Postmetamorphic Development Research Articles

White muscle differentiation in the eel (Anguilla anguilla L.): changes in the myosin isoforms pattern and ATPase profile during post-metamorphic development

Abstract Myosin isoforms and their light and heavy chains subunits were studied in the white lateral muscle of the eel during the post metamorphic development, in relation with the myosin ATPase profile. At elver stage VI Al the myosin isoforms pattern was characterized by at least two isoforms, FM3 and FM2. The fast isomyosin type 1 (FM1) appeared during subsequent development. It increased progressively in correlation with the increase in the level of the light chain LC3f. FM1 became predominant at stage VI A4. At the elver stage VI Al, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed at least two heavy chains, namely type II-l and II-2. The type II-l heavy chain disappeared in the yellow eel white muscle, and V8-protease peptide map showed the appearance of a minor heavy chain type II-3 as early as stage VI B. Comparison of myosin heavy chains and myosin isoforms patterns showed the comigration of different myosin isoforms during white muscle development. The myosin ATPase profile was characterized by a uniform pattern as far as stage VI A4. A mosaic aspect in white muscle was observed as early as stage VI B, showing the appearance of small acid labile fibers. This observation suggests that the type II-3 heavy chain is specific to the small fibers.

Development of functional sex differences in the larynx of Xenopus laevis

Three laryngeal properties associated with the production of masculine song--laryngeal muscle tension, fiber twitch type, and fiber recruitment--are markedly sexually dimorphic in adult Xenopus laevis frogs. To elucidate the pattern of sexual differentiation, tension and fiber recruitment in male and female larynges and fiber twitch type in male larynges were examined throughout postmetamorphic development. Masculinization of male laryngeal properties begins early in postmetamorphic development and continues until adulthood. In contrast, tension and fiber recruitment in females do not change after the end of metamorphosis. Laryngeal muscle tension and fiber type are gradually and progressively masculinized; the temporal pattern of masculinization is very similar for these properties. Fiber recruitment, on the other hand, appears to masculinize in a stepwise manner. Masculinization of all three properties is highly correlated with larynx weight in males. We have used this relation to divide postmetamorphic development into seven stages associated with key events in sexual differentiation. This staging scheme provides an important experimental tool for studying the hormonal regulation of sexual differentiation, the subject of the accompanying paper.

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.

Post-metamorphic cooling history of the Indian Plate crystalline thrust stack, Pakistan Himalaya

A post-metamorphic Himalayan cooling history for the crystalline internal zones of the northern Indian plate is defined by amphibole and mica K-Ar and Ar-Ar geochronology. Hornblende, muscovite and biotite cooling ages from cover sequences metamorphosed during the Himalayan orogeny are 35–40, 30 to 23, and 29 to 23 Ma respectively. The mica ages, together with those derived from zircon and apatite fission track data demonstrate a cooling rate of about 30°C/Ma during the late Oligocene to early Miocene. This rapid cooling was initiated during the post-metamorphic development of the Indian Plate south-verging crustal-scale thrust stack. Most of the cooling occurred during the stripping of some 10 ± 2 km of overburden through a combination of erosion, recorded in the Miocene molasse sediments of the foreland basin, and major crustal extension within upper levels of the Indian Plate and the Main Mantle thrust zone. Both erosion and extension were the direct consequence of the evolution of the thrust stack.

Androgen regulation of muscle fiber type in the sexually dimorphic larynx of Xenopus laevis

We used histochemical techniques [assays for adenine triphosphatase (ATPase) and succinate dehydrogenase (SDHase) activity] to identify muscle fiber types in the larynx of Xenopus laevis. Male muscle is made up of one fiber type, medium-sized fibers (approximately 9 microns2) that stain lightly for acid-stable ATPase and intensely for SDHase activity. In contrast, the female has 3 fiber types: small fibers (approximately 6 microns2) that stain intensely for ATPase and SDHase, medium-sized fibers (approximately 13 microns2) with moderate staining for ATPase and dark staining for SDHase, and large fibers (approximately 15 microns2) with little SDHase or ATPase activity. Long-term castration (6 months) has no effect on histochemical staining of adult male fibers. Long-term testosterone treatment (5 months) increases the proportion of medium-sized, moderately staining fibers in adult females, and reduces the proportion of both the small, darkly staining fibers and the large, lightly staining fibers. At metamorphosis, both males and females have 3 fiber types whose ATPase activity is similar to that of the adult female. However, no SDHase activity is observed. Treatment of juveniles for 3 weeks with testosterone results in nearly complete masculinization of muscle fibers, as judged by increased cross-sectional area, homogeneous ATPase staining, and a marked increase in SDHase activity. Thus, juvenile muscle is considerably more responsive to testosterone than is adult female muscle. We propose that the uniform metabolic properties of male laryngeal muscle contribute to the production of the rapid (66 Hz) mate call vocalizations characteristic of this species. Further, our results suggest that androgens direct the masculinization of laryngeal muscle fibers during postmetamorphic development.

The sexually dimorphic larynx of Xenopus laevis: development and androgen regulation.

The aims of this study were to characterize sexual dimorphism in the larynx of adult Xenopus laevis and to determine how sex differences arise during postmetamorphic development. The larger male larynx is a result of greater cell numbers in both cartilage and muscle. The dilator laryngis muscle of the male larynx has 6-7 times more muscle fibers than that of the female. At metamorphosis, the larynx is sexually monomorphic and feminine in phenotype. The DNA content of the male larynx doubles during the first 6 months following metamorphosis; there is no net DNA increase in the female larynx during this time. Both sexes experience a marked increase in laryngeal DNA content and mass between 6 months and adulthood. The number of muscle fibers in the male larynx increases at an average rate of 150 fibers a day during the first 10 months of postmetamorphic development. There is no net change in fiber numbers in the female larynx from metamorphosis to adulthood. Administration of the antiandrogen Flutamide to metamorphic frogs prevents the net addition of laryngeal muscle fibers in males. Thus, we propose that addition of postmetamorphic laryngeal muscle fibers in males is dependent upon the presence of circulating androgens. Exogenous testosterone administration results in an increase in laryngeal mass, DNA content, and cellular proliferation in juvenile frogs. Using [3H]thymidine injections to probe ongoing, as well as testosterone-induced, cell proliferation, we conclude that cellular proliferation is regulated differently in males and females during development. Thus androgen-induced proliferation is one cellular mechanism responsible for the sexual dimorphism observed in adults.

Changes in antibody and complement production in Xenopus laevis during postmetamorphic development revealed in a primary in vivo or in vitro antibody response

Xenopus laevis (G-line) mounts a primary plaque forming cell (PFC) response either in vivo or in vitro following challenge with foreign erythrocytes. Methods are described for generating and assaying the response, which specify criteria such as antigen dose, antigen choice, response kinetics, and complement source. The results suggest that at the peak of the primary response (approximately day 6), animals of different ages produce predominantly different 'classes' of antibody which display markedly different complement-fixing characteristics. Antibodies produced by larvae and 4-month-old postmetamorphic animals appear here to be unable to fix either guinea pig complement (GPC') or adult Xenopus complement, but can readily fix complement from 6-month-old Xenopus. The proportion of spleen PFC's producing antibody capable of fixing GPC' progressively increases from about six months to 18 months of age. Possible explanations for such ontogenetic changes are discussed.

Quantitative light and scanning electron microscopic study of the developing auditory organs in the bullfrog: implications on their functional characteristics.

During postmetamorphic development in the bullfrog, there is a downward shift in the distributions of best excitatory frequencies (BEFs) of the three populations of primary auditory fibers. This decrease in BEF distribution suggests that concurrent morphological changes occur in the peripheral auditory system during postmetamorphic growth. The postmetamorphic development of the auditory organs in the bullfrog was quantitatively investigated with light and scanning electron microscopy. In the basilar papilla, there are dramatic increases in the lumen volume, contact membrane area, and mass of the tectorial membrane (TM). The area of the sensory epithelium and the total number of hair cells also increase slightly. In the amphibian papilla, the mass of the TM is spatially graded in a step-wise fashion along the length of the organ in both juvenile and adult bullfrogs, but there is an increase in the absolute mass of the TM throughout the papilla with age. The height of the tallest stereocilia of the predominant hair cell type systematically decreases caudally in the juvenile amphibian papilla, but is uniform throughout the adult papilla. The increase in stereociliary height in the caudal end of the organ presumably results in a decrease in stereociliary stiffness with postmetamorphic age. The length of the amphibian papilla sensory epithelium and the number of hair cells also increase during postmetamorphic development. Theoretically, the observed morphological changes alter the micromechanical tuning properties of the auditory organs so that there is a decrease in the BEFs of the auditory fibers that innervate the two papillae.

Post-Metamorphic Development of the Frequency Selectivities and Sensitivities of the Peripheral Auditory System of the Bullfrog, Rana Catesbeiana

ABSTRACT Acoustic response characteristics of single fibres were studied in the VIIIth cranial nerve of adult and early post-metamorphic bullfrogs (Rana catesbeiana). Based on the distribution of units’ best excitatory frequencies, three populations of auditory fibres were found in each group of frogs. The sharpness of the tuning curves and temporal firing patterns of primary fibres were similar in both adults and froglets. However, the distributions of the populations were different between the two groups, and it was found that froglets responded to higher frequencies than did adults. There were also differences in the distributions of thresholds of excitation between the froglets and adults. The excitation thresholds of low-frequency selective and high-frequency selective fibres tended to be higher in froglets. Low-frequency selective fibres in both groups of frogs exhibited two-tone inhibition, and the best inhibitory frequencies were higher in froglets than in adults. These results demonstrate that changes in the response properties of primary auditory fibres occur during the development of the bullfrog. These functional changes presumably reflect morphological changes which may occur in the peripheral auditory system.

Changes in ganglion cell density during post-metamorphic development in a neotropical tree frog Hyla raniceps

In the frog, as in many other vertebrates, the eye and, consequently the retina, continues to increase in size throughout life. In this paper ganglion cell densities in the retina of a Brasilian tree frog, Hyla raniceps, have been measured from methylene blue stained whole mounts. From a series of retinae, taken from individuals with body sizes ranging from that of a post-metamorphic juvenile to sexually mature adult, isodensity maps of cell density across the whole retina have been prepared. Concomitant measurement of the extent of the visual field of these animals allowed cell counts per mm 2 to be converted to cells per unit visual angle. Changes in both cell density and distribution were observed with increasing body size and in central retina these changes resulted in an increasing number of cells representing each degree of visual field. In contrast, in the inferior and superior periphery the angular separation of ganglion cells remained approximately constant. The developmental, physiological and ecological significance of these findings is discussed.

Alterations in physical parameters and proteins of lens from Rana catesbeiana during development

Lens proteins and lens gross morphology were examined during tadpole and adult development of the bullfrog, Rana catesbeiana. Significant increases in the lens physical parameters of diameter, wet weight, dry weight (94–97% protein), and percent water were observed to accompany both natural and thyroxine-induced metamorphosis. These increases in lens parameters were not accompanied by growth of tadpoles during metamorphic change. Lens proteins were isolated from whole lenses and also from specified layers within whole lenses by a new fractionation method. Electrophoretic examination of whole lenses showed that the lens proteins did not change rapidly, one for another, prior to or during metamorphosis. However, changes became apparent during post metamorphic development. These changes included an increase in the relative concentration and mobility of alpha crystallin, a decrease in the relative concentration of gamma crystallin and an increase in the relative concentration of beta crystallin. Examination of specified layers within tadpole and frog lenses demonstrated that changes in the patterns of lens protein synthesis and modification may occur during development. Rapid and reproducible methods for quantitating changes in lens gross morphology and lens proteins, and for fractionating both tadpole and frog lenses into a number of definable layers were devised in the course of this study.

Genetic transcription during the development and metamorphosis of the tunicate Ascidia callosa

Large, synchronous cultures of embryos of the tunicate Ascidia callosa were prepared and grown through metamorphosis in the laboratory. 3H-uridine is taken up from sea water during all stages of development and metamorphosis. RNA synthesis during development and metamorphosis was studied by following the time course of 3H-uridine incorporation into TCA precipitable compounds. To the time of hatching, there is little, if any, RNA synthesis. Detectable RNA synthesis begins only after the larvae have been swimming for several hours. Once begun the level of synthesis increases rapidly through metamorphosis. Acrylamide gel electrophoresis analysis demonstrates that heterodisperse, ribosomal and transfer RNAs begin synthesis nearly simultaneously in the swimming tadpole. Early in the synthetic period there is a greater emphasis on heterodisperse RNA synthesis than after metamorphosis, when ribosomal and transfer RNA synthesis predominate. RNA-DNA binding efficiency studies show that tadpole RNA binds to DNA 3 times as efficiently as post-metamorphic RNA, reflecting the proportionally greater transcription of ribosomal cistrons in the older stage. RNA-DNA binding competition studies indicate that part of the genome active in the swimming tadpole was also active during oogenesis. The RNA populations present in the unhatched tadpole and unfertilized egg are identical when compared to radioactive tadpole RNA, lending further support to the incorporation studies. The RNA population transcribed by the swimming tadpole is similar to the RNA populations of newly metamorphosed individuals and adults, suggesting that RNA synthesized by the swimming tadpole may be important to the post-metamorphic development of the ascidian.

Memoirs: The Development of the Amphibian Kidney. Part III. The Post-Metamorphic Development of the Kidney, and the Development of the Vasa Efferentia and Seminal Vesicles in Rana Temporaria

The Post-Metamorphic Development of the Kidney. 1. The principal changes are in the method of attachment of malpighian unit to archinephric duct. 2. In the anterior region of the kidney there are few functional units attached to the straight tubules. (Text-fig. 1, A, C, F.)3. In the middle region of the kidney there are many functional units attached to the straight tubules. (Text-fig. 1, B, D, G.)4. Additional units are produced, after the end of the first year, only in the posterior region of the kidney. 5. There are no straight tubules in this newly formed posterior region so that the units become attached to outgrowths from the archinephric duct. (Text-fig. 1, E, H.)6. These outgrowths, termed collecting-trunks, branch and anastomose. 7. Accessory peritoneal funnels are formed from blastema lying in that part of the kidney which adjoins the gonad. (Text-fig. 2, C.) 8. These funnels are furnished with longer tails than those previously produced. (Text-fig. 2, D.) 9. The tails are in close association with renal arterioles which subsequently pass to excretory units. 10. It is suggested that the arterial supply of the ‘tail’ may purify the coelomic fluid which the funnels return to the renal venules. The Development of the Vasa Efferentia. 11. The origin of our present conception of the formation of the vasa efferentia is traced to Gaupp's adoption of Beissner's compromise between the views of Hoffman and Spengel. (Pp. 454 to 458.) 12. The gonadic ridge is primitively connected to the region of the developing kidney by a sheet of kidney blastema. (Textfig.3, A.) 13. This sheet breaks up into (a) a series of rudimentary vasa efferentia; (b) an anterior prolongation connecting the anterior end of the gonad to the anterior end of the kidney. (Text-fig. 3, B, C.) 14. The rudimentary vasa efferentia ends in a mass of blastema lying along the edge of the kidney. 15. This mass, from which Bidder's canal subsequently develops, is itself connected to the abortive unit which terminates the straight tubules. (Figs. 9a and b, Pl. 21, Fig. 4, Pl. 20.) 16. The anterior prolongation ends in a mass of kidney tubules at the anterior end of the kidney. (Fig. 8, Pl. 21.) 17. It is suggested that these anterior units are without functional significance. 18. The testis end of the anterior prolongation grows downwards into the testis and becomes connected to the tubules and vasa efferentia. (Text-fig. 3, D.) 19. The kidney connexions of the vasa efferentia remain unaltered but the anterior prolongation degenerates (Text-fig. 3, E.) The Development of the Seminal Vesicles. 20. In a four-legged tadpole there is a mass of mesenchyme, containing aggregations of kidney blastema, on the side of the archinephric duct between the points where this leaves the kidney and enters the rectum. (Fig. 26, Pl 23.) 21. The mesenchyme forms connective tissue and the blastema aggregations form short tubules. 22. During the third year after metamorphosis these tubules branch and coil. 23. It is emphasized that the seminal vesicle derives nothing from the archinephric duct, and suggested that it may represent the remnants of the most posterior units of the sexual kidney.