Abstract The apical membrane of epithelial cells in the intestine play critical roles in physiology and homeostasis. Many specialized functions of intestinal epithelial cells, such as nutrient absorption, depend on faithfully sorting proteins to the apical membrane during biosynthetic delivery. However, the cellular mechanisms driving apical membrane protein sorting and how it matures during development are poorly defined. Also unknown is what controls the onset of apical endocytosis in Lysosome Rich Enterocytes (LREs). To discover mechanisms of apical membrane polarization in vivo, we performed a forward genetics screen for factors required for sorting of a model apical cargo (O-glycan-GFP, Og-GFP) in the zebrafish intestine. We isolated distinct classes of mutants affecting apical Og-GFP sorting. Our preliminary data has uncovered a pH-dependent mechanism for sorting membrane proteins in subapical endosomes either to the degradative pathway or to the apical membrane. We also discovered a role for regulated pre-mRNA intron splicing in activating apical membrane transport pathways crucial for polarized protein delivery in enterocytes and for apical endocytosis in LREs of the developing intestine. Our studies will uncover genetic and cellular mechanisms crucial for the maturation of a fully polarized and physiologically active epithelium in the vertebrate gut.