Diverse developmental programs of Xenopus laevis metamorphosis are inhibited by a dominant negative thyroid hormone receptor

Abstract

Metamorphosis of anuran tadpoles is controlled by thyroid hormone (TH).Here we demonstrate that transgenic Xenopus laevis tadpoles expressing a dominant negative form of TH receptor-␣ are resistant to a wide variety of the metamorphic changes induced by TH. This result confirms that TH receptors mediate both early and late developmental programs of metamorphosis as diverse as growth in the brain, limb buds, nose and Meckel's cartilage, remodeling of the intestine, and death and resorption of the gills and tail.R ising thyroid hormone (TH) levels produced by the thyroid gland of a growing tadpole orchestrate the sequential changes of metamorphosis in the majority of tadpole organs. These morphological changes range from growth and cell differentiation (limbs) to cell death and tissue resorption (tail and gills), and include the remodeling of tadpole organs into their adult forms (intestine, skin, and brain). With the discovery that the TH receptors (TRs) are transcription factors (1, 2), these varied developmental programs have been studied as complex changes in gene expression initiated by TH (3). Despite the many experiments that prove the requirement of TH in metamorphosis, there is only one demonstration to date showing conclusively that TH acts by way of TRs in a particular metamorphic program. The asymmetrical replication of the ventral retina in Xenopus laevis is inhibited by expression of a dominant negative form of the TR (TRDN; ref. 4).All vertebrates studied to date, including X. laevis (5), have two highly conserved TR isoforms called TR␣ and TR␤. In X. laevis, tadpole TR␣ is constitutive and distributed widely in tissues even before the organism forms a thyroid gland (6). Because TR␤ is a direct response gene of TH (6, 7), the amount of TR␤ in cells increases along with the rise in endogenous TH that occurs as metamorphosis proceeds (6,8). During premetamorphosis when the early events of tadpole development (such as limb growth and DNA replication in the brain) occur, the TH concentration and the TR␤ levels are very low. TR␤ and TH rise to a peak at the climax of metamorphosis when the final changes (such as gill and tail resorption and intestinal remodeling) occur. We will show that TR␣ is required for the precocious response of young tadpoles to exogenous TH in their rearing water. We have taken advantage of the new transgenesis method (9) to express green fluorescent protein (GFP) fused to a dominant negative form of TR-␣ (GFP-TRDN␣) driven by two different widely expressed promoters. Metamorphic changes that are inhibited by the GFP-TRDN␣ include brain development, limb and Meckel's cartilage growth, intestinal remodeling, gill resorption, and death of cells in the tail including muscle. Materials and MethodsPlasmids, Transgenesis, and 3,5,3 Triiodothyronine (T3) Treatment.Constructs were prepared in pCS2 ϩ -based vectors and used either the cytomegalovirus (CMV) promoter͞enhancer (10) or a 6-kb ␣2(1) mouse collagen enhancer fused to a minimal ␣2 (1) promoter (Col; ref. 11). Constructs were made ...

Thyroid hormone promotes neurogenesis in the Xenopus spinal cord

“…This promotion may occur by two mechanisms, the relative importance of which is not clear. First, as suggested by the increase in spinal cord size, TH may stimulate the proliferation of neuronal precursors, confirming previous observations in frog spinal cord (Tusques, 1949;Weiss and Rossetti, 1951;Baffoni, 1957;Reynolds, 1966;Pollack and Towbin, 1975;Kollros, 1981;Goldberg and Pollack, 1989;Clorfene and Pollack, 1994;Schreiber et al, 2001) and other parts of the nervous system (Kaltenbach and Hobbs, 1972;Beach and Jacobson, 1979;Kollros, 1981;Marsh-Armstrong et al, 1999;Schreiber et al, 2001). Second, TH may directly promote the expression of neuronal determination or differentiation genes.…”

“…Due to the resorption of yolk granules between stages 42 and 44 (which probably sequester TH at earlier stages, preventing its effective binding to xTR␣ in overexpression experiments), maternally supplied TH should be freely available throughout spinal cord cells at larval stages. This hypothesis predicts that TH is not only sufficient but also necessary for the initiation or regulation of the larval peak of neurogenesis in the Xenopus spinal cord, but additional studies (e.g., loss of function studies as in Schreiber et al, 2001) are required to test this prediction.…”

“…1). Thyroid hormones (TH) in amphibians are known to be sufficient and in some cases necessary for promoting proliferation of neuronal precursors (Tusques, 1949;Baffoni, 1957;Reynolds, 1966;Kaltenbach and Hobbs, 1972;Pollack and Towbin, 1975;Beach and Jacobson, 1979;Kollros, 1981;Goldberg and Pollack, 1989;Clorfene and Pollack, 1994;Marsh-Armstrong et al, 1999;Schreiber et al, 2001) and neuronal differentiation (Beaudoin, 1956;Race, 1961;Reynolds, 1963;Hughes, 1966;Decker, 1976;Kollros, 1981;Hauser and Gona, 1984;Kollros and Bovbjerg, 1990;Denver et al, 1997;Denver, 1998;Marsh-Armstrong et al, 1999). As these effects of THs are thought to be mediated by TH recep- Nieuwkoop and Faber, 1967).…”

Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis

“…This antibody has been effective as a marker for early stages of cartilage formation in Xenopus laevis (Seufert et al, 1994). Several specimens were also stained with an anti-phospho-histone-3 polyclonal mitosis marker (1: 500; Upstate Biotechnology, Lake Placid, NY) to assess the survival of the injected cell lineage, or the anticaspase-3 apoptosis marker (1:500; BD-Biosciences, Franklin Lakes, NJ) to assess apoptosis in the morpholinoinjected cells (after Schreiber et al, 2001). Hatchlings were fixed in 4% PFA and stored in PBS prior to immunohistochemistry.…”

A Method for Generating Transgenic Frog Embryos

“…This technique has been used for many applications. For example, transgenesis has been used to express wild type and mutant forms of genes in specific regions of the embryo or at distinct times of development (for examples, see: [1][2][3][4][5][6][7]). In addition, transgenic lines that express fluorescent proteins ubiquitously have been established, and these have proved useful for lineage studies [8][9][10][11][12].…”