Background & aims: During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown.
Methods: By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy.
Results: Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking.
Conclusions: Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.
Keywords: AEC, apical endocytic complex; AP, alkaline phosphatase; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/cas9 endonuclease; EDTB, endotubin; EEA1, early endosomal antigen 1; Endosomes; Endotubin; G, guide; GFP, green fluorescent protein; GTPase, guanosine triphosphatase; KO, knockout; LAMP1, lysosome-associated membrane protein 1; MAMDC4, MAM domain containing 4; MVID, microvillus inclusion disease; P, postnatal day; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Rab; SDS, sodium dodecyl sulfate; TBST, tris-buffered saline with 0.1% tween-20; TEM, transmission electron microscopic; TJ, tight junction; Tight Junction; Trafficking.