Cells produce and secrete proteins to the extracellular space that assemble to form an extracellular matrix (ECM). The ECM is essential for growth, wound healing, and structural development. Defects in the ECM proteins themselves or their associated secretory machinery often manifest in clinical phenotypes. Much of the secretory machinery was discovered in yeast. Yeast, as single-cell organisms, do not secrete ECM proteins. Thus, the role of secretory machinery in ECM secretion has remained largely unexplored. A protein known as Rgp1 had been shown in yeast and mammalian cells to bind Ric1 and function as a guanine nucleotide exchange factor (GEF) complex for Rab6. However, the role of Rgp1 has not been examined in situ in multicellular organisms, and no vertebrate model was available to study its function. To understand the role of Rgp1 in a vertebrate, I used CRISPR/Cas9 genome editing to generate a stable rgp1-null (rgp1-/-) zebrafish line. My preliminary studies have shown that in the absence of Rgp1, zebrafish embryos manifest with malformed craniofacial cartilage, a tissue primarily composed of chondrocytes. The rgp1-deficient chondrocytes accumulate collagen intracellularly and adopt a biconcave discoid morphology, suggesting a more complex function of Rgp1 in multicellular organisms. The sequences of zebrafish and human RGP1 sequences are highly conserved; therefore, I predict that findings in zebrafish will inform on Rgp1 biology in humans and other vertebrates. The aims of this project are to assess the role of Rgp1 in ECM secretion and to determine the role of Rgp1 in chondrocyte cell shape development. This project uses genetic manipulations, immunofluorescence, and live cell imaging experiments to determine subcellular functions of Rgp1 to better understand how seemingly disconnected trafficking and cell shape pathways are regulated through a single protein. This study will help explain a connection between protein trafficking, ECM deposition, cell shape changes, and craniofacial skeleton development.