Fig. 1. Embryonic phenotypes of sll mutants. (A) Cuticle phenotype of a sll^7E18 homozygous embryo derived from heterozygous parents. Note occasional fusion of denticle belts (arrow). (B) Cuticle phenotype of a sll^7E18 homozygous embryo derived from a female carrying sll^7E18 germ-line clones (referred to as sll null mutant hereafter). Note the complete absence of smooth cuticle on the ventral side. (C) Wg expression in a wild-type embryo at stage 10. (D) En expression in a wild-type embryo at stage 11. (E) Wg expression in thoracic and abdominal segments of a sll^7E18 null mutant is completely abolished at stage 10. (F) En expression in thoracic and abdominal segments of a sll^7E18 null mutant is dramatically decreased at stage 11. (G) Expression of a UAS-wg transgene using a prd-Gal4 driver line in sll^7E18 null mutants. Note the development of smooth cuticle in alternating segments. (H) Expression of a UAS-hh transgene using a prd-Gal4 driver line in sll^7E18 null mutants. Note the development of smooth cuticle in alternating segments. Anterior is to the left and dorsal side up in all panels, except (A), which is a ventral view.

Fig. 3. Molecular identification of sll. (A) Cuticle phenotype of sll^l(3)24533 maternal and zygotic mutant larva. Note the occasional development of smooth cuticle in some segments, indicating that sll^l(3)24533 is not a null allele. (B) Pair-rule segmentation phenotype of sll^7E18 null mutant larvae expressing a UAS-sll transgene under the control of a prd-Gal4 driver line. (C) Gene structure of sll. The P-element insertions in the sll transcript are located 304 nucleotides [sll^l(3)142214] and 35 nucleotides [sll^l(3)24533], respectively, upstream of the start codon. The sll^7E18 allele has a C to T transition creating a nonsense mutation in the 42nd codon (nucleotide 127) of the reading frame. (D) The sll cDNA encodes a putative hydrophobic protein of 465 amino acids (52 kDa) with at least 10 predicted transmembrane spanning regions. (E) Homology alignment of Sll with a putative human protein (39.4%) (Protein Data Bank accession No. AK075456), a putative mouse Sll-like protein (41.1%) (BC036992), Caenorhabditis elegans gene M03F8.2 (28.8%) (NM072147), a putative human galactose transporter UGTREL1 (28.6%) (NM005827), a putative mouse galactose transporter (29.2%) (NM016752) and the Drosophila gene CG5802 (21.3%). Amino acid identity to Sll is indicated by percentage values in brackets. Protein accession numbers are in brackets.

Fig. 5. Expression of sll mRNA during embryonic development. (A) Northern blot showing expression of sll transcripts at different time intervals during embryogenesis. (B) Maternal expression of sll RNA in the nurse cells of the germline during oogenesis. (C–F) In situ hybridization of sll RNA to wild-type (C–E) and sll^7E18 mutant (F) embryos. (C) Maternal expression of sll RNA in the syncytial blastoderm embryo (stage 4). (D) Zygotic expression of sll RNA is first detected in the salivary gland placodes at stage 11. (E) Salivary gland specific expression of sll RNA at stage 14. (F) Expression of sll RNA in the salivary gland placodes of a sll^7E18 null mutant at stage 11. (G) Expression of Sll protein in the salivary glands of a wild-type embryo. (H) Expression of Sll protein in a prd-Gal4/UAS-sll embryo showing that the α-Sll antibody recognizes Sll specifically. Arrowheads indicate expression in epidermal stripes in the abdominal region. (C, D, F and H) Lateral view. (E and G) Ventral view. Anterior is to the left.

Fig. 2. Role of sll during wing development. (A and B) Examples of Wg distribution (red) in wing imaginal discs carrying sll^7E18 mutant clones. (A, center and right panel) Same as left panel showing only Wg expression. Compare Wg levels adjacent to the D/V boundary inside (arrow) and outside the clone. Note the reduction of Wg protein levels in sll^7E18 mutant cells. The D/V boundary often shows a change in direction associated with sll^7E18 mutant clones (arrowhead in center panel). (B, center and right panel) Same as left panel showing only Wg expression. Note bifurcation of the D/V boundary inside the sll^7E18 mutant clone (arrowhead in center panel). Homozygous sll^7E18 mutant cells are marked by the absence of GFP (green) expression in left panels and by the dashed outlines in center panels of (A), (B) and (C). (C) Ci distribution (purple) in wing imaginal discs carrying sll^7E18 mutant clones. The center and right panels are the same as the left panel showing only Ci expression. Note that Ci expression is moderately reduced in sll^7E18 mutant cells receiving the Hh signal. The arrowhead in the right panel indicates the region where strongest reduction is seen. (D–G) Wing phenotypes associated with sll^7E18 mutant clones. (D) Wing exhibiting a gap in the anterior margin. (E) Bifurcation of the anterior wing margin (arrow). (F) Wild-type wing. (G) Wing showing reduction in region between wing veins L3 and L4 (arrowhead). (H) Wing of dally⁵²⁷/dally^gem transheterozygote. Note truncation of wing vein L5 (arrowhead). (I) Wing of sll^l(3)142214/sll^7E18 transheterozygote showing gaps in the margin (arrow) and truncation of wing vein L5 (arrowhead) very similar to (H).

Fig. 4. sll encodes a PAPS transporter. (A) Transport activity of Sll for various nucleotide–sugar substrates and PAPS. The microsomes from sll-transfected S.cerevisiae were prepared and assayed for transport of the designated nucleotides at a concentration of 5 mM, as described in Materials and methods. Transport activity is shown in picomoles of substrate transported per milligram of microsomes per minute. Transport of GDP-Man is shown on a 10-fold higher scale compared with all other substrates. (B) Effect of nucleotide addition on PAPS transport into microsomal vesicles. Microsomal vesicles were prepared from sll-transfected S.cerevisiae and assayed for PAPS ³⁵S-transport activity in the presence of various nucleotide monophosphates at 100 µM concentration. In (A) and (B), the gray bars indicate transport by microsomes isolated from S.cerevisiae and transfected with empty vector. Black bars indicate transport by microsomes isolated from S.cerevisiae and transfected with a sll-expression vector. (C) The left panel shows the expression of Sll in salivary glands. Note the perinuclear localization of the staining. The arrow points to the nucleus of one cell visible as a dark area. The bar is 10 µm long. The center panel shows the expression of a Drosophila Golgi specific protein in salivary glands. The right panel was obtained by merging the left and center panels. Note the extensive co-localization of staining. (D) Genetic interaction between dally and sll in loss of the posterior postalar bristle (pPA). (E) Western blot analysis of HA–Dally from S2 cells incubated with buffer (–) or dsRNA specific for sugarless (sgl), or slalom (sll). Upper arrows indicate the size of HA–Dally in untreated cells. The lower arrow indicates the size of HA–Dally after RNAi treatment against sgl (center lane) or sll (right lane). (F) Amplification of sgl or sll RNA by RT–PCR from S2 cells incubated with buffer (–) or dsRNA (+) specific for sugarless (sgl) or slalom (sll). M, DNA size marker.

Fig. 6. Role of sll in the follicle cell epithelium. (A and B) Examples of cuticle phenotypes of embryos derived from females carrying sll^7E18 mutant clones in the follicle cell epithelium. Note the absence of ventral denticle belts in posterior abdominal segments (arrow in A) and the complete dorsalization of the cuticle in (B). (C) Twi expression in wild type at the cellular blastoderm stage (stage 5). (D–F) Examples of alterations in expression of the ventral marker Twi in embryos derived from females carrying sll^7E18 mutant clones in the follicle epithelium. (D) Dorsalization in the abdominal region. (E) Dorsalization in the gnathal and thoracic region. (F) Complete dorsalization of the entire embryo. Anterior is to the left, dorsal side up.