Embryonic and adult tissue were processed for hematoxylin and eosin (H and E) staining, Herovici’s collagen stain and, immunohistochemistry/immunofluorescence (IHC) as previously described (Dina et al., 2015 ; Durst et al., 2015 ; Sauls et al., 2015 ). IHC of cilia stains to look at expression and measure cilia length were done on 15 μm thick sections to insure measurement of full cilia length. Histology and IHC were preformed using 5 μm thick sections from E11,13,15,17, P0, and Adult (4month) aortic valves. Herovici stains were preformed using Herovici’s Collagen Stain Kit Procedure (American MasterTech, LODI, CA, Cat#KTHERPT). For immunohistochemistry (IHC): Antigen retrieval was performed for 1 minute using antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA, Cat#H-3300) by pressure cooker (Cuisinart, Stamford, CT, USA). The following are the antibodies and their dilutions; Acetylated Tubulin (Sigma, Cat#T6793, 1:500), Gamma Tubulin (Abcam, Cambridge, MA, USA, Cat#ab11317, 1:1000), Versican (gift from Stan Hoffman, Medical University of South Carolina, 1: 250), Collagen (gift from Stan Hoffman, Medical University of South Carolina, 1: 250), MF20 (DSHB, Iowa City, IA, USA, Concentrate, I:50), Ki67 (Abcam, Cat#ab16667, 1:250), Phospho-histone H3 (EMD Millipore, Darmstadt Germany, Cat#06-570, 1:250), Smoothened (LSBio, Seattle WA, Cat#LS-A2666, 1:250), Gli3 (Origene, Rockville MD, Cat#TA337186, 1:250). Primary antibody was detected using fluorescent secondary antibody, Goat anti-Mouse IgG Alexa fluor 488 (Cat#A-11029, 1:100), Goat anti-Rabbit Alexa fluor 488 (Cat#A-11034, 1:100) anti-Mouse Alexa fluor 568 (Cat#A-11004, 1:100), anti-Rabbit Alexa fluor 568 (Cat#A-11036, 1:100) Cy5 goat anti-Mouse (Cat#A-10524, 1:100) and Cy5 goat anti-Rabbit (Cat#A-10523, 1:100) (Life Technologies, Rockville, MD, US). Nuclei were counterstained with Hoechst (Life Technologies, Cat #H3569, 1:10,000) for 10 minutes and slides were cover slipped with Slow Fade mounting medium (Life Technologies, Cat#S36937). Fluorescence imaging was preformed using Zeiss Axioimager M2 and Leica TCS SP5 AOBS Confocal Microscope System (Leica Microsystems, Inc., 410 Eagleview Boulevard, Suite 107, Exton, PA 19341). Z-stacks were set by finding the highest and lowest depth with visible fluorescence and using the system optimized setting to determine steps. Z-stacks were then compiled to form maximum projection images.