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First published online 24 November 2005
doi: 10.1242/dev.02168


Development 133, 43-51 (2006)
Published by The Company of Biologists 2006


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MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

Daniel R. Marenda1, Alysia D. Vrailas1, Aloma B. Rodrigues1, Summer Cook1, Maureen A. Powers1, James A. Lorenzen2,3, Lizabeth A. Perkins2 and Kevin Moses1,*,{dagger}

1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
2 Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School Boston, MA 02114, USA.
3 Department of Pediatric Gastroenterology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.



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Fig. 1. pMAPK antigen, MAPK/GAL4 (MG)-driven GFP, DNA and bs:lacZ expression in wild-type larval wings. Anterior upwards, dorsal leftwards. Magnifications are equal in A and F, C and D, and E and G. (A) pMAPK is in red, MG-driven GFP is in green. (B) Confocal section of large boxed area at arrow in A. pMAPK is in red, MG-driven GFP is in green. MG-driven GFP is absent from margin cells (arrowheads). An example of a MG-driven GFP responsive nuclei is indicated by an arrow. (C,D) A single confocal section of vein tissue (C) through small boxed area at arrowhead in A, and lateral cross-section of margin tissue (D) at boxed area in B. pMAPK in red, DNA in blue. pMAPK is predominantly cytoplasmic in both regions (arrowhead indicates pMAPK staining next to pMAPK-negative nucleus at arrow). Bracket in D separates two columns of pMAPK stains at wing margin (arrowheads in B). (E) Confocal section in the wing pouch of the wing margin and veins L3, L4 and L5. pMAPK is in red, MG-driven GFP is in green, actin is in blue. Cells positive for pMAPK (arrows) and GFP (arrowheads) are indicated. (F,G) bs:lacZ is in red, MG-driven GFP is in green. GFP-positive nuclei devoid of lacZ (arrow in G), and GFP and lacZ nuclei are indicated (arrowhead in G). Broken white lines define area of bs:lacZ expression in G.

 


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Fig. 2. pMAPK antigen, MAPK/GAL4 (MG)-driven GFP and DNA expression in wild-type pupal wings. Anterior is upwards, distal is rightwards. Hours APF is indicated in the upper right-hand corner. pMAPK is in red, MG-driven GFP is in green, DNA is in blue in all panels. (A) pMAPK antigen is predominant in pupal veins (asterisk). (B) At 28 hours APF, pMAPK expression falls in the veins (compare asterisks in A and B). (C) High magnification of distal wing margin at arrowhead in B. MG-driven GFP is present in two lines of cells flanking pro-veins (arrows). (D) pMAPK returns to veins (compare asterisks in B and D). (E) High magnification of boxed area at arrow in A. (F) High magnification of boxed area at arrow in B. pMAPK is predominantly cytoplasmic in both E and F; arrowheads indicate pMAPK staining next to pMAPK-negative nuclei at arrows.

 


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Fig. 3. Overcoming MAPK cytoplasmic hold in the developing wing. Stages are indicated on the left. Genotypes indicated. (A-D) Adult wings after a 1-hour heat induction at 20-24 APF (A,B) and 24-27 APF (C,D). (E,F) Bar graphs showing comparison of wing-surface areas (E) and cell counts/hair density (F) between genotypes depicted in A-D. The hs:rho; hs:msk wing area is larger in E; P<10–7 in each case. hs:NM wings contain more cells in F (P<10–4 in each case). (G-K) Twenty-four hour APF pupal wings (anterior leftwards, distal upwards), pMAPK antigen white. (H-K) One-hour heat induction, then recovery for the time indicated top right in hours. Magnifications are equal in A-D and G-K. Elevated pMAPK at asterisks in H and I compared with G, J and K. P values in E are: hs:M to hs:NM=0.3, hs:M to hs:rho=0.6, hs:M to hs:rho; hs:msk=4.4x10–8, hs:NM to hs:rho=0.8, hs:NM to hs:rho; hs:msk=9.1x10–9, and hs:rho to hs:rho; hs:msk=5.3x10–7. P values in F are: hs:M to hs:NM=1.3x10–4, hs:M to hs:rho=0.03, hs:M to hs:rho; hs:msk=0.1, hs:NM to hs:rho=1.0x10–5, hs:NM to hs:rho; hs:msk=1.5x10–5 and hs:rho to hs:rho; hs:msk=0.2.

 


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Fig. 4. Posterior ectopic Msk affects MAPK expression. Late larval wings, anterior upwards, dorsal leftwards. Antigens and GFP are listed in the bottom left-hand corner, the expression genotype is listed in the bottom right-hand corner. (A-C) hsp70-driven expression of HSV epitope-tagged MAPK (hs:M) after a 1-hour induction. (B,C) en::msk is added to hs:M. Yellow boxed area in B is expanded in C. The epitope tag antigen is elevated where Msk is co-expressed (in the posterior, en domain in B). (C) Lamin D antigen (green) stains the nuclear envelope. Much of this MAPK is now in nuclei (arrows in C). Arrowheads in A,B indicate the AP boundary. (D-F) en::GFP (D) and en:GAL4 driving UAS:GFP and UAS:msk together (E,F). pMAPK expression that is normally associated with veins and margin (arrowheads in D) is suppressed in the posterior compartment wing pouch in E: compare non-suppressed anterior wing margin pMAPK (arrows in D,E) with suppressed posterior wing margin pMAPK and L5 pro-vein (arrowheads in D,E). The GFP expression domain expands towards the anterior (arrow in F). Magnifications are equal in A,B,D-F.

 


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Fig. 5. MAPK nuclear translocation and cell cycle in the developing wing. (A-D) Larval wing discs expressing hs:MG (A-C) or hs:NMG (D) after 4 hours of recovery after heat shock, showing GFP (green), bromo-deoxyuridine (BrdU, magenta in A) and phosphorylated Histone H3 (pH3, magenta in B, and white in C,D). (C,D) Confocal projections through the wing pouch of each genotype, showing each pH3-positive nuclei in the pouch. BrdU and GFP are largely restricted from non-proliferative zone in hs:MG discs (brackets in A), as are pH3 and GFP (brackets in B,C). pH3 nuclei are now observed in non-proliferative zone in hs:NMG discs (brackets in D). (E-H) Cell-sorting analysis of larval wing discs. GFP+ traces and numbers are in red, GFP are in blue. All are from wing discs expressing GFP (UAS:GFP), driven by the genetic elements indicated. Recovery times are as indicated. DNA content on the x-axis and fraction of the cells in each sample at each DNA value on the y-axis. Major DNA content peaks (2N and 4N, corresponding with G1 and G2/M) indicated. Inset tables: percentage of cells at the stages indicated (G1, S or G2/M). (I-P) Larval discs stained for expression of cell cycle and/or apoptosis markers as indicated. (I-L) Wild type. (M-P) Ectopic Msk expressed in the posterior compartment, under the control of en:GAL4 (same domain as is green in Fig. 4D). Arrowheads in I and M indicate the AP boundary. Where Msk is ectopically expressed, Cyclin E is elevated (arrow in M), stg:lacZ is elevated (arrow in N), DNA synthesis (BrdU) is slightly elevated (arrow in O) and cell apoptosis is induced (activated Caspase 3, arrow in P). Magnifications are equal in A,B; C,D; and I-P.

 


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Fig. 6. Msk prevents vein fate. (A-J) Adult wings, anterior upwards. (A-H) Broken lines in A-C and F-H indicate the AP boundary; magnifications are equal in all panels; genotypes/driven elements are indicated. Arrows indicate Twiggy wing phenotype in A. Asterisks indicate lost veins in B,C,H. There is enhancement of vein loss by Msk (arrows in G,H) and enhanced vein formation by msk loss-of-function mutation (compare at arrows in D and I, E and J).

 


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Fig. 7. Ectopic Msk vein loss is not due to cell death. (A-D) Larval wings, anterior upwards, dorsal rightwards. Magnifications are equal in all panels, genotypes are as indicated. Arrows and arrowheads indicate pMAPK antigen expression or its loss. (E-H) Adult wings, anterior upwards, broken lines indicate the AP boundary; magnification is equal in all panels and genotypes are as indicated. Asterisks indicate lost veins in G,H. Arrow indicates the Twiggy wing phenotype in F.

 





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