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Mosaic analyses reveal the function of Drosophila Ras in embryonic dorsoventral patterning and dorsal follicle cell morphogenesis

¹ Program in Genetics,
² Molecular and Cellular Biology Program, Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195-7730, USA

View larger version (80K): [in a new window]   Fig. 1. Wild-type pipe-lacZ expression is dynamic during oogenesis. (A-D) Confocal projections of egg chambers from females homozygous for the pipe-lacZ reporter construct. lacZ expression was detected using an anti-ß-gal antibody. Lateral views are shown, except where noted, with anterior to the left and dorsal at top. (A) At stage 8, pipe-lacZ is expressed in the anterior follicle cells that stretch to compensate for posterior migration (small white arrow) and in a sagittal ring (bracket) at the anterior margin of the posteriorly migrating group. Posterior follicle cells, and some posterior ventral follicle cells, express pipe-lacZ, with a gap in ventral expression about halfway across the columnar epithelium (arrowhead). (B) Dorsolateral view. As posterior follicle cell migration is completed at the end of stage 9, stretch cell and sagittal ring expression are maintained and ventral expression becomes contiguous. (C) Ventrolateral view. During stage 10A, the sagittal ring pattern gradually disappears, beginning dorsally and proceeding ventrally, such that expression protrudes dorsally from the anterior end of the ventral expression domain (arrowhead). (D) By stage 10B, dorsal sagittal ring expression has completely disappeared, while expression remains in the ventral, stretch (not shown), and posterior follicle cells. (E) Schematic diagram depicting wild-type pipe-lacZ expression in stretch (blue) and columnar (purple) follicle cells over time.

View larger version (107K): [in a new window]   Fig. 2. Ras and Egfr are required cell-autonomously for dorsal repression of pipe-lacZ. Confocal projections of control (A), RasC40b (B,D,E), and EgfrCO (C) follicle cell clones, marked by the absence of the myc epitope. Clones are outlined in yellow. pipe-lacZ expression was detected by ß-gal antibody. Anterior is to the left in all panels. (A) Dorsal view of a large control clone in which dorsal pipe-lacZ repression is unaffected. The posterior expression seen in this egg chamber is normal and is contiguous with the normal ventral region of pipe-lacZ expression (out of the plane of focus). Dorsal appendages from an adjacent S14 egg chamber abut the posterior half of this egg chamber. (B) pipe-lacZ is cell-autonomously derepressed in a dorsal Ras follicle cell clone. (C) Dorsolateral view of two Egfr clones: dorsal clone (top) and ventrolateral clone (bottom) both cell-autonomously derepress pipe-lacZ. (D,E) Lateral views of Ras mosaic egg chambers. In both egg chambers, the edge of the normal ventral and posterior domains of pipe-lacZ expression can be seen (small arrows), as can cell-autonomous pipe-lacZ derepression in the clones. (D) A patch of lateral follicle cells ventral/posterior to a large dorsal/lateral Ras clone still represses pipe-lacZ (large arrowhead). (E) Ventral pipe-lacZ expression spreads dorsally (large arrowhead) into the lateral region in this egg chamber with a dorsal Ras clone. This expansion, however, is also seen in wild-type (Fig. 1).

View larger version (69K): [in a new window]   Fig. 3. Twist protein is normal in most embryos derived from egg chambers with dorsal Ras clones. (A-D) Confocal projections (black/white-inverted) of anti-Twist fluorescence in cellularizing embryos hand-picked based upon dorsal appendage phenotype. Anterior is to the left in all panels. VM, ventral midline; DM, dorsal midline. (A) Twist protein in an embryo derived from a control clone egg chamber. 21 of 26 embryos derived from egg chambers with dorsal Ras clones were indistinguishable from controls. (B-D) Three examples of embryos derived from egg chambers with dorsal Ras clones that did display ectopic Twist protein (arrowheads), which always contacted the normal ventral Twist domain.

View larger version (93K): [in a new window]   Fig. 4. Dorsal appendage phenotypes resulting from Ras follicle cell clones. (A) The ‘extra nub’ phenotype: eggshell has an extra nub of chorion (arrowhead) in addition to two wild-type dorsal appendages. (B) The ‘nub’ phenotype: a nub of chorion material (white arrowhead) replaces one of the dorsal appendages. The other phenotypically wild-type appendage (a) is out of the plane of focus. The appendage of a neighboring egg can also be seen (black arrowhead). (C) The ‘two nubs’ phenotype: nubs replace both appendages (arrowheads). (D) Some chorion nubs are shaped like a teardrop (arrowhead), with a bulbous appendage base tapering to an anterior point. The other phenotypically wild-type appendage is out of the plane of focus (a). (E) Drawings, with corresponding frequencies, of the various chorion phenotypes seen in late stage Ras mosaic egg chambers. Frequencies are calculated from the number of eggs displaying a particular phenotype divided by the total number of eggs with chorion defects (n=177). Phenotypes labeled with (*) were selected for embryonic analysis.

View larger version (128K): [in a new window]   Fig. 5. Ras mutant cells do not participate in dorsal appendage formation. (A-D) Bright-field and confocal images of dissected stage 14 eggs with associated Ras follicle cell clones. Clones are identified by their lack of myc expression (outlined in yellow). (A) Chorion nub on a mosaic eggshell (black arrowhead). The other appendage on this egg (a) extends out of the plane of focus. An appendage from an adjacent egg also extends into the field (white arrowhead). (B) Confocal image of the egg chamber in A. A Ras clone covers a large portion of the dorsal anterior portion of the egg chamber, but the chorion nub is made by wild-type, myc+ cells neighboring the Ras clone. (C) Enlargement of the box in B. The dark hole is filled with the chorion making up a nub, which was secreted by about 8 wild-type cells. Though not visible in this projection, these cells cover the entire nub. (D) Confocal projection of a mosaic egg chamber with ‘teardrop’-shaped chorion nub. A Ras clone resides to the anterior of the group of wild-type cells making the nub, and appears to block the progress of the migrating cells. (E) Confocal projection of the anterior tip of a stage 14 control egg chamber showing that control clone cells are capable of migrating to the full anterior extent (arrowhead).

View larger version (93K): [in a new window]   Fig. 6. Ras mutant cells within the dorsal appendage primordia fail to undergo a reduction in apical diameter. Confocal projections of apicolateral anti-E-cadherin (E-Cad) fluorescence in stage 12 egg chambers highlight the apical morphology of wild-type (A) and Ras mosaic (B) dorsal appendage primordia. DM, dorsal midline. (A) The bright apices of wild-type follicle cells undergoing dorsal appendage morphogenesis are uniformly small (arrowhead) unlike their neighbors, which have rather large apical surfaces. (B) Dorsolateral view. The left appendage primordium of a Ras mosaic egg chamber is disrupted. (C-E) Higher magnification views of the disrupted appendage primordium, boxed in B, reveal that Ras mutant cells – those lacking the myc epitope (C) – exhibit large apical footprints (D, arrowhead). Although the wild-type cells adjacent to the clone have small apical diameters, their overall organization is less uniform than expected.

View larger version (166K): [in a new window]   Fig. 7. Basal localization of E-Cad is disrupted in a Ras clone on the dorsal midline. Basal confocal projections of E-Cad fluorescence in (A) wild-type and (B-E) Ras mosaic egg chambers reveal a novel, Ras-dependent pattern of basal E-Cad localization. Anterior is to the left in all panels. DM, dorsal midline (confirmed by position of oocyte nucleus, not shown). (A) During dorsal appendage morphogenesis, E-Cad is localized basally in an anterior patch of dorsal midline follicle cells as well as several rows of anterior follicle cells encircling the egg chamber. Note that all cells exhibit some degree of basal E-Cad fluorescence, but the level of basal localization in midline and anterior cells is much higher. (B) This stage 10B mosaic egg chamber bears two Ras clones, marked by lack of myc. The anterior clone resides in the dorsal midline region that normally displays basal E-Cad. (C,D) Basal E-Cad is greatly reduced in the Ras mutant cells. (E) An enlargement of the boxed region in C reveals the cell non-autonomous effect of the Ras mutant clone on basal E-Cad protein in neighboring wild-type cells, which have lost basal E-Cad on cell surfaces abutting the Ras clone (arrowhead).

View larger version (1K): [in a new window]   Fig. 8. (A) Ras is required for pipe repression and dorsal appendage morphogenesis. Higher levels of Ras protein are required for the dorsoventral patterning of the eggshell (black arrow) than for embryonic dorsoventral patterning, which begins with the cell-autonomous repression of pipe by Ras signaling (gray line) in dorsal and lateral follicle cells. This eggshell vs. embryo difference in requirement for Ras may be due to an eggshell-specific requirement for higher levels of Ras during Egfr signal amplification. Ras is required for dorsal follicle cell morphogenesis either through transcriptional activation of morphogenesis genes (solid black arrow) or through the direct cytoplasmic activation of cytoskeletal or adhesion molecules (dashed black arrow). One outcome of Ras signaling is the basal localization of E-Cad in dorsal midline cells. (B) A hypothesis for embryonic dorsoventral patterning: dorsal Egfr signaling restricts pipe mRNA (purple) to the ventral-most 40% of follicle cells. Pipe-positive cells activate a serine protease cascade in the perivitelline space separating the vitelline membrane (orange) and the oocyte. This cascade culminates in cleavage of the Spz zymogen to produce the ventral determinant C-Spz (green asterisks), the activity of which is negated by N-Spz and/or other inhibitors (red lines). This inhibition process normally reduces the ventral-determining region to 20% of the embryonic circumference, and, in embryos from Ras mosaic egg chambers, can completely overcome isolated regions of ectopic ventral activity caused by a Ras clone. This ‘ventral-most’ 20% region then instructs the nuclear Dorsal gradient along the dorsoventral axis (blue nuclei), and highest levels of nuclear Dorsal result in Twist protein expression (red nuclei).