 18.14. Elongation sequence of Drosophila leg disc.

Figure 18.14

Elongation sequence of Drosophila leg disc. (A) Surface view of uneverted disc. (B, C) Longitudinal section through (B) elongating and (C) fully everted leg disc. T1, basitarsus; T2–5, tarsal segments 2–5. (D) Adult leg. (From Fristrom (more...)

Studies by Condic and her colleagues (1990) have demonstrated that the elongation of imaginal discs is due primarily to cell shape change within the disc epithelium. Using fluorescently labeled phalloidin to stain the peripheral microfilaments of leg disc cells, they showed that the cells of early third-instar discs are tightly compressed along the proximal-distal axis. This compression is maintained through several rounds of cell division. Then, when the tissue begins elongating, the compression is removed, and the cells “spring” into their rounder state. This conversion of an epithelium of compressed cells into a longer epithelium of noncompressed cells represents a novel mechanism for the extension of an organ during development.

The type of leg structure generated is determined by the interactions between several genes in the imaginal disc. Figure 18.15 shows the expression of three genes involved in determining the proximal-distal axis of the fly leg. In the third-instar leg disc, the center of the disc secretes the highest concentration of two morphogens, Wingless (Wg) and Decapentaplegic (Dpp). High concentrations of these paracrine factors cause the expression of the Distal-less gene. Moderate concentrations cause the expression of the dachshund gene, and lower concentrations cause the expression of the homothorax gene. Those cells expressing Distal-less telescope out to become the most distal structures of the leg—the claw and distal tarsal segments. Those expressing homothorax become the most proximal structure, the coxa. Cells expressing dachshund become the femur and proximal tibia. Areas of overlap produce the trochanter and distal tibia (Abu-Shaar and Mann 1998). These regions of gene expression are stabilized by inhibitory interactions between the protein products of these genes and of the neighboring genes. In this manner, the gradient of Wg and Dpp proteins is converted into discrete domains of gene expression that specify the different regions of the Drosophila leg.

Figure 18.15. The fates of the imaginal disc cells are directed by transcription factors found in different regions of the imaginal disc.

Figure 18.15

The fates of the imaginal disc cells are directed by transcription factors found in different regions of the imaginal disc. At the periphery, the homothorax gene (purple) establishes the boundary for the coxa. The expression of the dachshund gene (green) (more...)

Hormonal control of insect metamorphosis

Although the detailed mechanisms of insect metamorphosis differ among species, the general pattern of hormone action is very similar. Like amphibian metamorphosis, the metamorphosis of insects appears to be regulated by effector hormones, which are controlled by neurohormones in the brain (for reviews, see Gilbert and Goodman 1981; Riddiford 1996). Insect molting and metamorphosis are controlled by two effector hormones: the steroid 20-hydroxyecdysone and the lipid juvenile hormone (JH) (Figure 18.21). 20-hydroxyecdysone initiates and coordinates each molt and regulates the changes in gene expression that occur during metamorphosis. Juvenile hormone prevents the ecdysone-induced changes in gene expression that are necessary for metamorphosis. Thus, its presence during a molt ensures that the result of that molt produces another instar, not a pupa or an adult.

Figure 18.21. Regulation of insect metamorphosis.

Figure 18.21

Regulation of insect metamorphosis. (A) Structures of juvenile hormone, ecdysone, and the active molting hormone 20-hydroxyecdysone. (B) General pathway of insect metamorphosis. Ecdysone and juvenile hormone together cause molts to keep the status quo (more...)

The molting process is initiated in the brain, where neurosecretory cells release prothoracicotropic hormone (PTTH) in response to neural, hormonal, or environmental signals. PTTH is a peptide hormone with a molecular weight of approximately 40,000, and it stimulates the production of ecdysone by the prothoracic gland. This ecdysone is modified in peripheral tissues to become the active molting hormone 20-hydroxyecdysone. Each molt is initiated by one or more pulses of 20-hydroxyecdysone. For a larval molt, the first pulse produces a small rise in the hydroxyecdysone concentration in the larval hemolymph (blood) and elicits a change in cellular commitment. A second, large pulse of hydroxyecdysone initiates the differentiation events associated with molting. The hydroxyecdysone produced by these pulses commits and stimulates the epidermal cells to synthesize enzymes that digest and recycle the components of the cuticle.

Juvenile hormone is secreted by the corpora allata. The secretory cells of the corpora allata are active during larval molts but inactive during the metamorphic molt. As long as JH is present, the hydroxyecdysone-stimulated molts result in a new larval instar. In the last larval instar, however, the medial nerve from the brain to the corpora allata inhibits the gland from producing JH, and there is a simultaneous increase in the body's ability to degrade existing JH (Safranek and Williams 1989). Both these mechanisms cause JH levels to drop below a critical threshold value. This triggers the release of PTTH from the brain (Nijhout and Williams 1974; Rountree and Bollenbacher 1986). PTTH, in turn, stimulates the prothoracic glands to secrete a small amount of ecdysone. The resulting hydroxyecdysone, in the absence of high levels of JH, commits the cells to pupal development. Larva-specific mRNAs are not replaced, and new mRNAs are synthesized whose protein products inhibit the transcription of the larval messages. After the second ecdysone pulse, new pupa-specific gene products are synthesized (Riddiford 1982), and the subsequent molt shifts the organism from larva to pupa. It appears, then, that the first ecdysone pulse during the last larval instar triggers the processes that inactivate the larva-specific genes and prepare the pupa-specific genes to be transcribed. The second ecdysone pulse transcribes the pupa-specific genes and initiates the molt (Nijhout 1994). At the imaginal molt, when ecdysone acts in the absence of juvenile hormone, the imaginal discs differentiate, and the molt gives rise to the adult.


18.3 Insect metamorphosis.Four websites discuss (1) the experiments of Wigglesworth and others who identified the hormones of metamorphosis and the glands producing them; (2) the variations that Drosophila and other insects play on the general theme of metamorphosis; (3) the remodeling of the insect nervous system during metamorphosis, and (4) a microarray analysis of Drosophila metamorphosis wherein several thousand genes were simultaneously screened.

The molecular biology of hydroxyecdysone activity

Ecdysone receptors

20-hydroxyecdysone cannot bind to DNA by itself. Like amphibian thyroid hormones, 20-hydroxyecdysone first binds to receptors. These receptors are almost identical in structure to the thyroid hormone receptors. The receptors specifically binding 20-hydroxyecdysone are called the ecdysone receptors (EcR). An EcR protein forms an active molecule by pairing with an Ultraspiracle (Usp) protein, the homologue of the amphibian RXR that helps form the thyroid hormone receptor (Koelle et al. 1991; Yao et al. 1992; Thomas et al. 1993). Although there is only one type of gene for Usp in Drosophila, and only one type of gene for EcR, the EcR gene transcript can be spliced in at least three different ways to form three distinct proteins. All three EcR proteins have the same domains for 20-hydroxyecdysone and DNA binding but they differ in their N-terminal domains (Figure 18.22). The type of EcR in a cell may inform the cell how to act when it receives a hormonal signal (Talbot et al. 1993; Truman et al. 1994). All cells appear to have some of each type, but the strictly larval tissues and neurons that die when exposed to 20-hydroxyecdysone are characterized by their great abundance of the EcR-B1 form of the ecdysone receptor. Imaginal discs and differentiating neurons, on the other hand, show a preponderance of the EcR-A isoform. It is therefore possible that the different receptors activate different sets of genes when they bind 20-hydroxyecdysone.

Figure 18.22. Formation of the ecdysone receptors.

Figure 18.22

Formation of the ecdysone receptors. Alternative mRNA splicing of the ecdysone receptor (EcR) transcript creates three types of EcR mRNAs. These generate proteins having the same DNA-binding site (blue) and hydroxyecdysone-binding site (red), but with (more...)

Binding of 20-hydroxyecdysone to DNA

During molting and metamorphosis, certain regions of the polytene chromosomes of Drosophila puff out in the cells of certain organs at certain times (see Figure 4.13; Clever 1966; Ashburner 1972; Ashburner and Berondes 1978). These chromosome puffs represent areas where DNA is being actively transcribed. Moreover, these organ-specific patterns of chromosome puffing can be reproduced by culturing larval tissue and adding hormones to the medium or by adding hydroxyecdysone to an earlier-stage larva. When 20-hydroxyecdysone is added to larval salivary glands, certain puffs are produced and others regress (Figure 18.23). The puffing is mediated by the binding of hydroxyecdysone at specific places on the chromosomes; fluorescent antibodies against hydroxyecdysone find this hormone localized to the regions of the genome that are sensitive to it (Gronemeyer and Pongs 1980).

Figure 18.23. Hydroxyecdysone-induced puffs in cultured salivary gland cells of D.

Figure 18.23

Hydroxyecdysone-induced puffs in cultured salivary gland cells of D. melanogaster. The chromosome region here is the same as in Figure 4.13. (A) Uninduced control. (B-E) Hydroxyecdysone-stimulated chromosomes at (B) 25 minutes, (C) 1 hour, (D) 2 hours, (more...)

Hydroxyecdysone-regulated chromosome puffs occurring during the late stages of the third-instar larva (as it prepares to form the pupa) can be divided into three categories: “early” puffs that hydroxyecdysone causes to regress; “early” puffs that hydroxyecdysone induces rapidly; and “late” puffs that are first seen several hours after hydroxyecdysone stimulation. For example, in the larval salivary gland, about six puffs emerge within a few minutes of hydroxyecdysone treatment. No new protein has to be made in order for these puffs to be induced. A much larger set of genes are induced later in development, and these genes do need protein synthesis to become transcribed. Ashburner (1974, 1990) hypothesized that the “early” genes make a protein product that is essential for the activation of the “late” genes and that, moreover, this protein itself turns off the transcription of the early genes (Figure 18.24). These insights have been confirmed by molecular analyses. The three early puffs include the genes for EcR and two other transcription factors, BR-C and E74B.

Figure 18.24. The Ashburner model of hydroxyecdysone regulation of transcription.

Figure 18.24

The Ashburner model of hydroxyecdysone regulation of transcription. Hydroxyecdysone binds to its receptor, and this compound binds to an early puff gene and a late puff gene. The early puff gene is activated, and its protein product (1) represses the (more...)

The broad-complex (BR-C) gene is particularly interesting. Like the ecdysone receptor gene, the BR-C gene can generate several different transcription factor proteins through differentially initiated and spliced messages. It appears that the variants of the ecdysone receptor may signal particular variants of the BR-C protein to be synthesized. Organs such as the larval salivary gland that are destined for death during metamorphosis express the Z1 isoform; imaginal discs destined for cell differentiation express the Z2 isoform; and the central nervous system (which undergoes marked remodeling during metamorphosis) expresses all isoforms, with Z3 predominating (Emery et al. 1994; Crossgrove et al. 1996).

In addition to the restricted activities of the different isoforms of BR-C, there appear to be common processes that all of the isoforms accomplish. Restifo and Wilson (1998) provided evidence that these common functions are prevented by juvenile hormone. Deletions of the BR-C gene lead to faulty muscle development, retention of larval structures that would normally degenerate, abnormal nervous system morphology, and eventually the death of the larva (Restifo and White 1991). This syndrome is very similar to that induced by adding excess JH to Cecropia silkworm larvae (Riddiford 1972) or by adding juvenile hormone analogues to Drosophila. Thus, it appears that juvenile hormone prevents ecdysone-inducible gene expression by interfering with the BR-C proteins.

The BR-C proteins are themselves transcription factors, and their targets remain to be identified. However, we are beginning to get a glimpse at the molecular level of one of the most basic areas of all developmental biology—the transformation of a larva into a fly, butterfly, or moth.


18.4 Precocenes and synthetic JH. Given the voracity of insect larvae, it's amazing that any plant exists. However, many plants get revenge on their predators by making compounds that alter their metamorphoses and prevent the animals from developing or reproducing.

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Raising Tadpoles.

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Determination of the Wing Imaginal Discs.



One of the most spectacular movements of the eyes during metamorphosis occurs in flatfish such as flounder. Originally, the eyes are on opposite sides of the face. However, during metamorphosis, one of the eyes migrates across the head to meet the other eye on the same side of the fish. This allows the fish to dwell on the bottom, looking upward.

Since its discovery in 1954, when Butenandt and Karlson isolated 25 mg of ecdysone from 500 kg of silkworm moth pupae, 20-hydroxyecdysone has gone under several names, including β-ecdysone, ecdysterone, and crustecdysone.

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Copyright © 2000, Sinauer Associates.
Bookshelf ID: NBK9986


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