Retinoic acid signaling regulates KRT5 independently of stem cell markers in submandibular salivary gland epithelium

............................................................................................................. v List of Tables ..................................................................................................... ix List of Figures ..................................................................................................... x Chapter 1: Introduction ....................................................................................... 1 1.1 Salivary gland overview ...................................................................... 1 1.2 Salivary gland diseases ...................................................................... 3 1.3 Treatment of salivary gland diseases ................................................. 4 1.4 Development of salivary glands .......................................................... 5 1.5 RA mechanism of action ..................................................................... 9 Chapter 2: Materials and Methods ...................................................................... 13 2.1 Mice .................................................................................................... 13 2.2 SMG whole gland ex vivo culture ....................................................... 13 2.3 Culture of ER ...................................................................................... 14 2.4 Stain for RARE-LacZ reporter activity ................................................. 16 2.5 Whole mount immunostain ................................................................. 17 2.6 Frozen section immunostain ............................................................... 18 2.7 Quantification of mRNA by qPCR ....................................................... 19 2.8 Antibodies ........................................................................................... 20 2.9 Primers ............................................................................................... 21 viii 2.10Statistical Analysis ............................................................................ 22 Chapter 3: Results .............................................................................................. 23 3.1 RA signaling occurs in multiple tissues of developing SMG ............... 23 3.2 Standard curve Verification of qPCR primers in cultured ER ............. 34 3.3 Inhibition of RA signaling impairs branching morphogenesis and FGF10 activity in cultured ER .................................................................. 42 3.4 Inhibition of RA signaling leads to downregulation of proliferation activity ...................................................................................................... 45 3.5 Block of RA signaling upregulates expression of KRT5 ..................... 48 3.6 Addition of RA signaling impairs branching morphogenesis in cultured ER ............................................................................................................ 52 3.7 Block of RA signaling coordinately upregulates expression of Krt5 and Krt14 ........................................................................................................ 58 3.8 Stem cell marker Kit is regulated inversely to Krt5/Krt14 by RA signaling ................................................................................................... 61 3.9 Bioinformatic analysis of RAR and RXR binding motifs near keratin and stem cell genes ................................................................................. 63 Chapter 4: Discussion ........................................................................................ 64 Chapter 5: Summary and Conclusions ............................................................... 73 References ......................................................................................................... 74 Curriculum Vitae ................................................................................................. 86