Author ORCID Identifier

https://orcid.org/0000-0002-3974-4731

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Biology

First Advisor

Dr. Ashley W. Seifert

Abstract

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.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.480

Funding Information

Lab funding:

Collaborative Research: Immune tradeoffs during tissue regeneration in mammals:

This study was supported by the National Science Foundation IOS -1353713, Principal Investigator Ashley W. Seifert, Start Date:05/15/2014

Personal fellowships and awards:

2020 Biology Summer Fellowship, Department of Biology and the Graduate school, University of Kentucky-$2999

2020 Society for Developmental Biology travel fellowship; to attend MBL practical course developmental biology, Quintay, Chile- $500

2019 Sherr special opportunity award, Department of Biology, University of Kentucky; to attend MBL practical course developmental biology, Quintay, Chile-$1334

2019 EMBO Travel Grant to attend the EMBO Workshop: Limb Development and Regeneration: New Tools for a Classic Model System, Barcelona, Spain- 200 Euro

2019 Biology Summer Fellowship, Department of Biology and the Graduate school, University of Kentucky- $4667

2019 Travel award, Department of Biology and the Graduate school, University of Kentucky; to attend EMBO Workshop: Limb Development and Regeneration: New Tools for a Classic Model System, Barcelona, Spain- $1015

2018 Ribble mini grant, Department of Biology, University of Kentucky- $700

2018 Society for Developmental Biology student/postdoc travel award; to attend the SDB 77th annual Main Meeting, Portland, OR, USA- $200

2018 Travel award, Department of Biology and the Graduate School, University of Kentucky; Society for Developmental Biology 77th Annual Meeting at Portland, Oregon, USA- $876

2018 Travel award, Department of Biology and the Graduate School, University of Kentucky; Mid-Atlantic Regional Society for Developmental Biology Meeting held at the University of Virginia-$109

2018 Sherr special opportunity award, Department of Biology, University of Kentucky; to work at the Monaghan lab (Northeastern University, Boston, MA) to learn CRISPR based gene knockout technique in Ambystoma mexicanum-$1080

2018 Morgan Graduate Fellowship, Department of Biology, University of Kentucky-$12000

2017 Travel award, Department of Biology and the Graduate school, University of Kentucky; to attend the 14th International Conference on Limb Development and Regeneration, Edinburgh, United Kingdom- $1133

2016 Society for Developmental Biology student/postdoc travel award; to attend the SDB 75th annual Main Meeting, Boston, MA, USA- $200

2016 Travel award, Department of Biology and the Graduate School, University of Kentucky; to attend the SDB 75th annual Main Meeting, Boston, MA, USA- $900

Available for download on Wednesday, June 09, 2021

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