A UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferase Is Required for Epithelial Tube Formation

E Tian,Kelly G Ten Hagen

doi:10.1074/jbc.m606268200

Abstract

Epithelial tubes are essential for the proper function of a diverse array of eukaryotic organs. Here we present a novel class of genes required for maintaining epithelial cell shape, polarity, and paracellular barrier function in the Drosophila embryonic tracheal system. Mutations in one member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family (pgant35A) are recessive lethal and result in tracheal tubes that are irregular in diameter and morphology. Further analysis of the pgant35A mutants reveals diminished levels of the apical determinant Crbs and the luminal marker 2A12, concomitant with increased staining in cytoplasmic vesicles within tracheal cells. GalNAc-containing glycoproteins are severely diminished along the apical region of the tracheal system as well. Tracheal cells become irregular in size and shape, and septate junction proteins are mislocalized to a more apical position. Most notably, paracellular barrier function is lost in the tracheal system of the mutants. Overexpression of wild type pgant35A under control of the trachea-specific breathless (btl) promoter results in partial rescue of the lethality. We propose a model where pgant35A is required to establish proper apical composition of tracheal cells by influencing apical delivery of proteins/glycoproteins. Disruption of the normal apical content results in altered cell morphology and loss of paracellular barrier function. These studies demonstrate a previously unrecognized requirement for mucin-type O-glycosylation in epithelial tube integrity and have obvious implications for epithelial morphogenesis in higher eukaryotes, since a unique ortholog to pgant35A exists in mammals.

Highlights

Epithelial tubes are crucial components of higher eukaryotic organs, such as the kidney, lung, salivary gland, and vascular system
The Drosophila tracheal system has served as a model system for epithelial tube formation and has aided in elucidating factors important for tube morphogenesis in mammalian systems [1,2,3,4]
Septate junctions influence cell shape and tube size, since some septate junction proteins have been found to limit the extent of apical cell surface formation [16, 20], and others severely alter tracheal tube morphology [2, 19, 21]

Summary

EXPERIMENTAL PROCEDURES

Drosophila Crosses—The mutants used in this study were described previously [24] and are as follows: pgant35AHG8, pgant35ASF32, and pgant35A3775 (Fig. 1C). Rescue experiments were performed using transgenic flies (w; P{UAS-pgant35A#5}/P {UAS-pgant35A#5}) with insertions on the third chromosome and the following stocks: w; btl-Gal4/TM3, Ser [29] All stocks used in the rescue experiments were first crossed into pgant35Amutant backgrounds to generate both Gal driver lines and P{UASpgant35A} transgenic lines heterozygous for pgant35Amutant/ CyO These heterozygous lines were crossed as follows, and rescue of the pgant35A lethality was assessed by the presence of straight winged, Sbϩ progeny: pgant35Amutant/ CyO; P{UAS-pgant35A#5}/P{UAS-pgant35A#5} females ϫ pgant35Amutant/CyO; P{tubP-GAL4}LL7/TM6C, cu, Sb, e, ca males. Whole Mount Antibody Staining and Lectin Staining—Embryos homozygous for pgant35A mutations were selected by lack of GFP fluorescence and fixed as previously described [30].

RESULTS

Larvae that pupate

Findings

DISCUSSION