LIMB DEVELOPMENT IN SALAMANDERS: AN EVOLUTIONARY PERSPECTIVE TO THE TETRAPOD LIMB

Vertebrate limb is an ideal model to study growth, patterning and morphogenesis and the interplay between these processes. The developing limb bud is a three-dimensional structure and its outgrowth depends on the interaction between 2 important signaling centers: the Apical Ectodermal Ridge (AER) at the junction of the dorsal and ventral halves of the limb bud and the Zone of Polarizing activity (ZPA) in the posterior mesenchyme. These centers produce their respective key molecules and the close interplay between them specifies structures along the anterior-posterior (thumb to pinky), proximal-distal (shoulder to fingertips) and dorsal-ventral (knuckle to palm) axes. Developmental biologists have extensively studied the limb using amniotes like chicken and mouse and have put forward comprehensive models for limb development based on experimental embryology and molecular genetics. Although decades of studies have produced a molecular model for tetrapod limb development, urodeles (salamanders/tailed amphibians) deviate from all other tetrapods in at least two key respects: their limbs exhibit pre-axial skeletal specification where skeletal chondrification proceeds anterior to posterior within the zeugopod and autopod and their limb buds do not develop an apical ectodermal ridge (AER). In this dissertation, I have specifically looked into forelimb development in Mexican axolotl (Ambystoma mexicanum) by characterizing the spatial domains and analyzing the functional role of the centers required for patterning along the 3 axes. Chapter 3 dissects the spatio-temporal domains and the functional role of AER- Fgfs and the ZPA molecule Shh during axolotl limb development. Functional studies were done using pharmacological inhibitors: Fgf-receptor inhibitor SU5402 and smoothened antagonist cyclopamine. Chapter 4 further examines the role of Shh during axolotl limb development using a small molecule inhibitor BMS-833923 and CRISPR/Cas9 system. Chapter 5 is a spatio-temporal characterization of the dorsal-ventral polarity genes: Wnt7a, Rfng, Lmx1b and En-1. Finally, chapter 6 discusses the significance of the results and focuses on the evolutionary aspects