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Transcriptional Profiles of Differentiating Periocular Neural

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Transcriptional Profiles of Differentiating Periocular Neural ABSTRACT Transcriptional profiles of differentiating periocular neural crest cells and the function of Nephronectin during chick corneal development by Lian Bi During eye formation, periocular neural crest cells (pNC) migrate and differentiate to form the anterior ocular structures. In the chick cornea, this process involves two waves of migration that result in the formation of the corneal endothelium and stroma. Abnormalities in pNC migration lead to corneal malformation, such as anterior segment dystrophy. Corneal dystrophies, infections, and injuries can lead to corneal blindness, one of the major causes of blindness. Alternative treatments are developed because of the limitation of traditional corneal transplantation. These treatments benefit from the study of the molecular basis of corneal development and regeneration. However, corneal development is not fully understood. The purpose of this work was to elucidate the gene expression profiles during pNC migration and to examine the function of a highly regulated gene, Nephronectin (NPNT), during corneal formation. By performing RNA-seq analysis comparing pNC to the derived corneal structures, I analyzed differentially expressed genes and examined differentiated pathways during corneal formation. This project was designed to summarize the transcriptional regulation that happens at three levels: signaling pathways, transcription factors, and the downstream endothelial and stromal genes, providing gene candidates involved in corneal formation for future studies. From the RNA-seq analysis, I identified novel upregulation of NPNT among the extracellular matrix (ECM) proteins of the cornea. NPNT has been studied in other developmental processes but has not been linked to the corneal formation. I report that NPNT is distributed in the primary stroma during pNC migration. Its receptor, Integrin (ITG) α8, is expressed in the pNC that migrate on the primary stroma. Thus, I hypothesized that NPNT interacts with ITGα8β1 to promote pNC migration during corneal development. I performed functional studies by the RCAS- RNAi system. The knockdown of either NPNT or ITGα8 resulted in the reduction of corneal stromal thickness. Further studies revealed that NPNT overexpression upregulated cell numbers in the corneal stroma but did not increase cell proliferation. Inhibition of ITGα8 in vivo and in vitro both reduced pNC migration. Together, the functional studies link NPNT/ITGα8β1 signaling to pNC migration during corneal formation. This study reveals a previously unknown ECM-receptor pathway in corneal formation, suggesting a potential gene target or culture matrix in corneal development and regeneration. Collectively, this work depicts the transcription profiles during chick embryonic corneal development and investigates the function of a candidate ECM protein, NPNT, in this process. This comprehensive analysis served as a foundation of the molecular mechanisms underlying pNC migration, proliferation, and differentiation, providing potential clinical targets during corneal development and induction signals for corneal regeneration. Acknowledgments First and foremost, I would like to thank Dr. Peter Lwigale for his guidance and mentorship in my graduate research and thesis writing. I also want to express my love for my fantastic labmates, Justin, Anna, and Ruda, and great thanks to their help and encouragement. Our friendship will last forever. Thanks to previous Lwigale lab members and undergrads who mentored or assisted me in my research. I also wish to thank my committee members, BCB professors, and BCB faculty. The support I received from you was precious. This work could not have been completed without the support of many other people. Over the past five years, I made friends with wonderful people within or outside of Rice Biosciences. They are always there to help. Thank my friends Siyu and Wenbo, who are in different cities but stay connected all the time. Thank my parents, who are far away but always behind me to provide unparalleled support. Special thanks go to my fluffy kitties, Peanut and Brucie, for being the best companions during my writing. Lastly, best wish to my best roommate Yidan who is also defending in April. Contents Acknowledgments ..................................................................................................... iv Contents .................................................................................................................... v List of Figures ............................................................................................................ ix List of Tables ............................................................................................................. xi Abbreviations ............................................................................................................xii Chapter 1: Literature review ....................................................................................... 1 1.1. Overview of cornea .................................................................................................. 1 1.1.1. Structure and function of the cornea ................................................................ 1 1.1.2. Disease, damage and traditional treatment of the cornea ............................... 3 1.1.3. Innovative treatments for corneal disease........................................................ 5 1.2. Overview of corneal development ........................................................................... 7 1.2.1. Cranial neural crest cells .................................................................................... 7 1.2.2. Periocular neural crest migration and anterior segment formation ................. 9 1.2.3. Corneal formation in model organisms ........................................................... 11 1.3. Overview of corneal ECM ....................................................................................... 12 1.3.1. ECM proteins in corneal development ............................................................ 13 1.3.2. Role of ECM in corneal wound healing ............................................................ 15 1.3.3. Role of ECM in corneal diseases ...................................................................... 17 1.4. Overview of ECM protein Nephronectin ................................................................ 18 1.4.1. Potential receptors of NPNT ............................................................................ 19 1.4.2. Npnt/Itgα8β1 signaling is critical in mouse kidney formation ........................ 21 1.4.3. Function and regulation of Npnt in osteoblast differentiation ....................... 22 1.4.4. Function of NPNT in other tissue formation processes ................................... 23 1.4.5. Clinical application of Npnt .............................................................................. 25 1.5. Hypotheses and Objectives .................................................................................... 26 Chapter 2: Materials and Methods ........................................................................... 27 2.1. Animals ................................................................................................................... 27 2.1.1. Manipulation of the embryo ........................................................................... 27 vi 2.1.2. Tissue collection............................................................................................... 27 2.1.3. Egg windowing for in ovo manipulation .......................................................... 28 2.2. Cell and tissue culture ............................................................................................ 29 2.2.1. DF-1 cell maintaining ....................................................................................... 29 2.2.2. Viral production with DF-1 cells ...................................................................... 29 2.2.3. In vivo explant culture ..................................................................................... 30 2.2.4. BrdU staining ................................................................................................... 30 2.3. Histology ................................................................................................................. 31 2.3.1. Modified Carnoy’s fixative ............................................................................... 31 2.3.2. PFA fixation ...................................................................................................... 31 2.3.3. Methanol fixation ............................................................................................ 31 2.3.4. Paraffin embedding and section ...................................................................... 32 2.3.5. Cryo-sectioning ................................................................................................ 32 2.3.6. Hematoxylin-eosin staining ............................................................................. 33 2.4. In situ Hybridization ............................................................................................... 33 2.4.1. Probe synthesis ................................................................................................ 33 2.4.2. Section and whole-mount in situ Hybridization .............................................. 34 2.5. Section and whole-mount immunostaining ........................................................... 35 2.6. Reverse transcription
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