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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.01.038 Byline: Sandeep Mukhi (a), Marko E. Horb (b)(c), Donald D. Brown (a) Keywords: Amphibian metamorphosis; Pancreas; [beta] cells; Thyroid hormone; Organ remodeling Abstract: Insulin-producing [beta]-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the [beta]-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the [beta]-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-[beta]-cell transdifferentiation, but rather is due to the aggregation of pre-existing [beta]-cells. The total number of [beta]-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by the elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of [beta]-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate [beta]-cells to form islets like controls. These results demonstrate that TH controls the remodeling of [beta]-cells through cell-cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene. Author Affiliation: (a) Department of Embryology, Carnegie Institution, 3520 San Martin Drive, Baltimore, MD 21218, USA (b) Laboratory of Molecular Organogenesis, Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada (c) Departement de Medecine, Universite de Montreal, Canada Article History: Received 10 October 2008; Revised 13 January 2009; Accepted 28 January 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.03.021 Byline: Donald D. Brown, Liquan Cai Keywords: Amphibian metamorphosis; Thyroid hormone; Thyroid hormone receptor; Deiodinase; Gene expression; Xenopus leavis; Tadpole Author Affiliation: Department of Embryology, Carnegie Institution, 3520 San Martin Dr., Baltimore, MD 21218, USA Article History: Received 1 February 2007; Revised 9 March 2007; Accepted 18 March 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.12.031 Byline: Liquan Cai, Biswajit Das, Donald D. Brown Keywords: Xenopus laevis; Metamorphosis; Thyroid hormone; Gene expression; Muscle development; Transgenesis Abstract: Transgenic Xenopus laevis tadpoles that express a dominant negative form of the thyroid hormone receptor (TRDN) controlled by the cardiac actin muscle promoter (pCar) develop with very little limb muscle. Under the control of the tetracycline system the transgene can be induced at will by adding doxycycline to the rearing water. Pre-existing limb muscle fibers begins to disintegrate within 2 days after up-regulation of the TRDN transgene. The muscle cells do not die even after weeks of transgene exposure when the myofibrils have degenerated completely and the tadpole is nearing death. A microarray analysis after 2 weeks of exposure to the transgene identified 24 muscle genes whose expression was altered in such a way that they might cause the muscle phenotype. These candidate genes are normally activated in growing limb muscle but they are repressed by the TRDN transgene. Several of these genes have been implicated in mammalian myopathies. However, the expression of only one of these genes, calsequestrin, is down-regulated in 1 day and therefore might initiate the degeneration. Calsequestrin is one of several affected genes that encode proteins involved in calcium sequestration, transport and utilization in muscle suggesting that uncontrolled calcium influx into the growing limb muscle fibers causes rhabdomyolysis. Many of the same genes that are down-regulated in the tail at the peak of metamorphic climax just before it is resorbed are suppressed in the transgenic limb muscle in effect turning the limb growth program into a tail resorption program. Author Affiliation: Carnegie Institution, 3520 San Martin Dr. Baltimore, MD 21218, USA Article History: Received 7 August 2006; Revised 14 November 2006; Accepted 13 December 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.032 Byline: Biswajit Das (a), Liquan Cai (a), Mark G. Carter (b), Yu-Lan Piao (b), Alexei A. Sharov (b), Minoru S.H. Ko (b), Donald D. Brown (a) Keywords: Thyroid hormone; Metamorphosis; Tadpole; Xenopus laevis; Tail resorption; Limb growth; Brain ventricle proliferation; Mitochondrial electron transport chain; Cell cycle; Transcriptional regulation Abstract: Thyroid hormone (TH) controlled gene expression profiles have been studied in the tail, hind limb and brain tissues during TH-induced and spontaneous Xenopus laevis metamorphosis. Amplified cRNA probes mixed with a universal standard were hybridized to a set of 21,807-sense strand 60-mer oligonucleotides on each slide representing the entries in X. laevis UniGene Build 48. Most of the up-regulated genes in hind limb and brain are the same. This reflects in part the fact that the initial response to TH induction in both tissues is cell proliferation. A large number of up-regulated genes in the limb and brain programs encode common components of the cell cycle, DNA and RNA metabolism, transcription and translation. Notch is one of the few genes that is differentially expressed exclusively in the brain in the first 48 h of TH induction studied in these experiments. The TH-induced gene expression changes in the tail are different from the limb and brain programs. Distinct muscle and fibroblast programs were identified in the tail. Dying muscle fibers in tail (marked by active caspase-3) up-regulate a group of genes that include proteolytic enzymes. At the climax of metamorphosis, tail muscle down-regulates more than half of the genes that encode the glycolytic enzymes in the cytoplasm and the tricarboxylic acid pathway and all five complexes of the electron transport system in mitochondria. These changes in gene expression precede the activation of caspase-3. Some of these same energy metabolism-related genes are up-regulated in the limb and brain programs by TH. A prominent feature of the tail fibroblasts is the down-regulation of several collagen and other extra cellular matrix genes and the up-regulation of hydrolytic enzymes that are responsible for dissolving the notochord and resorbing the tail. Author Affiliation: (a) Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA (b) Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD 21224, USA Article History: Received 30 September 2005; Revised 8 December 2005; Accepted 14 December 2005

The thyroid gland synthesizes thyroxine (T4), which passes through the larval tadpole's circulatory system. The enzyme type II iodothyronine deiodinase (D2) converts thyroxine (T4) to the active hormone 3,5,3'-triiodothyronine (T3) in peripheral tissues. An early response to thyroid hormone (TH) in the Xenopus laevis tadpole is the stimulation of cell division in cells that line the brain ventricles, the lumen of the spinal cord, and the limb buds. These cells express constitutively high levels of D2 mRNA. Exogenous T4 induces early DNA synthesis in brain, spinal cord, and limb buds as efficiently as T3. The deiodinase inhibitor iopanoic acid blocks T4- but not T3-induced cell division. At metamorphic climax, both TH-induced cell division and D2 expression decrease in the brain. Then D2 expression appears in late-responding tissues including the anterior pituitary, the intestine, and the tail where cell division is reduced or absent. Therefore, constitutive expression of D2 occurs in the earliest target tissues of TH that will grow and differentiate, while TH-induced expression of D2 takes place in late-responding tissues that will remodel or die. This pattern of constitutive and induced D2 expression contributes to the timing of metamorphic changes in these tissues. Keywords: Metamorphosis; Xenopus laevis; Thyroid hormone; Deiodinase; Gene expression; Cell division; Spinal cord; Intestine; Tail; Brain

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. Key Words: metamorphosis; Xenopus; notochord; slow muscle.

Four genes were identified in a screen for thyroid hormone-induced down-regulation of gene expression in Xenopus laevis tadpole tails. All four encode extracellular glycoproteins that are expressed exclusively in the apical cell layer of the entire tadpole epidermis, which is the equivalent of the mammalian fetal periderm. The onset of the four novel genes' expression late in embryogenesis, their activity throughout the life of the tadpole, their repression by exogenously added thyroid hormone, and the spontaneous cessation of their expression at the end of tadpole life are closely coordinated. These facts suggest that the protein products of these genes form a novel albeit temporary barrier or other structure in the tadpole epidermis that functions in lieu of the cornified, stratified epithelium of the adult epidermis. We have exploited the cloning of these genes for use as cell-specific markers to follow the appearance and loss of apical cells during development. We were able to demonstrate directly that the apical cells are derived from a stratification of the embryonic ectoderm at the onset of the formation of a true epidermis. The apical cells uniformly cover the surface of the tadpole until metamorphosis, when the expression of the four larval epidermis-specific genes is lost coordinately over the entire tadpole. In contrast, the adult epidermis develops with a distinct regional specificity: adult keratin is first expressed up to a line separating the body and tail epidermis and finally appears in the tail only at metamorphic climax. Finally, our analysis reveals that the TH-induced down-regulated gene expression program during metamorphosis is very different from the previously described up-regulated program which involves multiple cell types and several waves of gene expression changes. The down-regulated program only consists of the repression of a small number of genes which are expressed in larval cells preprogrammed to die during the larval to adult transition at metamorphosis.