DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during Drosophila metamorphosis.

Pulses of the steroid hormone ecdysone activate genetic regulatory hierarchies that coordinate the developmental changes associated with Drosophila metamorphosis. A high-titer ecdysone pulse at the end of larval development triggers puparium formation and induces expression of the DHR3 orphan nuclear receptor. Here we use both a heat-inducible DHR3 rescue construct and clonal analysis to define DHR3 functions during metamorphosis. Clonal analysis reveals requirements for DHR3 in the development of adult bristles, wings, and cuticle, and no apparent function in eye or leg development. DHR3 mutants rescued to the third larval instar also reveal essential functions during the onset of metamorphosis, leading to lethality during prepupal and early pupal stages. The phenotypes associated with these lethal phases are consistent with the effects of DHR3 mutations on ecdysone-regulated gene expression. Although DHR3 has been shown to be sufficient for early gene repression at puparium formation, it is not necessary for this response, indicating that other negative regulators may contribute to this pathway. In contrast, DHR3 is required for maximal expression of the midprepupal regulatory genes, EcR, E74B, and betaFTZ-1. Reductions in EcR and betaFTZ-F1 expression, in turn, lead to submaximal early gene induction in response to the prepupal ecdysone pulse and corresponding defects in adult head eversion and salivary gland cell death. These studies demonstrate that DHR3 is an essential regulator of the betaFTZ-F1 midprepupal competence factor, providing a functional link between the late larval and prepupal responses to ecdysone. Induction of DHR3 in early prepupae ensures that responses to the prepupal ecdysone pulse will be distinct from responses to the late larval pulse and thus that the animal progresses in an appropriate manner through the early stages of metamorphosis.     

Size assessment and growth control: how adult size is determined in insects

Size control depends on both the regulation of growth rate and the control over when to stop growing. Studies of Drosophila melanogaster have shown that insulin and Target of Rapamycin (TOR) pathways play principal roles in controlling nutrition-dependent growth rates. A TOR-mediated nutrient sensor in the fat body detects nutrient availability, and regulates insulin signaling in peripheral tissues, which in turn controls larval growth rates. After larvae initiate metamorphosis, growth stops. For growth to stop at the correct time, larvae need to surpass a critical weight. Recently, it was found that the insulin-dependent growth of the prothoracic gland is involved in assessing when critical weight has been reached. Furthermore, mutations in DHR4, a repressor of ecdysone signaling, reduce critical weight and adult size. Thus, the mechanisms that control growth rates converge on those assessing size to ensure that the larvae attain the appropriate size at metamorphosis.     

The Drosophila NR4A nuclear receptor DHR38 regulates carbohydrate metabolism and glycogen storage

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.     

Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

Effects of temperature on survival, development, growth and feeding of larvae of Yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes)

To understand the physiological and ecological responses of marine fishes to the change of water temperature, newly-hatched larvae of Yellowtail clownfish Amphiprion clarkii were reared in captivity at water temperatures of 23, 26 and 29 °C till they completed the metamorphosis to juvenile phase, and larval survival, development, growth and feeding were evaluated during the experimental period. The results showed that water temperature influenced the physiological performance of larvae of A. clarkii significantly. The survival and growth rates of larvae of A. clarkii increased significantly with the increase of water temperature from 23 to 29 °C (P < 0.05). Water temperature also influenced larval development of A. clarkii significantly and larvae reared at 23 °C took longer time for post-larval development and metamorphosis compared to 26 and 29 °C (P < 0.05). Total length and body weight for post-larval development and metamorphosis decreased with the increase of water temperature from 23 to 29 °C (P < 0.05). Q₁₀ in developmental rate was higher than in daily growth rate at the same rearing temperature, indicating that at water temperature had greater influence on larval development than on growth. Water temperature also influenced larval feeding of A. clarkii significantly with feed ration (FR) and feed conversion efficiency (FCE) increased with the increase of water temperature from 23 to 29 °C (P < 0.05). There was a positive correlation between FR and specific growth rate (SGR) (P < 0.05) but not between FCE and SGR (P > 0.05), indicating that FR influenced growth rate significantly in larvae of A. clarkii. This study demonstrated that the physiological responses of larvae of A. clarkii to the change of water temperature and confirmed that water temperature influenced larval survival, development, growth and feeding significantly. This study suggests that the decline of larval survival and growth rates, extension of pelagic larval duration and reduction of larval feeding at lower temperature have ecological impacts on larval dispersal and metamorphosis, juvenile settlement and population replenishment in A. clarkii in the wild.     

Thyroid hormone-responsive genes mediate otolith growth and development during flatfish metamorphosis

Flatfish begin life as up-right swimming, bilaterally symmetrical larvae that metamorphose into asymmetrically shaped juveniles that swim with a highly lateralized posture. We have previously shown that TH induces abrupt growth and mineralization of one component of the vestibular system, the otoliths, during early larval development and metamorphosis. Here we report that four of five vestibular-specific genes that we tested (alpha-tectorin, otogelin, otolith matrix protein, and otopetrins 1 and 2 that are known to be associated with otolith development in other vertebrates are up-regulated 1.5- to 7-fold in larval flatfish during spontaneous metamorphosis and/or following 72 h of TH treatment. These findings suggest that otolith growth and development are mediated by diverse TH-responsive genes during flatfish metamorphosis.     

Models of metamorphic timing: an experimental evaluation with the pond-dwelling salamander Hemidactylium scutatum (Caudata: Plethodontidae)

Amphibian larvae vary tremendously in size at metamorphosis and length of larval period. We raised pond-dwelling four-toed salamander (Hemidactylium scutatum) larvae to test two models that predict a larva’s age and size at metamorphosis. The Wilbur-Collins model proposes that the developmental rate of a larva responds to changes in growth rate in an adaptive manner throughout the larval period, and that metamorphosis can be initiated after a minimum size has been reached. The Leips-Travis or fixed-rate model states that developmental rate is set early in the larval period, perhaps by early growth rate or food availability and their positive correlation with developmental rate, and that changes in growth rate during the larval period affect size at metamorphosis, but have no effect on the age of an individual at metamorphosis. A modified version of the Wilbur-Collins model suggests that a larva’s developmental rate becomes fixed about two-thirds of the way through the larval period, with changes in growth rate after that point only affecting size at metamorphosis. Larvae were raised on eight different feeding regimes which created two constant and six variable growth histories. Growth history did significantly affect size at metamorphosis. However, an a posteriori statistical test revealed a group of seven and an overlapping group of six treatments with indistinguishable lengths of larval period, indicating a general picture of a fixed developmental rate regardless of growth history. This result is unique among similar studies on invertebrates, fish, and frogs. There was no association between early growth or food level and development rates. Neither the Wilbur-Collins nor the Leips-Travis fixed-rate models were supported. The invariable developmental rate of Hemidactylium and recent osteological evidence from the literature suggest that larvae begin the process of metamorphosis as soon as they hatch, probably a trait selected for by strong predation pressure in the aquatic environment. A variety of different approaches (ecological, developmental, phylogenetic) are necessary to fully evaluate the adaptive nature of the timing of transitions between life cycle stages. Received: 3 June 1999 / Accepted: 18 March 2000     

Variation in growth and instar number in field and laboratory Manduca sexta

The tobacco hornworm Manduca sexta has been an important model system for understanding physiological control of growth, development and metamorphosis of insects for more than half a century. Like all Manduca, M. sexta typically has five larval instars, with developmental commitment to metamorphosis occurring early in the 5th (final) instar. Here we show that M. sexta from a field population in North Carolina (USA) shows substantial intraspecific variation in the number of larval instars when feeding on a modified artificial diet. Individuals with six instars consistently exhibited slower growth rates during early larval development than individuals with five instars. The frequency of individuals with six instars decreased with increased rearing temperature. In contrast, M. sexta from a laboratory colony consistently had five instars, and had more rapid larval growth rates than M. sexta from the field. We identify a threshold body size at the start of the 5th instar that predicts whether an individual will have five (greater than 600mg) or six instars (less than 600mg). Variation in field populations in Manduca provides an important resource for understanding physiological control, developmental plasticity and evolution of growth rate, body size and instar number.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

Effects of temperature on survival, development, growth and feeding of larvae of Yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes)

To understand the physiological and ecological responses of marine fishes to the change of water temperature, newly-hatched larvae of Yellowtail clownfish Amphiprion clarkii were reared in captivity at water temperatures of 23, 26 and 29 °C till they completed the metamorphosis to juvenile phase, and larval survival, development, growth and feeding were evaluated during the experimental period. The results showed that water temperature influenced the physiological performance of larvae of A. clarkii significantly. The survival and growth rates of larvae of A. clarkii increased significantly with the increase of water temperature from 23 to 29 °C (P < 0.05). Water temperature also influenced larval development of A. clarkii significantly and larvae reared at 23 °C took longer time for post-larval development and metamorphosis compared to 26 and 29 °C (P < 0.05). Total length and body weight for post-larval development and metamorphosis decreased with the increase of water temperature from 23 to 29 °C (P < 0.05). Q₁₀ in developmental rate was higher than in daily growth rate at the same rearing temperature, indicating that at water temperature had greater influence on larval development than on growth. Water temperature also influenced larval feeding of A. clarkii significantly with feed ration (FR) and feed conversion efficiency (FCE) increased with the increase of water temperature from 23 to 29 °C (P < 0.05). There was a positive correlation between FR and specific growth rate (SGR) (P < 0.05) but not between FCE and SGR (P > 0.05), indicating that FR influenced growth rate significantly in larvae of A. clarkii. This study demonstrated that the physiological responses of larvae of A. clarkii to the change of water temperature and confirmed that water temperature influenced larval survival, development, growth and feeding significantly. This study suggests that the decline of larval survival and growth rates, extension of pelagic larval duration and reduction of larval feeding at lower temperature have ecological impacts on larval dispersal and metamorphosis, juvenile settlement and population replenishment in A. clarkii in the wild.     

Chronic malnutrition favours smaller critical size for metamorphosis initiation in Drosophila melanogaster

Critical size at which metamorphosis is initiated represents an important checkpoint in insect development. Here, we use experimental evolution in Drosophila melanogaster to test the long-standing hypothesis that larval malnutrition should favour a smaller critical size. We report that six fly populations subject to 112 generations of laboratory natural selection on an extremely poor larval food evolved an 18% smaller critical size (compared to six unselected control populations). Thus, even though critical size is not plastic with respect to nutrition, smaller critical size can evolve as an adaptation to nutritional stress. We also demonstrate that this reduction in critical size (rather than differences in growth rate) mediates a trade-off in body weight that the selected populations experience on standard food, on which they show a 15-17% smaller adult body weight. This illustrates how developmental mechanisms that control life history may shape constraints and trade-offs in life history evolution.     

A conditional rescue system reveals essential functions for the ecdysone receptor (EcR) gene during molting and metamorphosis in Drosophila

In Drosophila, pulses of the steroid hormone ecdysone trigger larval molting and metamorphosis and coordinate aspects of embryonic development and adult reproduction. At each of these developmental stages, the ecdysone signal is thought to act through a heteromeric receptor composed of the EcR and USP nuclear receptor proteins. Mutations that inactivate all EcR protein isoforms (EcR-A, EcR-B1, and EcR-B2) are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heat shock promoter drives expression of an EcR cDNA, we have employed temperature-dependent rescue of EcR null mutants to determine EcR requirements at later stages of development. Our results show that EcR is required for hatching, at each larval molt, and for the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone-responsive genes is blocked and normal ecdysone responses of both imaginal and larval tissues are blocked at an early stage. These results show that EcR mediates ecdysone signaling at multiple developmental stages and implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis.     

Generating,growing and transforming skeletal shape: insights from amphibian pharyngeal arch cartilages

Amphibians that undergo a metamorphosis provide an unparalleled opportunity to investigate how skeletal shape is generated, preserved, and transformed in development. Their pharyngeal arch (PA) cartilages, which support breathing and feeding behaviors, form embryonically from cranial neural crest cells, grow isometrically at larval stages, and abruptly change shape during metamorphosis. Further, the shape changes occur in three different ways: some adult cartilages form de novo, others emerge from within resorbing larval cartilages and some larval cartilages reshape themselves at the cellular level. Isometric growth followed by abrupt shape change is unique to amphibian PA cartilages, which suggests that the origin and evolution of amphibian metamorphosis has been influenced by the tissue properties of cartilage. This essay reviews the functional role of the PA skeleton in frogs and salamanders and presents a mechanistic framework for understanding how its shape is generated, preserved, and transformed at the levels of cell behavior and specification mechanisms.     

Zebrafish puma mutant decouples pigment pattern and somatic metamorphosis

The genetic and developmental bases for trait expression and variation in adults are largely unknown. One system in which genes and cell behaviors underlying adult traits can be elucidated is the larval-to-adult transformation of zebrafish, Danio rerio. Metamorphosis in this and many other teleost fishes resembles amphibian metamorphosis, as a variety of larval traits (e.g., fins, skin, digestive tract, sensory systems) are remodeled in a coordinated manner to generate the adult form. Among these traits is the pigment pattern, which comprises several neural crest-derived pigment cell classes, including black melanophores, yellow xanthophores, and iridescent iridophores. D. rerio embryos and early larvae exhibit a relatively simple pattern of melanophore stripes, but this pattern is transformed during metamorphosis into the more complex pattern of the adult, consisting of alternating dark (melanophore, iridophore) and light (xanthophore, iridophore) horizontal stripes. While it is clear that some pigment cells differentiate de novo during pigment pattern metamorphosis, the extent to which larval and adult pigment patterns are developmentally independent has not been known. In this study, we show that a subset of embryonic/early larval melanophores persists into adult stages in wild-type fish; thus, larval and adult pigment patterns are not completely independent in this species. We also analyze puma mutant zebrafish, derived from a forward genetic screen to isolate mutations affecting postembryonic development. In puma mutants, a wild-type embryonic/early larval pigment pattern forms, but supernumerary early larval melanophores persist in ectopic locations through juvenile and adult stages. We then show that, although puma mutants undergo a somatic metamorphosis at the same time as wild-type fish, metamorphic melanophores that normally appear during these stages are absent. The puma mutation thus decouples metamorphosis of the pigment pattern from the metamorphosis of many other traits. Nevertheless, puma mutants ultimately recover large numbers of melanophores and exhibit extensive pattern regulation during juvenile development, when the wild-type pigment pattern already would be completed. Finally, we demonstrate that the puma mutant is both temperature-sensitive and growth-sensitive: extremely severe pigment pattern defects result at a high temperature, a high growth rate, or both; whereas a wild-type pigment pattern can be rescued at a low temperature and a low growth rate. Taken together, these results provide new insights into zebrafish pigment pattern metamorphosis and the capacity for pattern regulation when normal patterning mechanisms go awry.