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First published online 27 July 2005
doi: 10.1242/dev.01962

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Drosophila Pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans

¹ Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA
² Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

View larger version (22K): [in a new window]   Fig. 1. Structure of the pipe locus and location of EMS-generated pipe mutations. (A) Intron structures of the various pipe isoforms are shown. On the far left are the isoform designations of the BDGP (PA-PK) and of Sergeev et al. (Sergeev et al., 2001). Box 10 is pipeST2. All mutations except pipe3 are associated with pipeST2-specific exons. pipe3 is located in the third common exon. (B). Structure of the coding region of PipeST2 showing the location of the eleven EMS-generated mutations. Green vertical bars indicate the position of exon/exon boundaries.

View larger version (92K): [in a new window]   Fig. 2. The pipeC14 allele leads to the loss of pipe RNA and protein expression in the ovary and the embryo. (A-C) In situ hybridization to a wild-type embryo (A) and to embryos homozygous for pipeC14 (B) and pipe2 (C). A pipe-ST2-specific probe was used. (D-F) An antibody directed against a peptide sequence present in the N terminus of all Pipe isoforms was used for immunostaining of wild-type (D), pipeC14 homozygous (E) and pipe2 homozygous (F) embryos. (G-I) In situ hybridization to stage 10 egg chambers from wild-type (G), pipeC14/Df(3L)pipeA13 (H) and pipe2/Df(3L)pipeA13 (I) females using pipe-ST2-specific sequences as a probe. (J) pipeC14/Df(3L)pipeA13 flies exhibit a growth defect relative to their heterozygous siblings. Similarly, windT6/windRP flies (K) are smaller than their wind/+ siblings. Both results suggest that zygotic expression of Pipe isoforms is required for normal growth.

View larger version (70K): [in a new window]   Fig. 3. Sodium chlorate influences embryonic dorsoventral patterning of the embryonic progeny of pipe7 mutant females Gastrulation patterns (A,C,E,G) and embryonic cuticles (B,D,F,H) of embryos produced by the following females: (A,B) Oregon R, (C,D) pipe7/Df(3L)A13untreated, (E,F) pipe7/Df(3L)A13 treated with sodium chlorate, (G,H) pipe1/Df(3L)A13.

View larger version (96K): [in a new window]   Fig. 4. Pipe and Windbeutel are required for Alcian Blue staining of embryonic salivary glands. (A) A stage 16 wild-type embryo stained with Alcian Blue. (B) The same embryo at higher magnification. Staging is according to Campos-Ortega and Hartenstein (Campos-Ortega and Hartenstein, 1985). Embryos heterozygous (C) and homozygous (D) for pipeC14 were stained with Alcian Blue, as were embryos heterozygous (E) and homozygous (F) for pipe3. Embryos homozygous for the RNA null allele pipeC14 or for the protein null allele pipe3 failed to stain with Alcian Blue. By contrast, embryos homozygous for pipe2, which affects only the Pipe-ST2 isoform, stain with Alcian Blue (G). Embryos homozygous for windT6 (H), windM88 (I) or windRP (J) do not produce Alcian Blue-staining material in their salivary glands.

View larger version (69K): [in a new window]   Fig. 5. Alcian Blue-staining in the embryonic salivary gland requires the activity of genes involved in biological sulfation. (A) Mutations affecting papss were identified by their increased sensitivity to the lethal effects of sodium chlorate. Concentrations of sodium chlorate are shown at top, as are the parental genotypes. At 25 mM and 30 mM sodium chlorate, few papss/+ pupae are present on the sides of the vials. (B-D) Embryos stained with Alcian Blue. Although papss2/+ embryos stain with Alcian Blue (B), embryos homozygous for papss2 lack Alcian Blue salivary gland staining (C), as do embryos homozygous for sll7E18 (D). (E-G) Staining of embryonic salivary glands with an antibody against Windbeutel. The salivary glands of an embryo heterozygous for papss2 stain for Windbeutel (E), as do the salivary glands of papss2 (F) and sll7E18 (G) mutant embryos.

View larger version (88K): [in a new window]   Fig. 6. The Alcian Blue-stained material in the embryonic salivary glands requires papss but not the expression of GAG-related genes. FLP-FRT-mediated recombination and the dominant female-sterile technique were used to generate adult females with germline clones homozygous for papss (A-C), sgl (D-F), sfl (G-I) and frc (J-L). For each of the four genes, embryos lacking both maternal and zygotic expression exhibited a typical segment polarity phenotype (C,F,I,L). Staining with an antibody directed against Windbeutel demonstrated that these embryos nevertheless developed salivary gland tissue (arrowheads in B,E,H,K). The salivary glands of embryos lacking both maternal and zygotic expression of papss failed to stain with Alcian Blue (A), but the salivary glands of embryos lacking maternal and zygotic expression of sgl (D), sfl (G) and frc (J) did stain.

View larger version (115K): [in a new window]   Fig. 7. GAGs are not required in the ovarian follicle cell layer for embryonic DV polarity. The technique of Nilson and Schüpbach (Nilson and Schüpbach, 1998) was used to generate marked mutant follicle cells whose position in the follicle layer could be determined by their altered chorion imprints. In all cases shown, mutant clones were located ventrally in the follicle cell layer, and are identifiable by the more transparent appearance of the chorion imprints in comparison to those made by wild-type cells. (A,B) Ventral clones of pipe mutant cells led to the production of embryos that were partially dorsalized, as indicated by the tail-up phenotype (A,B) and the narrow ventral denticle bands (A). By contrast, ventral follicle cells mutant for papss (C), sgl (D), sfl (E) and frc (F) did not lead to the production of dorsalized embryos.