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    Resolving embryonic blood cell fate choice inDrosophila: interplay of GCM and RUNX factors Laetitia Bataillé, Benoit Augé, Géraldine Ferjoux, Marc Haenlin and Lucas Waltzer

    There was an error published in Development 132, 4635-4644.

    In Fig. 2, the incorrect image was shown in panel K. Owing to an error during figure assembly, Fig. 2K is a duplication of the wild-type embryo image shown in Fig. 5A. The corrected Fig. 2 appears below.

    This error does not affect the conclusions of the paper. The authors apologise to readers for this mistake.

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    Introduction Understanding how cell lineages are established remains a central task in developmental biology and particularly in the field of hematopoiesis. Strikingly, it has become clear over the past few years that several aspects of hematopoiesis have been conserved between Drosophila and man (Evans et al., 2003). Thus, Drosophila may provide a valuable model system with which to gain insight into the mechanisms of blood cell lineage segregation in vivo. As in vertebrates, hematopoiesis in the fruit fly occurs in two waves: blood cell progenitors arise from the head mesoderm in the early embryo and from a specialised organ, the lymph gland, in the larva (Holz et al., 2003). These progenitors (prohemocytes) give rise to three terminally differentiated cell types (collectively called hemocytes), related to the vertebrate myeloid lineages: plasmatocytes, crystal cells and lamellocytes (Meister, 2004). Approximately 95% of hemocytes are plasmatocytes, which function as macrophages, engulfing apoptotic cells and small pathogens such as bacteria (Sears et al., 2003; Tepass et al., 1994). Crystal cells constitute a smaller population of blood cells (about 5%) that participate in melanisation, an insect-specific process involved in wound healing and the encapsulation of foreign invaders (Rizki et al., 1980). Finally, the lamellocytes encapsulate foreign bodies too large to be engulfed by macrophages (Crozatier et al., 2004). They are only generated during larval hematopoiesis, under specific conditions such as parasitisation of the larvae by wasp eggs.

    Despite the evolutionary distance between Drosophila and

    vertebrates, many of the molecular pathways governing hematopoiesis have been conserved (Evans et al., 2003). In particular, transcription factors of the GATA, FOG and RUNX families, which regulate several steps of hematopoiesis in vertebrates, also control Drosophila hematopoiesis (Fossett et al., 2001; Lebestky et al., 2000; Rehorn et al., 1996). In the embryo, all prohemocytes express the GATA transcription factor Serpent (SRP), which is required for blood cell precursor specification and maintenance (Rehorn et al., 1996). From this pool of prohemocytes, two populations of hematopoietic cells will emerge, plasmatocytes and crystal cells (Lebestky et al., 2000). SRP participates in their differentiation and remains expressed in the differentiated blood cells (Fossett et al., 2003; Waltzer et al., 2002). Furthermore, key lineage-specific factors are required for the differentiation into these two cell types. On the one hand, the RUNX transcription factor Lozenge (LZ) forms a functional complex with SRP to induce crystal cell formation (Waltzer et al., 2003), and in a lz mutant no crystal cells appear (Lebestky et al., 2000). On the other hand, the two related transcription factors Glial cells missing (GCM) and GCM2 (also known as Glide and Glide2) are jointly required for plasmatocyte differentiation (Alfonso and Jones, 2002; Bernardoni et al., 1997; Kammerer and Giangrande, 2001). In the absence of both gcm and gcm2 (gcm/gcm2), plasmatocytes do not differentiate normally and their number is strongly reduced, whereas crystal cell formation appears unaffected (Alfonso and Jones, 2002). In addition, enforced expression

    The differentiation of Drosophila embryonic blood cell progenitors (prohemocytes) into plasmatocytes or crystal cells is controlled by lineage-specific transcription factors. The related proteins Glial cells missing (GCM) and GCM2 control plasmatocyte development, whereas the RUNX factor Lozenge (LZ) is required for crystal cell differentiation. We have investigated the segregation process that leads to the formation of these two cell types, and the interplay between LZ and GCM/GCM2. We show that, surprisingly, gcm is initially expressed in all prohemocytes but is rapidly downregulated in the anterior- most row of prohemocytes, which then initiates lz expression. However, the lz+ progenitors constitute a mixed-lineage population whose fate depends on the

    relative levels of LZ and GCM/GCM2. Notably, we demonstrate that GCM/GCM2 play a key role in controlling the size of the crystal cell population by inhibiting lz activation and maintenance. Furthermore, we show that prohemocytes are bipotent progenitors, and that downregulation of gcm/gcm2 is required for lz-induced crystal cell formation. These results provide new insight into the mechanisms controlling Drosophila hematopoiesis and establish the basis for an original model for the resolution of the choice of blood cell fate.

    Key words: RUNX, GCM, Hematopoiesis, Cell fate choice, Drosophila

    Summary

    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 *Author for correspondence (e-mail: haenlin@cict.fr)

    Accepted 9 August 2005

    Development 132, 4635-4644 Published by The Company of Biologists 2005 doi:10.1242/dev.02034

    Research article

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    of GCM in crystal cells converts them into plasmatocytes (Lebestky et al., 2000). By contrast, ectopic expression of LZ in plasmatocytes induces crystal cell marker expression but does not repress plasmatocyte cell fate (Waltzer et al., 2003).

    It is proposed that, for their differentiation, crystal cells must express lz but not gcm/gcm2, whereas plasmatocytes have to express gcm/gcm2 but not lz (Lebestky et al., 2000). Interestingly, gcm/gcm2 expression is detected from stage 5 in the hematopoietic primordium (Alfonso and Jones, 2002; Bernardoni et al., 1997), whereas the onset of lz expression is only detected later (at stage 10) (Lebestky et al., 2000). These observations raise several questions: (1) do the crystal cell and the plasmatocytes precursors emerge from the same pool of prohemocytes; (2) when do the two populations segregate; and (3) what is the relationship between lz and gcm/gcm2 during blood cell lineage choice?

    To address these questions, we have undertaken an analysis of the mechanism of segregation of the two embryonic blood cell lineages. We find that gcm is expressed early on in all prohemocytes but is rapidly downregulated in the anterior- most cells of the hematopoietic primordium, which initiate lz expression by stage 7. Our results suggest that the coordinated repression of gcm and activation of lz in the anterior row of prohemocytes is a key step in the regulation of blood cell lineage choice. In the absence of both gcm and gcm2, we observe a striking increase in the number of crystal cells, indicating that gcm/gcm2 actually regulates crystal cell development. We further show that gcm/gcm2 inhibits crystal cell formation in two steps: first by regulating the number of cells that initiate lz expression, and second by interfering with lz maintenance in these cells. Furthermore, contrary to what has been reported during larval hematopoiesis (Duvic et al., 2002; Lebestky et al., 2003), we demonstrate that Notch signalling is neither sufficient nor required for crystal cell formation. Finally, our results indicate that prohemocytes are bipotent progenitors, and that the interplay between gcm/gmc2 and lz expression dictates the cell fate choice.

    Materials and methods Fly stocks Most Drosophila melanogaster lines were obtained from the Bloomington Drosophila Stock Center. The following strains were kindly provided by different laboratories: uas-gcm, uas-gcm2, gcm- lacZ (gcmrA87), Df(2L)200 and Df(2L)Gcm2 (B. Jones, University, MS, USA); uas-lz and lzR1 (U. Banerjee, Los Angeles, CA, USA); stg2 (M. Crozatier, Toulouse, France); N55e11 FRT10.1 (A. Martinez-Arias, Cambridge, UK). uas-srp, srp-gal4 and pg33 have been previously described by Waltzer et al. (Waltzer et al., 2002). To generate gcm- and gcm2-deficient germ-line clones, males carrying hs-flp; FRT40A ovoD1/CyO were crossed to Df(2L)200 FRT40A/CyO females. Larvae from this cross were heat shocked daily for 1 hour at 37°C for 3 days, and the emerging Df(2L)200 FRT40A/FRT40A ovoD1 adult females were crossed to Df(2L)200/CyO (twi-lacZ) males. Notch germ-line clone mutants were generated according to a similar protocol, using the N55e11FRT10.1 and the ovoD1FRT10.1; hs-flp stocks.

    Unless specified, crosses and embryo collections were performed at 25°C. To induce transient expression of GCM, hs-gal4; uas-gcm embryos were collected at 18°C for 3 hours, aged at 18°C for 6, 9 or 12 hours, heat shocked twice at 37°C for 20 minutes, and aged at 18°C for 16, 13 or 10 hours, respectively, before being processed for analysis.

    Plasmids and transgenesis The 1.5 kb upstream regulatory region of lz (nucleotides 234118 to 235562 on the genomic scaffold AE003446) was cloned into pCasper- hs43-lacZ to generate pLZ-lacZ. The corresponding P{lz-lacZ} transgenic lines were generated by s