Studies on tail regeneration and homeotic transformation in anuran tadpoles PRIYAMBADA MOHANTY-HEJMADI and PRAVATI KUMARI MAHAPATRA* Post Graduate Department of Zoology, Utkal University, Bhubaneswar, Odisha, India ABSTRACT Anuran tadpoles are excellent models for regeneration studies.The tail, an organ essen- tial for swimming for the aquatic tadpole, regenerates completely following injury or amputation. However, treatment with the morphogen, vitamin A or retinoic acid inhibits normal tail regenera- tion and induces homeotic transformation of tail to limbs. This phenomenon was discovered for the first time in the Indian marbled balloon frog Uperodon systoma in the Developmental Biology laboratory of Utkal University (Odisha, India) in the year 1992. In this paper, we present the results of morphological, histological, biochemical and molecular (immonohistochemistry) investigations of vitamin A induced homeotic transformation in different anuran species. In addition, we discuss the putative role of fibroblast growth factor 1 during spinal cord regeneration in the tadpoles of the Indian tree frog, Polypedates maculatus, an ideal model for regeneration studies in an Indian context. KEY WORDS: anuran tadpole, tail regeneration, homeotic transformation, vitamin A/retinoic acid Introduction Vitamin A induced homeotic transformation of tail to limbs in the marbled balloon frog Uperodon systoma was observed for the frst time in our laboratory (Mohanty-Hejmadi et al.1992). Fol- lowing the initial fnding, there were several reports on vitamin A induced inhibition of tail regeneration and homeotic transformation in different anuran species namely Polypedates maculatus, Bufo melanostictus (present name Duttaphrynus melanostictus), Micro- hyla ornata (Mahapatra, 1994; Mahapatra and Mohanty-Hejmadi, 1994, Mahapatra et al., 2001; Mohanty-Hejmadi and Crawford, 2003) and Rana tigerina (present name Hoplobatrachus tigerinus) (Das and Dutta, 1996). We investigated morphological, histologi- cal, biochemical and molecular aspects of tail regeneration in the anuran tadpoles. Collection of egg nests and rearing of tadpoles Egg nests of different anuran species were collected during the rainy season i.e., months of July to September from different breeding grounds inside the city of Bhubaneswar, Odisha, India (20.27° N, 85.84° E) and kept in enamel coated trays (45×60cm) containing 4 to 6 cm deep conditioned tap water (tap water stored and aerated for 72 hours). After hatching, rearing of tadpoles was done following standardized procedure (Mohanty-Hejmadi, 1977). The tadpoles were fed with boiled Amaranthus leaves ad libitum. Int. J. Dev. Biol. 64: 65-70 (2020) https://doi.org/10.1387/ijdb.190230pm www.intjdevbiol.com *Address correspondence to: Pravati Kumari Mahapatra. Post Graduate Department of Zoology, Utkal University, Bhubaneswar 751004, Odisha, India. E-mail: mpkzooluu@gmail.com - https://orcid.org/0000-0003-3188-901X Submitted: 22 July, 2019; Accepted: 19 August, 2019. ISSN: Online 1696-3547, Print 0214-6282 © 2020 UPV/EHU Press Printed in Spain Abbreviations used in this paper: DAB, diaminibenzidine; DPX, distyrene plasticiser xylene; FGF, fibroblast growth factor; FITC, fluorescein; HCl, hydrochloric acid; HSS-HRP, streptovidin conjugated to horseradish peroxidase; LS, longitudinal section; MS222, tricaine methanesulfonate; PBS, phosphate buffer saline; RA, retinoic acid; TS transverse section. Tail amputation and vitamin A / retinoic acid treatment Tadpoles of Gosner (1960) stages 26-28 (hind limb bud stage) were selected for tail amputation. The tadpoles were anaesthetized in 1:3000 solution of MS222 prior to amputation through the middle of the tail by keeping them laterally on a pre-sterilized porcelain plate. After operation, the tadpoles were transferred to amphib- ian ringer solution for about 10 minutes to prevent further loss of blood. Tadpoles of the control group were reared in conditioned tap water where as the tadpoles of the experimental group were treated with 10 to 30 IU/ml of vitamin A palmitate for different time periods ranging from 24 to 144 hours. In case of retinoic acid treatment, tadpoles were exposed to concentration of 125ng/ml to 750ng/ml of retinoic acid for 24 to 72 hours. Required amount of Vitamin A or retinoic acid was added to conditioned tap water in separate glass troughs (1000ml capacity) to make the volume 500ml where fve tail amputated tadpoles were reared (optimum rearing condition). Following treatment they were transferred to conditioned tap water keeping the volume same to avoid effect of crowding. Tail amputated tadpoles of both control and treated