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First published online 12 December 2007
doi: 10.1242/dev.008920

Development 135, 343-351 (2008)
Published by The Company of Biologists 2008
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Drosophila eggshell is patterned by sequential action of feedforward and feedback loops

Nir Yakoby1,*, Jessica Lembong1,*, Trudi Schüpbach2 and Stanislav Y. Shvartsman1,{dagger}

1 Department of Chemical Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, NJ 08544, USA.
2 Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, NJ 08544, USA.


Figure 1
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  		Fig. 1. Dynamics of EGFR and Dpp signaling in the Drosophila follicular epithelium. (A) Phase contrast image of a stage 10 egg chamber, showing the follicular epithelium (gray), the oocyte (white), the nurse cells (blue) and the stretch follicle cells. (B,C) In situ hybridization images of thickveins (tkv) expression at stage 10B of oogenesis (B, lateral view; C, dorsal view). (D) Schematic of the tkv pattern in a stage 10B egg chamber. (E-G) EGFR activation monitored by the nuclear localization of Capicua (Cic), a transcription factor excluded from the nucleus in response to EGFR signaling. (E) At stage 10A, Cic is excluded from nuclei of the dorsal anterior follicle cells (marked by a dotted white line). (F) At stage 10B, Cic is excluded from the nuclei within a broad patch of dorsal cells (marked by a dotted white line). (G) Dorsal view of a stage 12 egg chamber: Cic is excluded from the nuclei in and around two dorsolateral stripes of the follicle cells. At the same time, nuclear Cic reappears in the triangular patch of the dorsoanterior cells (marked by an asterisk). (H-J) Dpp signaling monitored by the phosphorylated Mad (P-Mad). P-Mad is first detected in the two rows of anterior follicle cells (H). Later, P-Mad is found in a single row of ventral cells, and in at least five rows of dorsal cells (marked by a solid white line) (I). After stage 11, the P-Mad pattern is split along the dorsal midline (J). (K-M) Merged images of Cic (red) and P-Mad (green). In all images anterior of the egg chamber is to the left; the yellow dashed line marks the anterior border of the oocyte; the arrowhead marks the dorsal midline. E,F,H,I,K,L are lateral views, and G,J,M are dorsal views, of the egg chamber. A, anterior; D, dorsal; FC, follicular epithelium; NC, nurse cells; P, posterior; SC, stretch follicle cells; V, ventral.

 

Figure 2
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  		Fig. 2. Dynamics of Dpp signaling in related fly species. (A) The evolutionary distance between Drosophila melanogaster (D. mel.) and three other Drosophila species with a different number of dorsal appendages. (B,C) Early and late patterns of P-Mad (green) and Br (red) in D. pseudoobscura (D. pse.). The eggshell has two appendages (D), as in D. melanogaster. (E,F) Early and late P-Mad and Br patterns in D. virilis (D. vir.). The eggshell has four dorsal appendages (G). (H-K) Dynamics of P-Mad and Br patterns in D. phalerata (D. pha.). (L-N) Dynamics of tkv expression in D. pha. The eggshell has three dorsal appendages (O). In all images the arrowhead marks the dorsal midline; anterior of the egg chamber is to the left.

 

Figure 3
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  		Fig. 3. The spatial pattern of tkv regulates the spatial pattern of Dpp signaling and is sensitive to EGFR signaling levels. (A-C) Dpp signaling is abolished in tkv- cells. tkv- cells are marked by the absence of GFP (green) and Dpp signaling is monitored by P-Mad staining (red). (D) Top: regulatory mechanism proposed for the formation of the spatial pattern of tkv expression: tkv is positively regulated by EGFR and negatively regulated by a midline repressor, potentially Pointed (Pnt), also controlled by EGFR. Bottom: schematic of the tkv patterns in the wild type, the EGFR hypomorph (QY1, anterior striped gray), and the mutant with extra copies of gurken (4PX, solid gray). The dashed line shows the boundary of the wild-type pattern of tkv; cells inside the red box express tkv in the wild-type background. In these cells, tkv expression is lost in both the 4PX and QY1 mutants. (E,F) tkv expression in the 4PX (E) and QY1 (F) backgrounds. The midline gap is marked by a double arrowhead and the midline is marked by an arrowhead. P-Mad (green) in 4PX (G) and QY1 (H) egg chambers, respectively. The wild-type patterns of Dpp signaling and tkv expression are shown in Fig. 1. Yellow dashed lines mark the anterior boundary of the oocyte.

 

Figure 4
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  		Fig. 4. Spatial and temporal correlation between the wild-type patterns of Dpp signaling and Br expression. (A-C) P-Mad (green) and Br expression (red) at different stages of oogenesis; see text for details. (D-G) The spatial pattern of br transcript at different stages of oogenesis. Stage 9, low level of expression throughout the follicular epithelium (D); stage 10A, repression in the anterior and dorsal midline follicle cells (E); stage 10B, expression in the roof cells (F); stage 11 and onward, no expression throughout the follicular epithelium (G). (H-J) A mosaic egg chamber with a large Medea- (Med-) clone that spans the lateral and anterior regions of the follicular epithelium; Med- cells are marked by the absence of GFP (H, green). The clone generates ectopic Br expression in the anterior cells, but does not disrupt the lateral expression of Br (I). (J) Enlarged view of Br expression in the boxed area in H. (K-M) A clone of Mad- cells, which spans both the roof cell domain and the dorsal midline, does not affect Br levels in the dorsal midline cells and does not disrupt Br expression in the roof cells. Br expression is shown in red; Mad- cells are marked by the absence of GFP (green). A similar effect can be seen with a lateral of clone Med- cells (N-P). Midline levels of Br are also not affected by removal of Med in the dorsal midline (not shown). In all images the dorsal midline is marked by an arrowhead, anterior is to the left, and the clones are outlined by a dotted line.

 

Figure 5
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  		Fig. 5. Dpp signaling limits the duration of br expression in the roof cells. (A) Schematic of the dynamics of br levels in the roof cells in the wild type (dark gray). We predicted that the pulse-like dynamics of br expression in the roof cells should become sustained in the absence of Dpp signaling (light gray). (B) Uniform expression of Dad, the inhibitory Smad, changes the spatial pattern of Br protein: Br is ectopically expressed in the anterior cells. (C) At the same time, the duration of br expression in the roof cells is increased, compared with the wild-type pattern of br expression (Fig. 4G). This agrees with the schematic prediction in Fig. 5A. (D,E) Egg chambers with uniformly inhibited Dpp signaling. (D) Changes in the spatial pattern of Br in the anterior cells generate a predictable change in the expression of rho, which was previously shown to be repressed by Br. (E) Schematic of rho (blue) and Br (red) patterns. (F) Patterning defects in egg chambers with uniformly inhibited Dpp signaling lead to defects in eggshell morphology: EM image of Dad overexpression eggshell shows flat dorsal appendages and reduced operculum size. (G) Wild-type rho expression; (H) schematic of wild-type rho (blue) and Br (red) patterns; (I) wild-type eggshell morphology. In B,C,D,G anterior is to the left; the dorsal midline is marked by an arrowhead; yellow dashed lines mark the anterior boundary of the oocyte. DA, dorsal appendages; op, operculum.

 

Figure 6
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  		Fig. 6. EGFR represses Br in the midline and is required for Br expression in the roof cells of Drosophila. (A-D) Experiments with genetic mosaics with GFP-marked clones of ras- cells reveal region-specific effects of EGFR signaling on Br expression. The dorsal midline is marked by an arrowhead in all images. (A) Merged image of Br expression (red) and GFP (green). (B) Midline ras- clones induce cell-autonomous ectopic expression of Br. (C,D) Lateral clones of ras- cells lead to cell-autonomous loss of Br expression in the roof cells. (E) A model proposed for the regulation of Br expression by EGFR: the spatial pattern of Br is established by the feedforward loop controlled by EGFR signaling. Br is induced in a wide dorsal domain. This effect is overridden in the dorsal midline cells, where Pointed, induced by high levels of EGFR signaling, represses Br.

 

Figure 7
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  		Fig. 7. Br controls Dpp signaling in the follicle cells by regulating the expression of tkv. (A-D) Uniform expression of Br-Z1 isoform in the follicular epithelium leads to ectopic expression of tkv and Dpp signaling. (A-C) Br (A) and P-Mad (B) patterns in a stage 12 egg chamber overexpressing Br-Z1. P-Mad signal is detected in the midline and anterior follicle cells, where it is absent in the wild-type egg chamber at this stage. The image in C is a merge of Br (red) and P-Mad (green) patterns. (D) Overexpression of the Br-Z1 isoform generates ectopic expression of tkv: tkv is expressed in the dorsal midline (denoted by the arrowhead), where it is repressed in the wild type (see also Fig. S3 in the supplementary material). (E-K) Clones of br- cells (marked by an arrow) generate defects in the spatial patterns of Dpp signaling and tkv expression. The arrowhead marks the dorsal midline. (E-J) P-Mad pattern (red) in mosaic epithelial layers with the GFP-marked clones of br- cells. (K) Defective pattern of tkv (denoted by the arrow) in the egg chamber with a clone of br- cells. Dorsal view; the arrowhead marks the midline. (L) Model summarizing the results of experiments in A-K: Br regulates tkv, which is essential for Dpp signaling. (M) ESEM image of the eggshell morphology induced by the overexpression of the Br-Z1 isoform.

 

Figure 8
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  		Fig. 8. Model for the dynamic regulation of Br by EGFR and Dpp pathways. (A) A revised model of Br regulation by EGFR and Dpp signaling. The rising phase of Br expression in the roof cells is due to a feedforward loop activated by EGFR signaling (blue). The shutdown of br expression in the roof cells is due to a negative-feedback loop (red), which is formed by Br and Dpp signaling. See text for details. (B) Schematic diagram of the spatial and temporal patterns of the main components in the model. See text for details.

 

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