Drosophila XBP1 expression reporter marks cells under endoplasmic reticulum stress and with high protein secretory load

Abstract

Expression of genes in the endoplasmic reticulum (ER) beyond its protein folding capacity activates signaling pathways that are collectively referred to as the Unfolded Protein Response (UPR). A major branch of the UPR pathway is mediated by IRE1, an ER-tethered endonuclease. Upon ER stress-induced activation, IRE1 splices the mRNA of XBP1, thereby generating an active isoform of this transcription factor. During normal Drosophila development, tissues with high protein secretory load show signs of IRE1/XBP1 activity indicative of inherent ER stress associated with those cell types. Here, we report that the XBP1 promoter activity itself is enhanced in secretory tissues of Drosophila, and it can be induced by excessive ER stress. Specifically, we developed a Drosophila XBP1 transcription reporter by placing dsRed under the control of the XBP1 intergenic sequence. DsRed expression in these xbp1p>dsRed transgenic flies showed patterns similar to that of xbp1 transcript distribution. In healthy developing flies, the reporter expression was highest in salivary glands and the intestine. In the adult, the male reproductive organs showed high levels of dsRed. These tissues are known to have high protein secretory load. Consistently, the xbp1p>dsRed reporter was induced by excessive ER stress caused by mutant Rhodopsin-1 overexpression. These results suggest that secretory cells suffer from inherent ER stress, and the xbp1p>dsRed flies provide a useful tool in studying the function and homeostasis of those cells.

Figure 1. XBP1 regulatory sequence drives dsRed expression in developing secretory tissues.

(A) Comparison of the xbp1 locus sequence between D. melanogaster, D. simulans, D. yakuba and D. erecta. Sequence conservation is not only found in the coding sequence of genes (purple) and UTRs (light blue), but also in the intergenic region (red) that may encode enhancers and promoters. The bracket (xbp1p) indicates the intergenic sequence that was placed upstream of the dsRed reporter. (B) xbp1 in situ hybridization in embryos. (C-E) xbp1_p>dsRed reporter expression in the embryo (C), the larval salivary gland (D) and the larval intestine (E). Images in (D, E) are composites of several frames taken with the 10x confocal microscope lens. There was no detectable xbp1_p>dsRed expression in the larval brain and imaginal discs (D). Abbreviations: Salivary Gland (SG), Mid Gut (MG).

Figure 2. Co-labeling of xbp1_p>dsRed with an IRE1-mediated XBP1 splicing reporter.

Shown is a composite image of multiple 10x confocal microscopy images of the 3^rd instar larval brain, eye discs and salivary glands. Labeled in red is the xbp1_p>dsRed reporter, and in green is the IRE1-mediated XBP1 mRNA splicing reporter. The latter (IRE1 reporter) was driven ubiquitously through the tubulin-Gal4 driver. (A) Double labeling of dsRed and GFP shows overlap in the salivary glands. On the other hand, the brain and the eye discs primarily show IRE1 activity without XBP1 expression. (B) A GFP only channel of the image shown in (A).

Figure 3. xbp1_p>dsRed expression in the male reproductive system.

Prominent dsRed signal was found in the adult abdomen of males (A), but not in females (B). Dissected adult tissues show the strongest dsRed signal in the accessory gland and the ejaculatory duct (C).

Figure 4. xbp1_p>dsRed expression is induced by misfolded membrane proteins.

Shown are larval eye imaginal discs with xbp1_p>dsRed (red). Control eye discs show no basal level of reporter activity (A). xbp1_p>dsRed is induced by the overexpression of a mutant membrane protein, Rhodopsin-1^G69D (green) (B), or the spliced xbp1 isoform xbp1-RB (D) in the eye using the GMR promoter. On the other hand, the unspliced xbp1 isoform, xbp1-RA, did not induce xbp1_p>dsRed expression (C). (E) ire1 is not required for xbp1_p>dsRed induction by mutant Rhodopsin-1^G69D. ire1 mosaic clones were generated in the eye discs and marked by the absence of GFP. (E’, E’’) show higher magnification images of the inset in (E). GMR promoter driven Rhodopsin-1^G69D (blue) induces xbp1_p>dsRed in both ire1+ and ire1 −/− cells, although the intensity of dsRed is moderately reduced in ire1 −/− clones. (E’’) is a dsRed only channel of (E’).

Figure 5. xbp1_p>dsRed is expressed in the salivary gland of ire1 −/− larva.

(A) Shown are 48 – 72 hour larvae of the ire1^f02170 −/− (left three) and ire1 −/+ (right two) genotypes. xbp1_p>dsRed was recombined to the ire1^f02170 chromosome to facilitate genotyping. The ire1+ chromosome is marked with GFP. No ire1 −/− larvae were found to grow beyond this 1^st instar larval stage. (B, C) A higher magnification view of an ire1 −/− larva. xbp1_p>dsRed expression in the salivary gland is visible (arrows) in the ire1 −/− zygotic (B), as well as maternal, zygotic mutants (C).