End Of Metamorphosis Research Articles

Remodeling of the intestine during metamorphosis of Xenopus laevis

Thyroid hormone controls remodeling of the tadpole intestine during the climax of amphibian metamorphosis. In 8 days, the Xenopus laevis tadpole intestine shortens in length by 75%. Simultaneously, the longitudinal muscle fibers contract by about the same extent. The radial muscle fibers also shorten as the diameter narrows. Many radial fibers undergo programmed cell death. We conclude that muscle remodeling and contraction play key roles in the shortening process. Shortening is accompanied by a temporary "heaping" of the epithelial cells into many layers at climax. Cells that face the lumen undergo apoptosis. By the end of metamorphosis, when the epithelium is folded into crypts and villi, the epithelium is a single-cell layer once again. Throughout this remodeling, DNA replication occurs uniformly throughout the epithelium, as do changes in gene expression. The larval epithelial cells as a whole, rather than a subpopulation of stem cells, are the progenitors of the adult epithelial cells.

Rearing of larvae of giant freshwater prawn, Macrobrachium rosenbergii up to postlarvae using different feeds

Ability to produce seed (postlarvae) of giant freshwater prawn, Macrobrachium rosenbergii, for stocking in various culture facilities was a breakthrough in aquaculture in Sri Lanka. However, high cost of Anemia cysts and their occasional scarcity are the major concerns as live Artemia nauplii are considered to be the only suitable larval food for M. rosenbergii. During the present study, a feed for postlarvae of M. rosenbergii was prepared and its performance was evaluated in relation to some other feeds with a view to reduce the use of live Artemia nauplii in the rearing of larvae of this species. No larvae survived beyond the 6th larval stage when they were fed 6 meal's day' with egg custard, egg and mussel meat custard, spray dried algal cells ofSchizochytrium sp., a commercially available liquid shrimp feed and the prepared feed separately, each as single feed. The ingredients were 20% powdered milk, 6.8% corn flour, 40% eggs, 30% wet shrimp, 1.0% cod liver oil, 1.0% vitamin mineral mixture, 1.0% agar powder and 0.2% tetracycline. The best feeding regime was 6 meals of Artemia nauplii per day followed by the feeding regime of 3 meals of Anemia nauplii and 3 meals of prepared feed per day when percentage survival (10.35% and 9.28% respectively), mean total length achieved at the end of metamorphosis (9.078 ± 2.828 mm and 7.896 ± 2.812 mm respectively) and time taken to complete metamorphosis by 50% of larvae (40.42 ± 1.38 days and 48.42 ± 2.23 days respectively) were considered, when compared to the two other feeding regimes tested, viz,Artemia nauplii plus spray dried algal cells and Artemia nauplii plus liquid shrimp feed. Therefore, the feeding regime of 3 meals of Artemia nauplii and 3 meals of feed prepared during this study could be recommended in order to reduce the heavy use of costly Artemia nauplii in rearing the larvae of giant freshwater prawn, M. rosenbergii.

Development and morphology of class II Kenyon cells in the mushroom bodies of the honey bee, Apis mellifera.

Class II Kenyon cells, defined by their early birthdate and unique dendritic arborizations, have been observed in the mushroom bodies of evolutionarily divergent insects. In the fruit fly Drosophila melanogaster, Class II (also called clawed) Kenyon cells are well known for their extensive reorganization that occurs during metamorphosis. The present account reports for the first time the occurrence of mushroom body reorganization during metamorphosis in holometabolous insect species outside of the Diptera. In the honey bee, Apis mellifera, Class II Kenyon cells show signs of degeneration and undergo a subtle reshaping of their axons during metamorphosis. Unlike in Drosophila, reorganization of Class II Kenyon cells in the honey bee does not involve the loss of axon branches. In contrast, the mushroom bodies of closely related hymenopteran species, the polistine wasps, undergo a much more dramatic restructuring near the end of metamorphosis. Immunohistochemistry, dextran fills, and Golgi impregnations illuminate the heterogeneous nature of Class II Kenyon cells in the developing and adult honey bee brain, with subpopulations differing in the location of dendritic arbors within the calyx, and branching pattern in the lobes. Furthermore, polyclonal antibodies against the catalytic subunit of Drosophila protein kinase A (anti-DC0) label an unusual and previously undescribed trajectory for these neurons. The observed variations in morphology indicate that subpopulations of Class II Kenyon cells in the honey bee can likely be further defined by significant differences in their specific connections and functions within the mushroom bodies.

The epithalamus of the developing and adult frog: calretinin expression and habenular asymmetry in Rana esculenta

Expression of the calcium binding protein (CaBP) calretinin (CR) was studied with immunohistochemistry in the pineal complex and habenular nuclei (HN) of the developing and adult frog Rana esculenta. The frog pineal complex is a medial structure formed by two interconnected components, the frontal organ and the pineal organ or epiphysis; the habenular nuclei are bilateral and are asymmetric due to subdivision of the left dorsal nucleus into medial and lateral components. In the pineal complex, calretinin immunostaining of cells and fibers was consistently observed in developing and adult frogs. In the habenulae, calretinin immunoreactivity exhibited instead marked variations during development, and was expressed only in cells of the medial subnucleus of the left dorsal habenula. In particular, calretinin was detected at larval stages, peaked during metamorphosis, was markedly downregulated at the end of metamorphosis, and was evident again in adulthood. This sequence of calretinin expression was confirmed by quantitative analysis of immunoreactive cells in the left habenula. In tadpoles, calretinin-positive cells exhibited a dorsoventral gradient of density, while in adulthood, they were distributed throughout the dorsoventral extent of the medial subnucleus. The study demonstrates a peculiar developmental pattern, with transient downregulation, of asymmetric calretinin expression in the frog epithalamus. The findings indicate that calcium and calcium buffering systems may play critical roles in neurogenetic and neuronal migration processes implicated in the formation of the asymmetric habenular portion in amphibians. In addition, the reappearance of calretinin expression in the adult frog supports a distinct functional role of the asymmetric habenular component in amphibians.

Calorimetrical and biochemical investigations on the influence of the parasitic mite Varroa destructor on the development of honeybee brood

The parasitic mite Varroa destructor is a serious pest to the western honeybee Apis mellifera. Here, we investigated the impact of mite infestation on the energy content, energy density, hemolymph volume and hemolymph protein concentration of drone and worker pupae. Mite infestation had a significant impact on the energy content of worker pupae at the end of metamorphosis, with an energy content of highly infested pupae 15% lower than non-parasitized pupae. The energy content of infested and uninfested drone pupae did not differ significantly. The energy density of drones and workers was not affected by mite infestation. Drones had a higher energy density compared to workers and lost more energy during metamorphosis. The heat production rates of drone pupae did not change due to mite parasitation. The total hemolymph volume of worker bees was significantly reduced in infested pupae, whereas the mites did not influence the hemolymph protein concentration. Although drones generally were less affected through mite infestation than workers, our results reveal a clear energetic impact of mite parasitation on honeybee pupae.

Appearance of Dendritic Cells in the Basal Layer of Corneal Epithelium during Metamorphosis in Rana temporaria L.

A population of dendritic cells was found at the light optic level in the basal layer of the corneal epithelium inRana temporaria L. The cells had wide short processes which penetrated between neighboring epithelial cells. The cell bases lied on the basal membrane while the bodies are slightly elongated perpendicularly to the epithelium surface. The appearance of dendritic cells coincided in time with the beginning of metamorphosis of the tadpoles and their number increased by the end of metamorphosis. The dendritic cells we described were similar in their morphology to the Langerhans cells.

Early temperature effects on muscle growth dynamics and histochemical profile of muscle fibres of sea bass Dicentrarchus labrax L., during larval and juvenile stages

Recently, it has been found that the thermal experience during the earliest phases of development could determine the larval and postlarval growth characteristics of teleosts. In order to investigate the effects of the early temperature regime on the advanced stages of growth of sea bass, Dicentrarchus labrax L., this species was reared during the vitelline phase at two temperatures: natural temperature (i15 jC) and 17.7F0.1 jC, and then larvae transferred to common temperature (natural temperature). Muscle growth was studied by morphometric and histochemical techniques (mATPase and NADH-TR). Body length and body mass were also measured. During the vitelline phase, muscle growth was similar in both experimental groups, but at 25 days, both hypertrophy and hyperplasia of white muscle fibres were greater in the prewarmed group (p<0.05). At the end of metamorphosis (80 days) and at 120 days, the average diameter of white muscle fibres, as well as the body length, were greater in the prewarmed group (p<0.05), but the number of white fibres did not differ significantly between groups. The morphological mosaic of white muscle fibres was observed at the end of metamorphosis, and the histochemical mosaic appeared gradually since the early postlarval stages. Thus, at 120 days, some specimens in both experimental groups showed three or four different mATPase staining white fibres: low (L), moderate (M), high (H) and/or very high (vH), whereas in other specimens, only L or M mATPase activity fibres were observed. Early T influenced the histochemical maturity of the

Functional genomics and sexual differentiation in amphibians

In Xenopus laevis the basic mechanisms underlying sexual differentiation were investigated by determining time courses of sexual steroids and their corresponding receptors during complete larval development from egg to juveniles. Androgens as well as estradiol (E2) are derived from maternal origin and accumulate in hatching tadpoles. Sexual steroid contents decreased rapidly after hatching and rose again at the end of metamorphosis indicating endogenous production. In parallel the mRNA expression for corresponding androgen (AR) and estrogen receptors (ER) was measured by means of semiquantitative RT-PCR. Both receptor mRNAs increased dramatically just after hatching and decreased only moderately until end of metamorphosis. In female juveniles E2 and ER-mRNA levels were higher compared with males. Treatment by exogenous E2 elevated both, ER- and AR-mRNA, indicating stimulatory functions of E2 for gene expression of both receptors. Effects on sexual differentiation during larval development were achieved by treatment with E2 and the antiandrogen cyproterone acetate both causing feminization, the antiestrogen tamoxifen resulting in neutralization, and the androgens, methyltestosterone and dihydrotestosterone, but not testosterone, leading to masculinization. The data presented are in accordance with further recent findings and suggest a new hypothesis for functional genomics in sexual differentiation of amphibians.

Control of Dopa decarboxylase gene expression by the Broad-Complex during metamorphosis in Drosophila

The induction of the Dopa decarboxylase gene ( Ddc) in the epidermis of Drosophila at pupariation is a receptor-mediated response to the steroid molting hormone, ecdysone. Activity is also dependent on the Broad-Complex ( BR-C), an early ecdysone response gene that functions during metamorphosis. BR-C encodes a family of zinc-finger protein isoforms, BR-C Z1–Z4. Genetic experiments have shown that the Z2 isoform is required for epidermal Ddc to reach maximum expression at pupariation. In this paper, we report that BR-C regulates Ddc expression at two different developmental stages through two different cis-acting regions. At pupariation, BR-C acts synergistically with the ecdysone receptor to up-regulate Ddc. DNase I foot printing has identified four binding sites of the predominant Z2 isoform within a distal regulatory element that is required for maximal Ddc activity. The sites share a conserved core sequence with a set of BR-C sites that had been mapped previously to within the first Ddc intron. Using variously deleted Ddc genomic regions to drive reporter gene expression in transgenic organisms, we show that the intronic binding sites are required for Ddc expression at eclosion. At both pupariation and eclosion, BR-C releases Ddc from an active silencing mechanism, operating through two distinct cis-acting regions of the Ddc genomic domain at these stages. Transgenes, bearing a Ddc fragment from which one of the cis-acting silencers has been deleted, exhibit β-galactosidase reporter activity in the epidermal cells prior to the appearance of endogenous DDC. Our finding that BR-C is required for Ddc activation at eclosion is the first evidence to suggest that this important regulator of the early metamorphic events, also regulates target gene expression at the end of metamorphosis.

Changes in contractile properties of skeletal muscle during developmentally programmed atrophy and death.

Skeletal muscle atrophy and death are protracted processes that accompany aging and pathological insults in mammals. The intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta are composed of giant fibers that undergo distinct hormonally-regulated programs of atrophy and death at the end of metamorphosis. Atrophy occurs during the 3 days preceding adult emergence and results in a 40% reduction of mass, whereas death takes place during the subsequent 30 h and results in the complete loss of the fibers. There are no significant changes in tetanic force or calcium sensitivity in skinned fiber preparations during atrophy. However, the size of caffeine-induced contractions fell by about 50%. With the onset of the death phase, dramatic reductions occur in ISM: tetanic force, twitch amplitude, resting potential, caffeine-induced contractions, calcium sensitivity, and Hill coefficients. Several lines of evidence suggest that ISM atrophy is caused by an increase in protein turnover without significant modification of fiber organization. In contrast, ISM death is accompanied by disorganization of the contractile apparatus and concomitant loss of contractile function.

Temperature effects on muscle growth in two populations (Atlantic and Mediterranean) of sea bass, Dicentrarchus labrax L.

Two genetically different populations of sea bass, Dicentrarchus labrax L., Atlantic (Atl.) and Mediterranean (Med.), were subjected to the following incubation/cultivation temperatures ( T): 15 °C/natural, 17 °C/natural, 15/19 °C, 17/19 °C (natural T averaging 15 °C and raising gradually). Muscle cellularity was measured at different larval stages for each T regime. During the vitelline phase, muscle growth was mainly due to muscle fibre hypertrophy. In Med. larvae, higher incubation T (17 °C) increased the area of white and red fibres at hatching, while in Atl. larvae there was no significant T effect at this stage. At mouth opening, the area of white fibres increased at 19 °C in Atl. larvae ( P<0.05), but in Med. larvae it was similar for all temperatures. Following yolk-sac reabsorption, hypertrophy and hyperplasia increased in both populations. In these stages (20–55 days), both parameters were greater at 19 °C ( P<0.05). Metamorphosis finished earlier at 19 °C. At this stage, Atl. larvae reared at 19 °C showed higher value of total myotomal area than at natural T, while in Med. sea bass, larvae reared at 19 °C showed a lower size of the myotome than at 17 °C/natural. In larvae from both populations reared at natural T, incubating T had a positive effect at the end of metamorphosis, thus the total myotomal area were higher at 17 °C/natural than at 15 °C/natural. Following metamorphosis, all groups showed a rapid growth, but higher at 19 °C ( P<0.05). The results indicate that muscle cellularity was clearly influenced by T, and that both populations had different levels of response. These differences can be apparently associated with their respective genetic background.

Morphological differentiation of taste organs in the ontogeny of Salamandra salamandra.

Morphological studies presented here provide additional cytological evidence that in the postnatal development of Salamandra salamandra there are two successive generations of taste organs: premetamorphic taste buds (TBs) in larval forms and taste disks (TDs) in postmetamorphic animals. The TBs have been found in the epithelium of the whole oropharyngeal cavity of larval forms, while in adults TDs appear only at the end of metamorphosis. The TDs can be papillary (or fungiform) on the soft (secondary) tongue and non-papillary outside the tongue. Two main cyto-morphological criteria distinguishing TDs from TBs have been established: (1) high differentiation of "nonsensory" components of a taste organ into several kinds of cells (often named "associate cells")--at least mucous cells and, separating them, wing cells; (2) a considerably larger area of the sensory epithelium than that in TBs, as the consequence of the large size of the mucous cells. In contrast to TDs each TB consists of longitudinally elongated supporting cells and taste cells, as well as of horizontally oriented basal cells, adjacent to the basement membrane. The sensory area in TBs measures 10-12 microm in diameter, while that in TDs has diameter of 45-90 microm. The anlage of the secondary tongue appears as a small folding of the floor epithelium just in front to the tip of the primary tongue in larvae 3 cm long, and is definitely formed in an animal with body length of about 6 cm.

Bifurcation Patterns and Branching Exponents in Branchial Vessels in Tadpoles of Xenopus laevis Daudin during Metamorphosis-3D-Measurements from Microvascular Corrosion Casts

IntroductionFluid transport systems of organisms [1] in general and the blood vascular system in particular are considered to be optimally designed. From Murray's laws [2] it is concluded that an arterial bifurcation where the diameter of the parent vessel (d0) relates to the larger (d1) and the smaller daughter vessel (d2) according to d03 = d13 + d23 is optimal. Interestingly, existing data predominantly refer to arterial branchings of the fully developed circulatory system of mammals. to the best of our knowledge there are no data available on arterial bifurcations and venous mergings in an initially growing but then regressing tubular system of blood vessels as it is found in the gill filter apparatus of the anuran tadpole where the highly complex three-dimensional vascular network totally disappears at the end of metamorphosis.

Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation

Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20-30% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.

Gill morphogenesis in the oyster Ostrea chilensis

Despite the importance of the gills in the acquisition of food by suspension-feeding bivalve mollusks, there is almost no information on gill organogenesis. By means of a series of stereoscan electron micrographs, this paper describes gill development in the Chilean oyster, Ostrea chilensis, from the brooded larval stages to 1-month-old spat. A single gill rudiment was observed on each side of the mantle at a shell length of 320 μm, and the rudiments increased in number and size until the end of the brooding period. During metamorphosis the gill filaments increased in number from 5 or 6 to between 7 and 9. The loss of the velum and the absence of functional gill filaments during metamorphosis are consistent with previous observations of weight loss during this critical period of the life history, because the newly settled juvenile lacks the ability to remove particles from suspension. The end of metamorphosis (100% of spat with dissoconch edge) was reached 36 h after larval settlement, when the gill filaments began to grow cilia, which increased in density and differentiated as the spat developed and acquired the capability of suspension-feeding, accounting for the increase in body weight previously recorded during this stage. The larval rudiments gave rise to the inner demibranchs. The outer demibranchs were observed 10 days after settlement, located between the inner demibranch and the mantle. In 1-month-old spat, the gill did not show differentiation between primary and secondary filaments, indicating that the heterorhabdic condition characteristic of adult oysters had yet to be attained.

Temperature effect on muscle growth of the axial musculature of the sea bass (Dicentrarchus labrax L.).

In order to determine the temperature effect on the axial muscle growth of sea bass, a stock of larvae was subjected to the following incubation and cultivation temperatures, respectively: 15 degrees C/ambient, 15/17 degrees C, 17 degrees C/ambient and 17/17 degrees C. In all groups the cross-sectional area of white and red muscles and the number and average area of the white and red muscle fibres were quantified. Results showed that the embryonic period, pre-larval phase and the end of metamorphosis were accelerated at higher temperatures. During the endogenous feeding period, muscle growth took place by fibrillar hypertrophy, and was not influenced by the temperature. Thereafter (external feeding) muscular hyperplasia began, and growth of all the muscular parameters was favoured by the effect of high incubation and cultivation temperatures, with the latter having higher influence. High incubation temperature had an slight effect on muscle growth and body length, which was only observed from 15 days. Metamorphosis finished at 3 +/- 0.4 cm in all the larvae, but this length was earlier reached at higher temperatures. At 120 days, the largest growth was obtained in the larvae maintained at a higher temperature.

Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile.

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL-R) gene expression has made the physiological importance of the PRL/PRL-R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL-R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL-R expressed in COS-7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL-R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL-R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.

Steroid regulation of octopamine expression during metamorphic development of the moth Manduca sexta.

Octopamine (OA), a biogenic amine similar to norepinephrine, has profound and well-documented actions on the nervous systems of invertebrates. In the insect, Manduca sexta, we examined the developmental plasticity of OA synthesis, studied its endocrine regulation, and observed previously undescribed OA-immunoreactive (ir) neurons. We found that levels of tyramine beta-hydroxylase (TbetaH), an essential enzyme for the biosynthesis of OA, increase during metamorphosis. Based on the established and influential roles of the steroid hormone 20-hydroxyecdysone (20-HE) during development, we tested the hypothesis that increases in TbetaH levels and OA immunoreactivity are regulated by the rise in 20-HE occurring during pupal-adult development. We determined that the levels of TbetaH in the terminal abdominal ganglion (neuromeres 6-9) remain at a constant level during pupal development and the early stages of adult development. Beginning at ca. pupal stage 8, however, the levels of TbetaH begin to rise, reaching a maximum level by pupal stage 12. By removing the source of ecdysteroid hormone through ligation, and by subsequent replacement of 20-HE via infusion, we found evidence indicating that the preadult rise of 20-HE is both necessary and sufficient for the increased levels of TbetaH. During the course of our study, we also identified previously unreported OA-ir neurons. In particular, adult-specific OA-ir lateral cells were found, as were relatively small OA-ir dorsal median pairs that doubled in size during adult development. Abdominal ganglia not exposed to the preadult rise in 20-HE possessed neither the OA-ir lateral neurons nor the somatic growth of the smaller OA-ir median neurons. These newly described OA-ir neurons probably contribute to the steroid-induced elevations of TbetaH observed at the end of metamorphosis.

Novel Structural Elements Identified during Tail Resorption in Xenopus laevis Metamorphosis: Lessons from Tailed Frogs

When the tail of the Xenopus laevis tadpole resorbs at the end of metamorphosis, various cell types, including muscle, fibroblasts, skin, and spinal cord, are lost at about the same time. However, feeding frogs with tails can be produced by inhibiting thyroid hormone production at the climax of metamorphosis with the goitrogen methimazole. These tails lose their fast muscle preferentially, showing that the different cell types of the tail have different fates and confirming that more than one cell death program is involved in tail resorption. Both normal and methimazole tails contain “cords,” novel structures that consist of two dorsal and two ventral parallel rows of slow muscle bundles joined by collagen fibers that run the length of the tail. The cords persist until the very end of tail resorption, being the last structure to dissolve. When thyroid hormone induces expression of proteolytic enzymes in the notochord sheath, the notochord, a structural rod that runs the length of the tail, begins to buckle, demonstrating that the tail is under tension. When sections of the tail that contain cords are surgically separated from the notochord, they contract in vitro, suggesting that the cords contribute to the tension that augments tail resorption.

Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation.

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C(2)C(12) myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved "switches" to block muscle differentiation and induce death.