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First published online 9 April 2008
doi: 10.1242/dev.020891

Development 135, 1745-1749 (2008)
Published by The Company of Biologists 2008
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Wollknäuel is required for embryo patterning and encodes the Drosophila ALG5 UDP-glucose:dolichyl-phosphate glucosyltransferase

Achim Haecker1, Mattias Bergman1, Christine Neupert2, Bernard Moussian3, Stefan Luschnig3,*, Markus Aebi2 and Mattias Mannervik1,{dagger}

1 Stockholm University, Wenner-Gren Institute, Developmental Biology, Svante Arrhenius Väg 16-18, SE-106 91 Stockholm, Sweden.
2 Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
3 Max-Planck Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstraße 35, D-72076 Tübingen, Germany.


Figure 1
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  		Fig. 1. Posterior patterning is disrupted in embryos derived from wol germline clones. Anterior is to the left in all figures. Genotypes, RNA probes and antigens are indicated. (A-C) Cuticle preparations of Drosophila embryos prior to hatching. Some posterior denticle belts are missing and the remainder are curled-up in wol embryos, as compared with wild type (wt). (D-I) Ventrolateral views of embryos undergoing germband elongation. Every other en stripe is missing in the posterior of wol embryos. (G,H) Eve protein (brown) labels the odd-numbered en stripes (blue). Arrowheads point to en stripes 7 and 9. (I) Posterior en stripes are missing in embryos from cad germline clones. (J-N) Lateral views of pre-cellular embryos showing changes in gt and kni expression in wol and cad embryos as compared with wild type. Arrow in N indicates a shift in position of the kni stripe towards posterior. (O-R) Maternal cad mRNA is present at similar levels in wild-type and wol mutant embryos, whereas the Cad protein gradient is reduced in wol embryos (R).

 

Figure 2
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  		Fig. 2. Reduced Dpp signaling causes dorsal-ventral phenotypes in wol1 germline clone embryos. (A,B) Ventrolateral views of cellularized Drosophila embryos. Expression of rho in dorsal cells occurs in wild type in response to Dpp signaling, but is lost in wol embryos (arrows). (C-E) Dorsal views of cellularized embryos showing expression of the Dpp target gene Race (C,D) or of Race and dpp (E). Ectopic dpp (purple) expressed from the Krüppel enhancer (Kr:dpp) was able to rescue Race expression (brown) in wol mutant embryos (E). (F,G) Lateral views of cellularizing embryos. Expression of dpp is reduced in wol embryos (G).

 

Figure 3
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  		Fig. 3. wol encodes the Drosophila ALG5 UDP-glucose:dolichyl-phosphate glucosyltransferase. (A) The gene CG7870/ALG5 consists of four exons encoding a 326 amino acid protein (coding region shaded dark gray). The lesions in the wol1 and wol2 alleles are shown. A transgene extending from -474 to +2340 was able to rescue the lethality and fertility of the wol alleles. (B-D) Expression of wol/ALG5 in wild-type embryos (lateral views in B,C, and a dorsal view in D). In cellularizing embryos (B), maternal ALG5 RNA is ubiquitous. At stage 11, zygotic exression begins in salivary gland precursor cells (arrowhead, C). Strong expression in salivary glands and part of the proventriculus is detected in stage 16 embryos (D). (E) Western blot analysis of carboxypeptidase Y (CPY). CPY glycoforms lacking one (-1) or two (-2) oligosaccharide chains can be detected in a {Delta}alg5 mutant yeast strain. Transformation of yeast (pScALG5) or fly ALG5 (pDmALG5) cDNA restores CPY glycosylation, whereas fly ALG5 cDNAs with either the wol1 or wol2 mutations fail to do so.

 

Figure 4
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  		Fig. 4. The unfolded protein response (UPR) is triggered in wol embryos. (A) Splicing of xbp1 mRNA was examined by RT-PCR. In wild-type Drosophila embryos, only the unspliced 127 bp product was detected, whereas some spliced 104 bp xbp1 message was found in wol1 embryos. In untreated S2 cells, mostly unspliced product is present. Tunicamycin (tm) or DTT treatment shifts the ratio of spliced/unspliced in favor of the spliced product. (B) Increased eIF2{alpha} phosphorylation in wol embryos. Western blot analysis of phosphorylated and total levels of eIF2{alpha}. The ratio of phosphorylated/total eIF2{alpha} was calculated from two independent experiments.

 

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© The Company of Biologists Ltd 2008