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First published online 10 July 2006
doi: 10.1242/dev.02467

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The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila

¹ The Gurdon Institute and Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
² Centre for the Molecular Genetics of Development, Research School of Biological Sciences, Australian National University, Canberra ACT 0200, Australia.

View larger version (25K): [in a new window]   Fig. 1. Genomic organisation of the dfer locus. (A) In GAL4MZ465, pGawB (red) is inserted upstream of dfer (green). The deletions dfer1 and dferex1 are depicted (purple). (B) Exon structure of the four predicted dfer isoforms (a section of the third intron is deleted for clarity). Exon 1 is non-coding. Additional exons in dferRA and dferRD are in pink. (C) Predicted protein structure of DFer isoforms. (D) Molecular map of the dfergof locus. pGawB (red) has undergone a rearrangement, duplicating and inverting GAL4, and deleting pBluescript (pBS) and most of mini-white (mw+). (E) dfergof produces three transcripts, splicing the first exon of mini white (red) to exons of dfer (green). Light green exons are spliced out. (F) One transcript (wex1-DFerRB) is predicted to encode a White-DFer fusion protein.

View larger version (112K): [in a new window]   Fig. 2. dfer is expressed in leading edge cells and the CNS. (A) dfer mRNA is expressed in the leading edge cells (arrowhead). (B) In the CNS, dfer is expressed in midline glial cells (arrowhead) and upregulated in a segmentally repeated, lateral, cell (C, arrowheads). (D) dfer is expressed in a pair of dorsal neurons, corresponding to the MP2 neurons (arrowheads). (E) dfer mRNA (black), Odd-skipped (brown), MP1s (single arrowhead), MP2s (double arrowhead).

View larger version (90K): [in a new window]   Fig. 3. DFer colocalises with DE-Cadherin and is apical to Fasciclin 3. (A) At stage 12, DFer protein localises to cell-cell junctions (arrow) in leading edge cells and the dorsal epidermis. (B) DFer is lost from the border with the amnioserosa by stage 14 (arrow). (C-E) DFer colocalises with DE-Cadherin in the dorsal epidermis at stage 14, but is largely absent from the adherens junctions in the amnioserosa. (F-H) At stage 13, DFer overlaps extensively with DE-Cadherin. (I,J) At stage 14, the cell-cell junction staining of (I) DFer (red, arrow) is apical to (J) Fas3 (green, arrow). Confocal sections in I and J are 1 µm apart.

View larger version (105K): [in a new window]   Fig. 4. DFer is required for normal leading edge morphology and normal rates of closure. (A,B) Late stage 16 wild-type embryos (A) have been dorsally closed for approximately three hours, whereas late stage 16 dfer1 embryos (B) are dorsally open (white, anti-phospho-tyrosine). (C) DFerRB rescues dorsal closure (late stage 16 UAS-DFerRB/+;daGAL4,dfer1/dfer1). (D) At stage 14, leading edge cells elongate dorsoventrally (arrow), develop a thick F-actin cable (up arrowhead), and extend filopodial and lamellipodial protrusions (down arrowheads). (E) In dfer1 embryos, the F-actin cable is greatly reduced (up arrowhead), but filopodia are still present (down arrowheads). (F) DFerRB expression largely restores the F-actin cable (arrowhead; white, Alexa568-Phalloidin; UAS-DFerRB/+;daGAL4,dfer1/dfer1 embryos). (G) At stage 14, the leading edge is enriched for P-Tyr, particularly at the contact points between adjacent leading edge cells (arrowhead). (H) In dfer1 embryos, P-Tyr staining is reduced (arrowhead). (I) In rescued embryos, P-Tyr staining is restored (arrowhead).

View larger version (149K): [in a new window]   Fig. 5. DFer and Src42A cooperate during dorsal closure. (A-C) Late stage 16 Src42Amyri embryos (A) are closed but have an irregular arrangement of epidermal cells, whereas Src42Amyri;dferex1 mutants (B) are still closing and Src42Amyri;dfer1 mutants (C) are dorsally open (white, anti-phospho-tyrosine). (D-F) The F-actin cable (arrowheads) is slightly disrupted in Src42Amyri mutants (D, compare with Fig. 4D), further disrupted in Src42Amyri;dferex1 mutants (E) and completely lost in Src42Amyri;dfer1 embryos (F; white, Alexa568-Phalloidin). (G-I) P-Tyr staining (arrowheads) is progressively reduced in Src42Amyri (G), Src42Amyri;dferex1 (H) and Src42Amyri;dfer1 (I) mutants. (J) Wild-type cuticle preparation. (K,N) Src42Amyri;dferex1 mutants typically show a small anterior hole (K; 95%, n=152; arrowhead), but are occasionally anterior open (N; 5%, arrowhead). (L,O) Src42Amyri;dfer1 mutants have a small to medium anterior hole (L; 59%, n=102, down arrowhead), often with scabs along the dorsal midline (up arrowhead), or dorsal closure fails (O), yielding an anterior open phenotype (30%, down arrowhead). (M) Src42Amyri mutants fail to hatch (63%, n=191), with 37% showing a small anterior hole near the mouthparts (arrowhead).

View larger version (191K): [in a new window]   Fig. 6. A dfer gain-of-function mutant affects dorsal closure, the morphology of leading edge and amnioserosal cells. (A,B) Late stage 16 wild-type embryo (A), stained for the cell adhesion molecule Fasciclin 3, is dorsally closed, whereas an equivalently staged dfergof embryo (B) is still open. (C) At stage 14, wild-type leading edge cells develop an actomyosin cable (up arrowhead; white, Alexa568-Phalloidin), and numerous filopodia (down arrowhead). F-actin is enriched at amnioserosal cell borders (double arrowhead; same embryo as is shown in Fig. 4D). (D) In dfergof mutants, the actomyosin cable is thinner (up arrowhead), and the filopodia less abundant (down arrowheads). Amnioserosal cells show weaker F-actin staining at cell-cell junctions (double arrowhead). Contraction of individual amnioserosal cells still occurs (up arrow). (E) In wild-type embryos, GFP-Actin highlights the actomyosin cable (up arrowhead), filopodia (down arrowhead) and cortical enrichment in amnioserosal cells (double arrowhead; UAS-GFP-Actin/+; daGAL4/+; stage 14). (F) In dfergof embryos, the actomyosin cable and filopodia are reduced, and amnioserosal cells show less cortical actin and a more motile morphology (arrowhead; UAS-GFP-Actin/+;daGAL4,dfergof/dfergof; stage 14).

View larger version (146K): [in a new window]   Fig. 7. The dfer gain-of-function mutant disrupts axon guidance at the ventral midline of the CNS. (A,B) Fillets of stage 16 embryos immunostained on the same slide show significantly higher levels of DFer protein in the CNS of dfergof mutants (B) than in wild type (A). (C) DFer protein is not expressed in the CNS of dferex1 mutant embryos. (D) In wild-type embryos, Fasciclin 2 (brown) is expressed on longitudinal axons. (E,F) In dfergof mutants, axons cross the midline aberrantly (E, arrowhead), a phenotype that is enhanced when the levels of DFerRB are increased (F, arrowheads; UAS-DFerRB/+;+;dfergof). (G) Expression of the JNK inhibitor Puc rescues the midline phenotype of dfergof mutants (UAS-Puc,dfergof/dfergof).

View larger version (64K): [in a new window]   Fig. 8. DFer interactions with the JNK pathway. (A) Stage 13 wild-type embryos express dpp in leading edge cells (arrowhead) and in the midgut. (B,C) In Src42Amyri embryos (B), dpp expression in leading edge cells (arrowheads) is slightly reduced and expression is almost lost in Src42Amyri;dfer1 double mutants (C). (D-G) dpp (E) and puc-lacZ (G) expression in leading edge cells of dfergof mutants (arrowheads) is comparable to wild type (D,F). (H,I) Phosphotyrosine staining in dfergof/dfergof (H) and UAS-puc, dfergof/dfergof (I) embryos. (I) Expression of the JNK inhibitor Puc with dfergof serving as the GAL4 driver, rescues the dorsal open phenotype of dfergof mutants.

View larger version (101K): [in a new window]   Fig. 9. ß-catenin levels and phosphorylation state are altered in dfer mutants. (A,B) ß-catenin immunoprecipitated from (A) dfer1 and (B) dfergof late stage embryo extracts, probed with anti-arm (ß-catenin) and anti phosphotyrosine. ß-catenin is hypophosphorylated in dfer1 embryos (21% relative to yw control embryos) and hyperphosphorylated in dfergof embryos (168% relative to yw). (C) Levels of ß-catenin are reduced in the epidermis of dfergof embryos. DE-Cadherin may also be reduced to a lesser degree. (D) Western blots on total embryo extracts confirm that ß-catenin is depleted in dfergof mutants. (E) Loading control (anti-actin).