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First published online 21 September 2005
doi: 10.1242/dev.02034

Development 132, 4635-4644 (2005)
Published by The Company of Biologists 2005
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Resolving embryonic blood cell fate choice in Drosophila: interplay of GCM and RUNX factors

Laetitia Bataillé, Benoit Augé, Géraldine Ferjoux, Marc Haenlin* and Lucas Waltzer

Centre de Biologie du Développement, CNRS UMR 5547, 118 route de Narbonne, 31062 Toulouse, France



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  		Fig. 1. The downregulation of gcm precedes the induction of lz in the anterior-most row of prohemocytes. (A,B) gcm (red) and srp (green) transcription in stage 5 (A) or stage 6 (B) embryos. (C) gcm transcript (red) and SRP protein (green) expression in stage 6 embryo. (D,E) lz-lacZ embryo processed to reveal gcm transcript (red) and ß-gal protein (green) at stage 7 (D) and at stage 10 (E). (F-J) lacZ expression in lz-lacZ (F) or lz-gal4/uas-gal4; uas-gal4/+; uas-gal4/uas-lacZ (G-J) embryos at stage 7 (F,G, ventral views), stage 11 (H, side views) or stage 14 (I, side views). (J) High magnification views of lacZ-expressing cells localised in the crystal cell cluster (left panel) or far from the cluster (right panels) in a stage 14 lz-gal4/uas-gal4; uas-gal4/+; uas-gal4/uas-lacZ embryo. (K-M) Side views of gcm-lacZ embryos processed to reveal DoxA3 mRNA (red) and ß-gal protein (green) at stage 11 (K), 14 (L) or 15 (M). (N,L) Side views of DoxA3 mRNA (red) and ß-gal protein (green) in srp-Gal4;uas-lacZ (N) or lz-lacZ (O) embryos at stage 15. Contrary to lz-lacZ or srp-lacZ, gcm-lacZ expression is progressively lost in the crystal cells during embryogenesis: 26 out of the 28 DoxA3+ cells are gcm-lacZ+ at stage 11 (K), against 9 out of 27 at stage 14 (L) and 0 out of 27 at stage 15 (M). lz-lacZ codes for a cytoplasmic ß-gal whereas gcm-lacZ and uas-lacZ code for a nuclear ß-gal.

 


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  		Fig. 2. GCM and GCM2 inhibit crystal cell formation. (A-D) Side views of DoxA3 expression in stage 11 (A,B), or 15 (C,D) embryos. (A,C) Wild-type embryos; (B,D) Df(2L)200 embryos. (E,F) Higher magnification views of DoxA3-expressing cells located in the crystal cell cluster in a wild-type embryo (E), or located ectopically in a Df(2L)200 embryo (F). (G) Side views of DoxA3 expression in a lzR1;Df(2L)200 mutant embryo at stage 15. (H,I) Side views of stage 17 embryos carrying the Bc1 mutation that induces spontaneous melanisation of the crystal cells. (H) Bc1, (I) Bc1,Df(2L)200. (J-P) Side views of DoxA3 expression in stage 15 embryos. (Q-S) Side views of Pxn expression in stage 15 embryos. Genotypes as indicated in the lower part of each panel.

 


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  		Fig. 3. GCM/GCM2 regulates both the number of lz+ progenitors and their subsequent differentiation. (A,B) gcm/gcm2 restricts lz activation in the prohemocytes. Ventral views of lacZ (dark purple) and gcm (red) transcripts in stage 7 lz-lacZ (A) and Df(2L)200;lz-lacZ (B) embryos. (C-F) gcm/gcm2 inhibits the differentiation of the lz+ progenitors into crystal cells. Side views of stage 13 lz-gal;uas-lacZ (C,E) and lz-gal4;Df(2L)200;uas-lacZ (D,F) embryos showing lacZ (dark purple) and gcm (blue) transcripts (C,D), or ß-gal (green) and DoxA3 (red) expression (E,F). (G,H) The lack of gcm/gcm2 induces an increase in the number of crystal cells even in the absence of cell proliferation. Side view of DoxA3 expression in stg2 (G) and Df(2L)200;stg2 (H) stage 13 embryos.

 


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  		Fig. 4. GCM inhibits lz induction and maintenance but not LZ activity. (A-E) Side views of lz expression in stage 11 embryos. (A) Wild type. lz expression is repressed by GCM when gcm expression is driven in the whole mesoderm (B), the hemocytes (C) or the prospective crystal cells (D), but not when it is overexpressed in the plasmatocytes (E). (F-J) Side views of lz-lacZ expression in stage 11 embryos. lz-lacZ transcription is repressed upon overexpression of GCM in the mesoderm (G). Pan-mesodermal expression of SRP (H) or LZ (I) induces restricted activation of lz-lacZ, whereas co-expression of LZ and SRP (J) induces synergistic activation throughout the mesoderm. (K-N) Side views of DoxA3 expression in stage 16 embryos. Crystal cell formation is repressed by heat-shock induced transient expression of GCM around stage 10 (L), 11 (M) or 12 (N). No repression was observed in the absence of heat-shock treatment (K). (O-R) Side views of DoxA3 expression in stage 11 embryos. srp-gal4-driven expression of GCM represses DoxA3 expression (P). srp-gal4-driven expression of LZ activates DoxA3 expression (Q) and relieves GCM-induced repression upon DoxA3 (R).

 


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  		Fig. 5. Notch is neither required nor sufficient for crystal cell formation in the embryo. (A-E) Side views of DoxA3 expression in stage 14 embryos. (A) Wild type; (B) N55e11 zygotic mutant; (C) N55e11 germ-line clone mutant; (D) srp-gal4;uas-Nintra; (E) pg33; uas-Nintra.

 


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  		Fig. 6. All the prohemocytes have the capability to develop as crystal cells. (A-H) Side views of stage 14 to 15 embryos processed to reveal DoxA3 (A-D), or DoxA3 (blue) and Pxn (black) expression (E-H). The arrows in C,D,G and H point to the ectopic activation of DoxA3 expression in the amnioserosa upon srp-gal4-induced expression of uas-lz in this tissue. (I-L) Higher magnification views of wild-type crystal cells expressing DoxA3 (I), wild-type plasmatocytes expressing Pxn (J), and DoxA3-expressing hemocytes upon srp-driven expression of LZ in a wild-type embryo (K) or in a Df(2L)200 embryo (L). Scale bar in I-L: 12 µm.

 


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  		Fig. 7. Schematic representation of blood cell fate resolution during Drosophila embryogenesis. Initially all the prohemocytes express gcm but not lz. Then gcm transcription is turned off and lz expression activated in the first row of prohemocytes but not in the others that subsequently differentiate into plasmatocytes. 60% of these lz+ progenitors manage to maintain lz expression through an autoactivation loop and differentiate into crystal cells, while in the remaining 40%, the presence of residual GCM interferes with lz expression and promotes plasmatocyte differentiation. In the absence of gcm/gcm2, more prohemocytes (potentially the second row) initiate lz expression and all the lz+ progenitors differentiate into crystal cells.

 

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© The Company of Biologists Ltd 2005