CHAPTER 18, POSTEMBRYONIC DEVELOPMENT: METAMORPHOSIS & REGENERATION ZOO3603 12/1/09 Developmental history of the leopard frog, Rana pipiensMetamorphosis & thyroid hormones in amphibian  Metamorphosis in amphibian is initiated by thyroid hormone such as thyroxine (T4) and tri-iodothyronine (T3). Thyroxine (T4) Tri-iodothyronine (T3) Circulating inactive form Active form Changes associated with metamorphosis  Morphology  Physiology  Behavior Fry! Jump! Water Land Morphological changes: Growth of new structures Eye migration and associated neuronal changes during metamorphosis of Xenopus laevis Contralateral projection Both ipsilateral & contralateral projection Projection of Retinal Neurons Wide, but not 3D 3D, but narrow Cell death during metamorphosis  Paddle-like tail  Oxygen-procuring gills  Conversion of red blood cells  Tadpole red blood cells Cell death 1. Apoptosis (program cell death) 2. Necrosis Regress by apoptosis Necrosis and Apoptosis  Necrosis = murder  Apoptosis = suicide  DNA fragmentation into the nucleosome unit  Detected by TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) method in situ. Goodlett & Horn, Alcohol Res & Health 25:175–184 (2001) Cell death during metamorphosis: Conversion of red blood cells Tomori & Wakahara, Int. J. Dev. Biol. 44: 373-380 (2000) Anti-larval hemoglobin Anti-adult hemoglobin Tadpole Blood cells Juvenile Blood Cells Juvenile Spleen Red, positive cells; blue, nuclei Cell death during metamorphosis: Conversion of red blood cells Tomori & Wakahara, Int. J. Dev. Biol. 44: 373-380 (2000) Anti-larval hemoglobin TUNEL Co-localization Apoptosis of the larval RBC in spleen at metamorphic climax Cell death during metamorphosis: Conversion of red blood cells Tomori & Wakahara, Int. J. Dev. Biol. 44: 373-380 (2000) Remodeling during metamorphosis: Changes the skull in Xenopus Before metamorphosis During metamorphosis New bones are made: Neural crest-derived cartilage in tadpole skull → Neural crest-derived bone in adult skull Open arrowheads, branchial arch cartilage; arrowheads, ceratohyal cartilage Alcian blue, cartilage; Alizarin red, calcified boneBiochemical Re-specification: Development of urea cycle during anuran metamorphosis  Ammonotelic, excreting ammonia in most freshwater fish including tadpoles.  Ureotelic, excreting urea in most terrestrial vertebrates including many of adult frogs. Ammonotelic ⇒ Ureotelic ↑T3 in LiverMajor features of the urea cycleThe emergence of urea cycle enzyme activities It correlates with metamorphic changes in Rana catesbeiana Hormonal control of amphibian metamorphosis  Fed powdered horse thyroid glands ⇒ Premature metamorphosis.  Removed or destroyed thyroid ⇒ Never metamorphosed  Increasing amount of thyroid hormone regulates anuran metamorphosis. Regulations of thyroid hormones http://www.creationofman.net/chapter3/chapter3_8.html Feedback loop Hypothalamic–pituitary–thyroid axis and thyroid hormone synthesis McPherson & Pincus, 2006 Thyroid Follicles Key aspects of thyroid iodine transport and thyroid hormone synthesis Kronenberg et al., Williams Textbook of Endocrinology (2008) NIS, Sodium-iodide symporter; T3, triiodothyronine; T4, thyroxine; Tg, thyroglobulin; TPO, thyroid peroxidase TSHR, thyrotropin receptor. (Modified from Spitzweg C, Heufelder AE, Morris JC. Thyroid iodine transport. Metabolism of thyroxine (T4) and tri-iodothyronine (T3) 1. Secretion of T4 into the circulation by thyroid gland. 2. Conversion of T4 into T3 by type II deiodinase in the target tissues. 3. Degradation of T3 by type III deiodinase in the target tissues.Metabolism of thyroxine (T4) and tri-iodothyronine (T3) (Part 2) 1. Secretion of T4 into the circulation by thyroid gland. 2. Conversion of T4 into T3 by type II deiodinase in the target tissues. 3. Degradation of T3 by type III deiodinase in the target tissues.The thyroid hormone pathway Dayan & Panicker, Nat Rev Endocrinol doi:10.1038/nrendo.2009.19 (2009) Thyroid hormone receptor hetero-dimerizes with retinoid X receptor (RXR) Location of functional regions in a typical steroid receptor Thyroid hormone receptor is the nuclear hormone receptor Activation of steroid/thyroid hormone receptors Homo-dimer Hetero-dimer Thyroid hormone receptor hetero-dimerizes with retinoid X receptor (RXR) Repression/activation of thyroid hormone receptors Repressed Transcription Activated Transcription Sachs et al., Gene Ther Mol Biol 5: 101-110 (2000) Basal Transcription Repressed Transcription Activated Transcription Developmental expression of TR genes suggests dual functions for TR in frog development Sachs et al., Gene Ther Mol Biol 5: 101-110 (2000) Pre-metamorphosis Pro-metamorphosis Climax Regional specificity during frog metamorphosis  The different regions of body can respond to thyroid hormones at different time.  The type of response is determined by other factors already present in the different tissues.  Proliferation  Apoptosis  MigrationRegional specificity during frog metamorphosis Tail tip transplanted to trunk Eye cups transplanted to tail Tail tip regress even when transplanted to trunk Eye cups remain even when transplanted to tail These responses are independent from the host place Skin remodeling during metamorphosis  The same regions of body can respond to thyroid hormones at different time.  During metamorphic climax, the larval skin is instructed to undergo apoptosis.  The tadpole head and body are able to generate a new epidermis from epithelial stem cells.  The tail epidermis lack these stem cells.How do they take the water? http://seawayblog.blogspot.com/2008/02/frogs-umbrella.html http://chem11.proboards.com Need to avoid the dehydration. Water intake in anurans Absorption Secretion Drink Ventral Dorsal Control 0.2 % Eosin 1 cm When frogs were placed in eosin solution, ventral skin absorption was indicated (pink color) while dorsal skin color remained unchanged. Control frogs showed no staining. Cutaneous Absorption of Eosin-containing Water in vivo Histological Analysis of Frog Skin Water Absorption