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Slow as Molasses is required for polarized membrane growth and germ cell migration in Drosophila

¹ Howard Hughes Medical Institute, Developmental Genetics Program, Skirball Institute at NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
² Department of Genetics, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA
³ Incyte Genomics, 3160 Porter Drive, Palo Alto, CA 94304, USA

View larger version (83K): [in a new window]   Fig. 1. slamwaldo mutants have a strong and penetrant germ cell migration phenotype. Anterior is towards the left in all panels. (A,B,D,E) Lateral views. (C,F) Dorsal views. (A-C) Wild-type embryos. (D-F) slamwaldo embryos. All embryos are labeled with -Vasa to mark germ cells in brown. Embryos in A,D are labeled with RACE RNA to mark the midgut in blue. Embryos in B,C,E,F are labeled with the 412 retrotransposon RNA to mark lateral mesoderm and SGPs (arrowheads) in blue. (A,D) By stage 11, germ cells in slamwaldo embryos have properly migrated through the gut epithelium; however, the midgut has not become mesenchymal and flattened out. (B,E) By stage 12, most of the germ cells in wild type (B) are in the mesoderm and in contact with three SGP clusters (arrowheads). Many germ cells in slamwaldo embryos (E) are still on the midgut (arrow), though some are correctly located in the mesoderm next to the two posterior SGP clusters (left two arrowheads in E). (C,F) At stage 14, germ cells and SGPs coalesce to form two round gonads. Though some germ cells in slamwaldo embryos are in the gonads (F), on average 50% are located at ectopic locations, either on the midgut (arrowheads) or in the posterior end of the embryo (arrows).

View larger version (67K): [in a new window]   Fig. 2. slamwaldo; Hmgcr double mutants have a different phenotype from either single mutant. Dorsal views of stage 14 embryos, stained with -Vasa to mark germ cells, anterior is towards the left. (A) Wild type, (B) slamwaldo, (C) Hmgcr, (D) slamwaldo; Hmgcr. In slamwaldo; Hmgcr, germ cells do not move off the gut, and the embryos are poorly differentiated.

View larger version (75K): [in a new window]   Fig. 3. slamwaldo M–Z– mutants have a cellularization defect. (A-D) Wild type, (E-H) slamwaldo M–Z– mutants, embryos are stained with -myosin in green to mark the advancing basal-most part of the cell membrane, and in red with DAPI to mark the nuclei. Arrows mark cellularization front. I-K are stained with -Vasa to mark germ cells. (A,E) Pre-cellularization embryos, the nuclei are lined up under the plasma membrane, which has not begun to invaginate. slamwaldo M–Z– is indistinguishable from wild type at this stage. (B,F) Mid-cellularization, nuclei are elongating in both wild type and slamwaldo M–Z–. The membrane has begun to invaginate in wild type, but not in slamwaldo M–Z–. (C,G) Mid-late cellularization, in wild type, the nuclei have continued to elongate as the membrane has advanced almost to the end of the nuclei. The membrane in slamwaldo M–Z– still has not begun to invaginate. (D,H) End of cellularization, in wild type, the membrane has now reached its final basal position, past the nuclei, and has pinched off individual cells. In slamwaldo M–Z–, the membrane has begun to invaginate, but it does not advance beyond the nuclei once it begins to pinch off. The nuclei are trapped in the pinching membrane. The appearance of two lines of basal Myosin staining is due to the section being slightly tangential, revealing apposing membranes. (I) slamwaldo M–/–Z–/– mutant. (J) M–/–Z–/+ slam cellularizes properly. (K) Germ cell migration is normal in M–/–Z–/+ slam, stage 14.

View larger version (70K): [in a new window]   Fig. 4. slam is encoded by CG9506 and is expressed in blastoderm embryos. (A) Male mitotic recombination mapping placed slam between k13720 and k07502, a region with 16 predicted genes. (B) Genomic structure of CG9506. Boxes represent exons. Sequencing of the slamwaldo alleles revealed a nonsense mutation at Q722 in slamwaldo1 and a glycine to lysine change at G879 in slamwaldo2. (C) slam RNA expression in a stage 2 embryo. There is no zygotic transcription at this stage, thus the weak expression in early embryos is maternal RNA contribution. (D) Stage 4. There are high amounts of slam in somatic nuclei, but not in germ cells, where zygotic transcription is repressed (inset). (E) Stage 6 embryo. By the end of cellularization, slam decays to three stripes in the blastoderm. (F) Stage 11 embryo. By the end of gastrulation, slam is no longer expressed.

View larger version (71K): [in a new window]   Fig. 5. Slam protein is expressed in the blastoderm embryo. Lateral views of embryos stained with -Slam, with accompanying transverse 2 µm sections. Dorsal upwards, anterior towards the left in whole mounts. Dorsal upwards in sections. (A) Pre-cellularization (arrow marks Slam staining at membrane, arrowhead marks Slam staining basal to the nuclei). (B) Mid-cellularization. (C) End cellularization. Note decreased expression on ventral side of section. (D) Beginning of gastrulation. Invaginating mesoderm has lost Slam staining while Slam is still present in the dorsal epithelium of the section. (E) Stage 10, end of gastrulation. (F,G) Peptide competition experiment. Slam antibody was pre-incubated with the peptide used to generate -Slam (F) or a control Nanos peptide (G) before incubation with wild-type embryos.

View larger version (71K): [in a new window]   Fig. 6. Slam localizes to the furrow canals during cellularization. Embryos are stained in green with -Neurotactin, which marks surfaces of membrane during cellularization. -Slam is in red; overlap is yellow. (A-D) Slam localizes to the basal-lateral domain of the invaginating plasma membrane from early cellularization (A) to the end of cellularization (D). Note the decrease of Slam expression in the ventral part of the embryo at the end of cellularization (D). (E,F) Posterior pole during early (E) and late (F) cellularization. Slam is not expressed in the germ cells. (G) Enlarged double-labeling of Slam and Neurotactin reveals Slam (red) preceding Neurotactin (green) in growing membrane during early cellularization. (H) Section (2 µm) through gastrulating embryo. White arrow indicates lack of Slam in the farthest invaginated cells. Black arrow indicates presence of Slam in the basal membrane of cells that will become adjacent to migrating germ cells at stage 10-11. White arrowhead indicates germ cells.

View larger version (21K): [in a new window]   Fig. 7. Slam is hypothesized to deposit a germ cell guidance cue early that will act later in embryogenesis. Slam expression, green; posterior midgut primordium, red; germ cells, yellow; hypothesized guidance cue, blue triangles. (A) Blastoderm embryo, Slam is expressed on the basal-lateral membrane of all cells. A guidance cue (blue triangles) is expressed in the posterior midgut (if not in other cells as well), which is deposited on the basal-lateral membrane by Slam. (B) Gastrulating embryo, Slam expression begins to decrease. (C) Stage 9-10, germ cells are exiting the midgut pocket, moving from the apical to the basal side of the midgut cells. Slam protein is no longer present, but the guidance cue remains. (D) Stage 11, germ cells are guided off the midgut into the mesoderm by the guidance cue that requires Slam.