Transient “deafness” accompanies auditory development during metamorphosis from tadpole to frog

During metamorphosis, ranid frogs shift from a purely aquatic to a partly terrestrial lifestyle. The central auditory system undergoes functional and neuroanatomical reorganization in parallel with the development of new sound conduction pathways adapted for the detection of airborne sounds. Neural responses to sounds can be recorded from the auditory midbrain of tadpoles shortly after hatching, with higher rates of synchronous neural activity and lower sharpness of tuning than observed in postmetamorphic animals. Shortly before the onset of metamorphic climax, there is a brief “deaf” period during which no auditory activity can be evoked from the midbrain, and a loss of connectivity is observed between medullary and midbrain auditory nuclei. During the final stages of metamorphic development, auditory function and neural connectivity are restored. The acoustic communication system of the adult frog emerges from these periods of anatomical and physiological plasticity during metamorphosis.

N-methyl-d-aspartate receptor activation and visual activity induce elongation factor-2 phosphorylation in amphibian tecta: A role for N-methyl-d-aspartate receptors in controlling protein synthesis

N-methyl-d-aspartate receptor (NMDAR) activation has been implicated in forms of synaptic plasticity involving long-term changes in neuronal structure, function, or protein expression. Transcriptional alterations have been correlated with NMDAR-mediated synaptic plasticity, but the problem of rapidly targeting new proteins to particular synapses is unsolved. One potential solution is synapse-specific protein translation, which is suggested by dendritic localization of numerous transcripts and subsynaptic polyribosomes. We report here a mechanism by which NMDAR activation at synapses may control this protein synthetic machinery. In intact tadpole tecta, NMDAR activation leads to phosphorylation of a subset of proteins, one of which we now identify as the eukaryotic translation elongation factor 2 (eEF2). Phosphorylation of eEF2 halts protein synthesis and may prepare cells to translate a new set of mRNAs. We show that NMDAR activation-induced eEF2 phosphorylation is widespread in tadpole tecta. In contrast, in adult tecta, where synaptic plasticity is reduced, this phosphorylation is restricted to short dendritic regions that process binocular information. Biochemical and anatomical evidence shows that this NMDAR activation-induced eEF2 phosphorylation is localized to subsynaptic sites. Moreover, eEF2 phosphorylation is induced by visual stimulation, and NMDAR blockade before stimulation eliminates this effect. Thus, NMDAR activation, which is known to mediate synaptic changes in the developing frog, could produce local postsynaptic alterations in protein synthesis by inducing eEF2 phosphorylation.

Overexpression of Xenopus laevis growth hormone stimulates growth of tadpoles and frogs

The role of growth hormone (GH) in amphibian metamorphosis is ambiguous based on experiments in which mammalian GH was administered to tadpoles and frogs. We have reexamined the effects of GH by producing transgenic Xenopus laevis that overexpress the cDNA encoding X. laevis GH. These transgenic tadpoles take the same length of time to reach metamorphosis as control tadpoles, but the transgenic tadpoles are twice as large. After metamorphosis, the transgenic frogs grow at a greatly accelerated rate and develop skeletal abnormalities reminiscent of acromegaly. The transgenic frogs are larger than mature frogs in a few months and die in about 1 year. At as early as 10 months of age, the males have mature sperm. We conclude that the growth-promoting effects of GH in this amphibian closely resemble those described for mammals. Although excess GH increases the size of the tadpole, it does not alter the developmental programs involved in metamorphosis.

Prolactin is not a juvenile hormone in Xenopus laevis metamorphosis

Prolactin (PRL) is widely considered to be the juvenile hormone of anuran tadpoles and to counteract the effects of thyroid hormone (TH), the hormone that controls amphibian metamorphosis. This putative function was concluded mainly from experiments in which mammalian PRL was injected into tadpoles or added to cultured tadpole tissues. In this study, we show that overexpression of ovine or Xenopus laevis PRL in transgenic X. laevis does not prolong tadpole life, establishing that PRL does not play a role in the life cycle of amphibians that is equivalent to that of juvenile hormone in insect metamorphosis. However, overexpression of PRL produces tailed frogs by reversing specifically some but not all of the programs of tail resorption and stimulating growth of fibroblasts in the tail. Whereas TH induces muscle resorption in tails of these transgenics, the tail fibroblasts continue to proliferate resulting in a fibrotic tail that is resistant to TH.

Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis

Two important features of amphibian metamorphosis are the sequential response of tissues to different concentrations of thyroid hormone (TH) and the development of the negative feedback loop between the pituitary and the thyroid gland that regulates TH synthesis by the thyroid gland. At the climax of metamorphosis in Xenopus laevis (when the TH level is highest), the ratio of the circulating precursor thyroxine (T4) to the active form 3,5,3′-triiodothyronine (T3) in the blood is many times higher than it is in tissues. This difference is because of the conversion of T4 to T3 in target cells of the tadpole catalyzed by the enzyme type II iodothyronine deiodinase (D2) and the local effect (cell autonomy) of this activity. Limb buds and tails express D2 early and late in metamorphosis, respectively, correlating with the time that these organs undergo TH-induced change. T3 is required to complete metamorphosis because the peak concentration of T4 that is reached at metamorphic climax cannot induce the final morphological changes. At the climax of metamorphosis, D2 expression is activated specifically in the anterior pituitary cells that express the genes for thyroid-stimulating hormone but not in the cells that express proopiomelanocortin. Physiological concentrations of T3 but not T4 can suppress thyrotropin subunit β gene expression. The timing and the remarkable specificity of D2 expression in the thyrotrophs of the anterior pituitary coupled with the requirement for locally synthesized T3 strongly support a role for D2 in the onset of the negative feedback loop at the climax of metamorphosis.

Xenopus Bcl-XL selectively protects Rohon-Beard neurons from metamorphic degeneration

Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-XL plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-XL homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal β-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.

The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis.

Genes that are up- and down-regulated by thyroid hormone in the tail resorption program of Xenopus laevis have been isolated by a gene expression screen, sequenced, and identified in the GenBank data base. The entire program is estimated to consist of fewer than 35 up-regulated and fewer than 10 down-regulated genes; 17 and 4 of them, respectively, have been isolated and characterized. Up-regulated genes whose function can be predicted on the basis of their sequence include four transcription factors (including one of the thyroid hormone receptors), an extracellular matrix component (fibronectin) and membrane receptor (integrin), four proteinases, a deiodinase that degrades thyroid hormone, and a protein that binds the hypothalamic corticotropin-releasing factor, which has been implicated in controlling thyroid hormone synthesis in Xenopus tadpoles. All four down-regulated genes encode extracellular proteins that are expressed in tadpole epidermis. This survey of the program provides insights into the biology of metamorphosis.

Dominant-negative mutant thyroid hormone receptors prevent transcription from Xenopus thyroid hormone receptor beta gene promoter in response to thyroid hormone in Xenopus tadpoles in vivo.

We describe a dominant-negative approach in vivo to assess the strong, early upregulation of thyroid hormone receptor beta (TR beta) gene in response to thyroid hormone, characteristic of the onset of natural and thyroid hormone-induced amphibian metamorphosis, 3,3',5-Triiodo-thyronine (T3) treatment of organ cultures of premetamorphic Xenopus tadpole tails coinjected in vivo with the wild-type Xenopus TR beta (wt-xTR beta) and three different thyroid responsive element chloramphenicol acetyltransferase (TRE-CAT) reporter constructs, including a direct repeat +4 (DR +4) element in the -200/+87 fragment of the xTR beta promoter, resulted in a 4- to 8-fold enhancement of CAT activity. Two human C-terminal TR beta 1 mutants (delta-hTR beta 1 and Ts-hTR beta 1), an artificial Xenopus C-terminal deletion mutant (mt-xTR beta), and the oncogenic viral homology v-erbA, none of which binds T3, inhibited this T3 response of the endogenous wt-xTR in Xenopus XTC-2 cells cotransfected with the -1600/+87 xTR beta promoter-CAT construct, the potency of the dominant-negative effect of these mutant TRs being a function of the strength of their heterodimerization with Xenopus retinoid X receptor gamma. Coinjection of the dominant-negative Xenopus and human mutant TR beta s into Xenopus tadpole tails totally abolished the T3 responsiveness of the wt-xTR beta with different TREs, including the natural DR +4 TRE of the xTR beta promoter.

A conserved family of elav-like genes in vertebrates.

A large family of genes encodes proteins with RNA recognition motifs that are presumed to bind RNA and to function in posttranscriptional regulation. Neural-specific members of this family include elav, a gene required for correct differentiation and maintenance of neurons in Drosophila melanogaster, and a related gene, HuD, which is expressed in human neuronal cells. I have identified genes related to elav and HuD in Xenopus laevis, zebrafish, and mouse that define a family of four closely related vertebrate elav-like genes (elrA, elrB, elrC, and elrD) in fish, frogs, and mammals. In addition to protein sequence conservation, a segment of the 3'-untranslated sequence of elrD is also conserved, implying a functional role in elrD expression. In adult frogs, elrC and elrD are exclusively expressed in the brain, whereas elrB is expressed in brain, testis, and ovary. During Xenopus development, elrC and elrD RNAs are detected by late gastrula and late neurula stages, respectively, whereas a nervous system-specific elrB RNA species is expressed by early tadpole stage. Additional elrB transcripts are detected in the ovary and early embryo, demonstrating a maternal supply of mRNA and possibly of protein. These expression patterns suggest a role for different elav-like genes in early development and neuronal differentiation. Surprisingly, elrA is expressed in all adult tissues tested and at all times during development. Thus, the widely expressed elrA is expected to have a related function in all cells.