Serpent, Suppressor of Hairless and U-shaped are crucial regulators of hedgehog niche expression and prohemocyte maintenance during Drosophila larval hematopoiesis

Owing to highly productive research efforts in recent years, Drosophila has emerged as a valuable organism for the discovery and analysis of genes controlling hematopoiesis (Evans et al., 2003; Sorrentino et al., 2005; Crozatier and Meister, 2007; Koch and Radtke, 2007; Martinez-Agosto et al., 2007). As in vertebrate animals, blood cell formation occurs in multiple spatiotemporal waves during Drosophila development. Transplantation experiments using single cells derived from blastoderm stage embryos have indicated that blood cell precursors arise from two distinct origins: cephalic mesoderm that generates embryonic hemocytes and a thoracic mesodermal region that will form lymph glands (Holz et al., 2003), the organ used for hematopoiesis during larval development (Lebestky et al., 2000). Differentiated hemocytes produced during both hematopoietic waves possess characteristics comparable to those found in vertebrate myeloid lineages (Meister and Langueux, 2003). Embryo-derived blood cells include plasmatocytes and crystal cells. Plasmatocytes constitute the major class, functioning as macrophage-like cells that mediate phagocytosis of bacterial pathogens and apoptotic bodies. Crystal cells make up the minor class, being involved in wound healing and large foreign body encapsulation. Together, they number ∼700 cells by the end of embryogenesis and persist into larval stages, when they undergo multiple rounds of mitosis, generating 6000-8000 hemocytes by the end of larval development (Sorrentino et al., 2007; Tokusumi et al., 2009b). A subpopulation of these cells will take up residence as sessile hemocytes in subepidermal locations within larvae, where they can be rapidly converted into lamellocytes, a third hemocyte type that is produced in response to wasp infestation (Markus et al., 2009; Honti et al., 2010). Lamellocytes are large, flat, adhesive cells that are crucial for the successful encapsulation of invasive species, such as the parasitic wasp egg.

The lymph gland hematopoietic organ is formed near the end of embryogenesis from two clusters of cells derived from anterior cardiogenic mesoderm (Crozatier et al., 2004; Mandal et al., 2004). About 20 pairs of hemangioblast-like cells give rise to three distinct lineages that will form the lymph glands and anterior part of the dorsal vessel. Notch (N) pathway signaling serves as the genetic switch that differentially programs these progenitors towards cell fates that generate the lymph glands (blood lineage), heart tube (vascular lineage), or heart tube-associated pericardial cells (nephrocytic lineage). An essential requirement has also been proven for Tailup (Islet1) in lymph gland formation, in which it functions as an early-acting regulator of serpent (srp), odd-skipped and Hand hematopoietic transcription factor gene expression (Tao et al., 2007).

By the end of the third larval instar, each anterior lymph gland is composed of three morphologically and molecularly distinct regions (Jung et al., 2005). The posterior signaling center (PSC) is a cellular domain formed during late embryogenesis due to the specification function of the homeotic gene Antennapedia (Antp) (Mandal et al., 2007) and the maintenance function of Collier (Col; Knot – FlyBase), the Drosophila ortholog of the vertebrate transcription factor early B-cell factor (Crozatier et al., 2004). PSC cells selectively express the Hedgehog (Hh) and Serrate (Ser) signaling molecules and extend numerous thin filopodia into the neighboring medullary zone (Lebestky et al., 2003; Krzemien et al., 2007; Mandal et al., 2007). This latter lymph gland domain is populated by undifferentiated and slowly proliferating blood cell progenitors (Mandal et al., 2007). Prohemocytes within the medullary zone express the Hh receptor Patched (Ptc) and the Hh pathway transcriptional effector Cubitus interruptus (Ci). Medullary zone cells also express components of the Jak/Stat signaling pathway. By contrast, the third lymph gland domain – the cortical zone – solely contains differentiating and mature hemocytes, such as plasmatocytes and crystal cells. Upon wasp parasitization, or in certain altered genetic backgrounds, lamellocytes will also appear in the cortical zone as a third type of differentiated hemocyte (Lanot et al., 2001; Sorrentino et al., 2002; Sorrentino et al., 2007; Gao et al., 2009; Markus et al., 2009; Tokusumi et al., 2009b).

Two independent studies have provided compelling data to support the contention that the PSC functions as a hematopoietic progenitor niche within the lymph gland, with this cellular domain being essential for maintaining normal hemocyte homeostasis (Krzemien et al., 2007; Mandal et al., 2007). These investigations showed that communication between the PSC and prohemocytes present in the medullary zone is crucial for the preservation of the progenitor population and to prevent these cells from becoming abnormally programmed to differentiate into mature hemocytes. Seminal findings from these studies can be summarized as follows (Crozatier and Meister, 2007): Col expression must be restricted to the PSC by the localized expression of Ser; Hh must be expressed selectively in the PSC, coupled with the non-autonomous activation of the Hh signaling pathway in prohemocytes of the medullary zone; and the PSC triggers activation of the Jak/Stat pathway within cells of the medullary zone. With the perturbation of any of these molecular events, the precursor population of the medullary zone is lost owing to the premature differentiation of hemocytes, which swell the cortical zone. Although the exact interrelationship of Ser, Hh and Jak/Stat signaling within the lymph gland is currently unknown, the cytoplasmic extensions emanating from PSC cells might facilitate instructive signaling between these niche cells and hematopoietic progenitors present in the medullary zone (Krzemien et al., 2007; Mandal et al., 2007). A more recent study showed that components of the Wingless (Wg) signaling pathway are expressed in the stem-like prohemocytes to reciprocally regulate the proliferation and maintenance of cells within the supportive PSC niche (Sinenko et al., 2009). The cellular organization and molecular signaling of the Drosophila lymph gland are remarkably similar to those of the hematopoietic stem cell niches of vertebrate animals, including several mammals (Gering and Patient, 2005; Yamashita et al., 2005; Martinez-Agosto et al., 2007; Orkin and Zon, 2008).

These findings have documented a complex role for distinct signaling pathways and a sophisticated organization of non-equivalent cell types within the hematopoietic progenitor-niche microenvironment of the Drosophila lymph gland. To gain mechanistic insights into the genetic control of one of these pathways, we pursued the transcriptional enhancer controlling the PSC-specific expression of the essential Hh signaling molecule. We rationalized that through a detailed analysis of the positive and negative inputs influencing this PSC-active regulatory module, enlightening information could be gained on those genes required for the precise expression of Hh in niche cells and on their role in the maintenance of the hematopoietic progenitor population. Here, we document crucial roles for the Srp, Su(H) and Ush transcriptional regulators in the control of Hh niche expression and verify their importance for normal blood cell homeostasis within the Drosophila larval hematopoietic organ.

We used the following lines in this study: ush^VX22 and ush^r24 (Cubadda et al., 1997) (M. Haenlin); P85col-Gal4 and UAS-col (Crozatier et al., 2004; Krzemien et al., 2007) (M. Crozatier); BcF6-GFP (Gajewski et al., 2007); eater-DsRed and MSNF9mo-mCherry (Tokusumi et al., 2009a); tepIV-Gal4 (Drosophila Genetic Resource Center, Kyoto Stock Center, Japan) and UAS-srp RNAi (two lines: GD12779, Vienna Drosophila RNAi Center; and HM05094, Transgenic RNAi Resource Project at Harvard Medical School). We also obtained several stocks from the Bloomington Stock Center (Indiana University): w¹¹¹⁸, N^1N-ts1, Antp²⁵, Antp¹⁷, Su(H)¹, Su(H)^IB115, UAS-gapGFP, CyO Act-GFP, UAS-hh and UAS-dTCF^DN.

Transgenic lines harboring hh DNA-GFP reporter fusions were generated as described previously (Rubin and Spradling, 1982; Tokusumi et al., 2009b). Briefly, we amplified hh genomic DNA fragments from a BAC clone using PrimeStar DNA polymerase (Takara-Millus), followed by DNA subcloning into the P-element vector pH Stinger (Barolo et al., 2000). For the analysis of essential cis-regulatory elements, we introduced point mutations into trans factor binding sites of the enhancer region with the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The sequence of the PSC-active 1020 bp hhF4f enhancer DNA is given in Fig. S1 in the supplementary material. The PSC-active hhF4l enhancer fragment corresponds to the 3′ 190 bp interval of the larger DNA. Potential Antp, Srp, Su(H) (Brou et al., 1994; Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995) and Col (Dubois et al., 2007) binding sites are highlighted within the hhF4f sequence, with the introduced nucleotide changes indicated. Sequence comparison of hhF4f DNA from D. melanogaster with the corresponding DNAs of 11 additional Drosophila species showed that the putative EBF/Col binding site was fully conserved in only six of 12 species (see Fig. S2 in the supplementary material). Following germline transformation of hh DNA-GFP reporter constructs, we established and analyzed at least ten independent lines.

The following primary antibodies were used to determine protein expression in lymph glands dissected from control or mutant animals: rabbit anti-Hh (1:100; T. Kornberg) (Tabata and Kornberg, 1994), rabbit anti-Ush (1:400) (Fossett et al., 2001), rabbit anti-Su(H) (1:1000; sc-25761, Santa Cruz), mouse anti-Antp (1:100; 4C3, Developmental Studies Hybridoma Bank), mouse anti-Ptc (1:200; Developmental Studies Hybridoma Bank), and mouse anti-plasmatocyte (P1) antibody (1:100; I. Ando) (Vilmos et al., 2004). Lymph glands were dissected from staged larvae, fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature, washed with PBS containing 0.1% Triton X-100 (PBST), and blocked with 5% goat serum in PBST. We then added diluted primary antibodies in PBST containing 5% goat serum, incubated overnight at 4°C, washed with PBST several times, and then incubated with Alexa 488- or 555-conjugated secondary antibodies (1:500; Molecular Probes) in PBST containing 5% goat serum for 1 hour at room temperature. After washing with PBST, samples were mounted in 50% glycerol in PBS. Immunostained samples were analyzed with a Zeiss Axioplan fluorescence microscope or a Zeiss LSM710 laser-scanning confocal microscope.

As Hh produced by the PSC is a crucial signaling molecule regulating the maintenance of hemocyte precursors within the medullary zone (Mandal et al., 2007), we were interested to elucidate the genetic mechanisms controlling hh cell-specific expression in the hematopoietic niche. Towards this goal, we localized the transcriptional enhancer controlling hh expression in the PSC. Initially, we scanned seven 3-kb DNA intervals, spanning 21 kb of hh upstream and intragenic sequence, for regulatory DNAs that could drive GFP reporter expression selectively in the PSC domain of the lymph gland. The overlapping hhF4 and hhF5 DNAs, derived from hh intron 1, possessed the ability to direct GFP expression exclusively in the PSC (Fig. 1A). The smaller, 1.0 kb hhF4f interval likewise directed GFP expression in niche cells, indicating that it harbored a regulatory module sufficient for hh transcription in the PSC (Fig. 1A,B). Double-labeling experiments verified the fidelity of the transcriptional enhancer, as the regulatory DNA was active in PSC cells that also expressed Antp (Fig. 1C) and Hh (Fig. 1D).

The functions of a few transcription factors have been shown to be vital for the generation and maintenance of the PSC cell population within the lymph gland: Antp specifies the PSC (Mandal et al., 2007), whereas Col maintains the PSC population (Crozatier et al., 2004). We monitored the activity of the hhF4f-GFP transgene in mutant backgrounds that altered the functions of these hematopoietic regulators. In Antp loss-of-function embryos, the PSC was lost and a corresponding absence of hhF4f-GFP expression was observed (Fig. 1E). By contrast, forced expression of Col during lymph gland development resulted in an overproduction of PSC cells, with the supernumerary cells being labeled with the GFP reporter driven by the hh enhancer (Fig. 1F). Wg signaling also positively regulates the proliferation and maintenance of these niche cells (Sinenko et al., 2009). Consistent with this observation, we failed to detect hhF4f-GFP-positive cells in lymph glands expressing a dominant-negative version of TCF (Pangolin – FlyBase), a downstream effector of Wg signaling (Fig. 1G). Together, these genetic studies confirm the fidelity of the hh regulatory module for hematopoietic niche cells.

Six additional 5′- or 3′-truncated versions of the PSC-active hhF4f DNA were generated and tested for their ability to drive GFP reporter expression in hematopoietic niche cells within lymph glands isolated from transgenic larvae. Through these analyses, we were able to further delimit the PSC regulatory module to the 190 bp hhF4l interval of hh intron 1 (Fig. 2A). A sequence comparison of this region of the Drosophila melanogaster genome with corresponding DNA intervals of 11 other sequenced Drosophila species revealed the precise conservation of four putative transcription factor binding sites: a possible Antp recognition element, two GATA sequences that could bind Srp, and a potential recognition site for the Su(H) protein (Fig. 2B; see Fig. S1 in the supplementary material). As mutation of the putative Antp binding site within the 1.0 kb hhF4f DNA (hhF4f mAntp construct) and 190 bp hhF4l DNA (hhF4l mAntp construct) did not affect hh enhancer function (Fig. 2A), we concluded that this sequence was not a direct target of Antp activation. Thus, we pursued Srp and Su(H) as potential transcriptional regulators of the hh enhancer.

A role for the hematopoietic factor Srp in the regulation of hh PSC expression was implicated by the finding that mutation of both conserved GATA recognition sequences (hhF4f mGATA construct) led to a complete loss of PSC enhancer activity (Fig. 2C). More direct evidence for Srp involvement in hh niche expression was found when RNAi knockdown of srp function resulted in a complete loss of hh enhancer activity (Fig. 2D) and an absence of Hh protein (Fig. 2E), as derived from expression of the endogenous gene, from niche cells of the PSC. RNAi knockdown of srp also resulted in defects in niche cell differentiation, based on the absence of filopodial extensions (Fig. 2G). As an important control for these experiments, we showed that PSC cell nuclei present in lymph glands dissected from P85col>UAS-srp RNAi animals failed to accumulate Srp protein at detectable levels (see Fig. S3 in the supplementary material). In summary, the enhancer mutagenesis and srp loss-of-function data demonstrate that this hematopoietic GATA factor serves as an activator of hh expression in the PSC niche.

As noted above, the minimal hh PSC enhancer present in the 190 bp hhF4l DNA contains an evolutionarily conserved GTGGGAA element, which is a potential recognition sequence for the transcriptional repressor Su(H). Using an antibody directed against Su(H), we demonstrated that this protein is expressed in lymph glands, with highest accumulation in hematopoietic progenitors within the medullary zone and a lower accumulation in differentiated hemocytes in the cortical zone (Fig. 3A). A double-labeling experiment to detect Su(H) and GFP driven by the hh PSC enhancer also showed a low, but detectable, presence of Su(H) in niche cells (Fig. 3B).

Given this demonstration of Su(H) expression in lymph glands, we mutated the conserved GTGGGAA element within the context of the 1.0 kb hhF4f PSC enhancer [hhF4f mSu(H) construct; Fig. 2A] and generated transgenic flies harboring this enhancer test DNA. Mutation of the prospective Su(H) binding site resulted in the de novo expression of the GFP reporter in prohemocytes of the medullary zone (Fig. 3C). Furthermore, in a Su(H) loss-of-function mutant background, we observed an expansion of hh PSC transcriptional enhancer activity to cells of the medullary zone (Fig. 3F) and ectopic expression of Hh protein in the same cells (Fig. 3G). In the same Su(H) mutant, Antp expression was not expanded, indicating the maintenance of a normal PSC cell population in this genetic background (Fig. 3H). It has been previously reported that Su(H) can function as a repressor of gene expression in an N-independent manner (Koelzer and Klein, 2003; Koelzer and Klein, 2006). We tested the activity of the hh-GFP transgene in lymph glands obtained from N^ts mutant larvae and determined that the PSC-active enhancer functioned normally in this altered genetic background (data not shown). These results demonstrated that Su(H) serves as a crucial negative regulator of hh transcription (in an N-independent mode), preventing both endogenous and transgenic hh expression in blood cell progenitors of the medullary zone.

Previous studies have shown that the disruption of Hh signaling from the PSC niche to hematopoietic precursor cells within the medullary zone results in a loss of the progenitor population and in enhanced differentiation of plasmatocytes and crystal cells throughout the lymph gland (Mandal et al., 2007). Since RNAi knockdown of srp resulted in an absence of Hh in the PSC, we investigated the effect of Srp perturbation on prohemocyte and hemocyte homeostasis. The Hh receptor Ptc serves as a marker of hemocyte precursors within the medullary zone (Mandal et al., 2007) (Fig. 4A) and we observed a strong reduction in the number of Ptc-positive cells in the induced srp mutant background (Fig. 4B). Also, in srp mutant lymph glands, a large increase in the number of differentiated plasmatocytes (Fig. 4E) and crystal cells (Fig. 4H) was observed based on the use of the P1 antibody and BcF6-GFP markers. Additionally, we determined the circulating hemocyte concentration in control versus P85col>UAS-srp RNAi third instar larvae and observed a 67% increase in blood cell number due to perturbation of srp function in niche cells (see Fig. S4 in the supplementary material). Thus, the knockdown of srp function in lymph glands, and the resulting loss of Hh expression in the PSC, culminated in a blood cell phenotype identical to that seen in hh mutant lymph glands, with a loss of the progenitor population and copious production of differentiated plasmatocytes and crystal cells.

Comparable analyses were undertaken with lymph glands obtained from Su(H) loss-of-function mutants, which showed the abnormal expansion of Hh expression to progenitor cells within the medullary zone. As with the disruption of srp function, Su(H) mutant tissues exhibited a loss of Ptc-positive prohemocytes (Fig. 4C) and the overproduction of differentiated plasmatocytes (Fig. 4F). By contrast, an increase was not observed in the crystal cell population in Su(H) mutant lymph glands (Fig. 4I), probably because this gene is required for lozenge activation and crystal cell differentiation (Duvic et al., 2002; Lebestky et al., 2003). Together, these findings demonstrated that Srp and Su(H) are required for prohemocyte maintenance and for the regulation of hemocyte differentiation in the lymph gland.

As all lymph gland cells express Srp (Jung et al., 2005; Sorrentino et al., 2007), the question arose as to the mechanism by which this hematopoietic GATA factor activates hh selectively in the PSC and not in cells throughout the remainder of the lymph gland. hh repression by Su(H) is certainly part of the answer to this question of regulatory control. In addition, we considered the possibility that the transcriptional regulator Ush functions with Srp in precisely regulating hh expression. Previous reports demonstrated that the SrpNC-Ush (SrpNC is an Srp isoform with N- and C-terminal zinc fingers) combination functions as a negative regulator of crystal cell lineage specification (Fossett et al., 2003; Muratoglu et al., 2007).

To address a potential role for Ush in hh regulation, we assayed its expression in lymph glands. Ush protein was expressed in most lymph gland cells, with the exception of the hhF4f-GFP-positive PSC cells (Fig. 5A). Forcing the expression of Ush in PSC cells resulted in a loss of hh PSC enhancer activity, while maintaining the Antp-positive niche cell population (Fig. 5B). Expressing Ush in the PSC also led to defects in niche cell differentiation, based on a lack of filopodia formation, processes that normally extend from PSC cells (Fig. 5C). Conversely, in lymph glands dissected from ush loss-of-function animals, an expansion of hhF4f-GFP transgene activity (Fig. 5D,G,H) and Hh protein accumulation (Fig. 5F,G) were observed, implicating Ush as an additional and crucial negative regulator of hh transcription in this hematopoietic organ. A reduction in ush function also culminated in a robust production of lamellocytes in the lymph glands (Fig. 5H) owing to its proven function as a positive regulator of prohemocyte maintenance and as a negative regulator of lamellocyte differentiation (Sorrentino et al., 2007; Gao et al., 2009).

The Drosophila lymph gland has emerged as an informative tissue for the study of cellular organization and intercellular signaling within a hematopoietic progenitor-niche cell microenvironment. Four signaling pathways have been identified thus far that control blood cell homeostasis within this larval hematopoietic organ: Hh, Jak/Stat, N and Wg (Crozatier et al., 2004; Krzemien et al., 2007; Mandal et al., 2007; Sinenko et al., 2009). Our current studies have focused on the regulation of Hh expression in the PSC owing to the vital role of the Hh signal sent from the niche cells to the hemocyte precursors, which maintains this progenitor population while preventing premature and precocious blood cell differentiation. The utilization of this strategy has allowed us to gain mechanistic insights into the mode of hh transcriptional control in the PSC and the crucial functions of its positive and negative regulators in prohemocyte maintenance.

Through detailed molecular and gene expression analyses we have identified the PSC-active transcriptional enhancer within hh intron 1 and delimited its location to a minimal 190 bp region. The hh enhancer-GFP transgene faithfully recapitulates the niche cell expression of Hh derived from the endogenous gene, as double-labeling experiments with the GFP marker and Antp or Hh show a clear co-expression in PSC domain cells. Appropriately, GFP expression is not detected in Antp loss-of-function or TCF^DN genetic backgrounds, which culminate in an absence of niche cells from the lymph gland. The hematopoietic GATA factor Srp serves as a positive activator of hh PSC expression, as mutation of two evolutionarily conserved GATA elements in the enhancer abrogates its function and Srp functional knockdown via srp RNAi results in hh enhancer-GFP transgene inactivity and the absence of Hh protein expression. An additional intriguing phenotype was observed in lymph glands expressing the srp RNAi transgene, that being a strong reduction in the number of filopodial extensions emerging from cells of the PSC. This phenotype suggests a functional role for Srp in the correct differentiation of niche cells, via a requirement for normal Hh presentation from these cells and/or the transcriptional regulation by Srp of additional genes needed for the formation of filopodia.

As Srp accumulates in all cells of the lymph gland (Jung et al., 2005; Sorrentino et al., 2007), a question arose as to how hh expression is restricted to cells of the PSC. This paradox could be explained by a mechanism in which hh expression is also under some means of negative transcriptional control in non-PSC cells of the lymph gland. This possibility proved to be correct, with our analyses identifying two negative regulators of hh lymph gland expression. The first is Su(H). Mutation of the evolutionarily conserved GTGGGAA element, a predicted recognition sequence for this transcriptional repressor, resulted in an expanded activity of the hh PSC enhancer-GFP transgene; that is, we observed the de novo appearance of GFP in prohemocytes of the medullary zone. Likewise, ectopic medullary zone expression of the wild-type PSC enhancer-GFP transgene and of Hh protein was seen in lymph glands mutant for Su(H). These findings, coupled with the detection of Su(H) in blood cell progenitors, strongly implicate this factor as a transcriptional repressor of the hh PSC enhancer, restricting its expression to niche cells.

Additional studies identified Ush as a second negative regulator of hh expression. Ush is expressed in most cells of the lymph gland, with the exception of those cells resident within the PSC domain. Previous research demonstrated that ush expression in the lymph gland is under the positive control of both Srp (Muratoglu et al., 2006) and the Jak/Stat signaling pathway (Gao et al., 2009). Why Ush protein fails to be expressed in the PSC remains to be determined. Forced expression of ush in niche cells resulted in inactivation of the hh PSC enhancer and reduced the formation of filopodia. We hypothesize that Ush might be forming an inhibitory complex with the SrpNC protein, changing Srp from a positive transcriptional activator to a negative regulator of hh lymph gland expression. Such a mechanism has been demonstrated previously in the negative regulation by Ush of crystal cell lineage commitment (Fossett et al., 2003; Muratoglu et al., 2007). The expansion of wild-type hh enhancer-GFP transgene and Hh protein expression to prohemocytes within the medullary zone and to differentiated hemocytes within the cortical zone in lymph glands mutant for ush is also supportive of Ush functioning as a negative regulator of hh expression.

Bringing these results together, a model can be proposed for the regulatory events that culminate in the precise expression of the vital Hh signaling molecule in niche cells (Fig. 6). Srp is a direct transcriptional activator of hh in the lymph gland and Hh protein is detected in niche cells due to this activity. hh expression is inhibited in prohemocytes of the medullary zone by Su(H) action, while a repressive SrpNC-Ush transcriptional complex prevents Srp from activating hh expression in prohemocytes and in differentiated hemocytes of the medullary zone and cortical zone. Together, these positive and negative modes of regulation would allow for the niche cell-specific expression of Hh and facilitate the localized presentation of this crucial signaling molecule to neighboring hematopoietic progenitors.

The identification of Srp and Su(H) as key regulators of Hh expression in the larval hematopoietic organ prompted an investigation into the functional requirement of these proteins in the control of blood cell homeostasis. Since Srp knockdown by RNAi leads to an absence of the crucial Hh signal, it was not surprising to find that normal Srp function is required for prohemocyte maintenance and the control of hemocyte differentiation within the lymph gland; that is, we observed a severe reduction of Ptc-positive hematopoietic progenitors and a strong increase in differentiated plasmatocytes and crystal cells in srp mutant tissue.

Likewise, Ptc-positive prohemocytes were lost and large numbers of plasmatocytes were prematurely formed in Su(H) mutant lymph glands. This disruption of prohemocyte maintenance occurred even though Hh protein expression was expanded throughout the medullary zone. This raised the question as to why expanded Hh protein and possible Hh pathway activation did not increase the progenitor population in Su(H) mutant lymph glands, instead of the observed loss of prohemocytes and appearance of differentiated plasmatocytes. One explanation might be that the PSC niche is not expanded in Su(H) mutant lymph glands (Fig. 3H) and Hh might only function in promoting blood cell precursor maintenance within the context of the highly ordered progenitor-niche microenvironment. It has been hypothesized that the filopodial extensions that emanate from differentiated niche cells are crucial for Hh signal transduction from the PSC to progenitor cells of the medullary zone (Krzemien et al., 2007; Mandal et al., 2007). The possibility exists that ectopic Hh protein, which is not produced or presented by niche cells, is unable to positively regulate prohemocyte homeostasis. An experimental result consistent with this hypothesis is that expression of UAS-hh under the control of the medullary zone-specific tepIV-Gal4 driver failed to expand the blood cell progenitor population (data not shown). A second possibility is that the Hh pathway transcriptional effector Ci might require the co-function of Su(H) in its control of prohemocyte maintenance. This model would predict that, in the absence of Su(H) function, Hh signaling would be less (or non) effective in controlling the genetic and cellular events needed for the maintenance of the prohemocyte state. Third, Su(H) might regulate additional target genes, the expression (or repression) of which is crucial for normal blood cell precursor maintenance and the prevention of premature hemocyte differentiation. Finally, we cannot rule out the possibility that the expression of ectopic Hh in medullary zone cells, in the context of the adverse effects of Su(H) loss of function in these cells, culminates in the disruption of normal Hh pathway signaling due to an unforeseen dominant-negative effect.

In summary, these findings add significantly to our knowledge of hematopoietic transcription factors that function to control stem-like progenitor maintenance and blood cell differentiation in the lymph gland. An additional conclusion from these studies is that the hh enhancer-GFP transgene can serve as a beneficial reagent to identify and characterize genes and physiological conditions that control the cellular organization of the hematopoietic progenitor-niche cell microenvironment. RNAi-based genetic screens could be undertaken using this high-precision marker to determine signaling pathways and/or environmental stress conditions that might alter niche cell number and function, leading to an alteration in hematopoietic progenitor maintenance coupled with the robust production of differentiated blood cells. Much remains to be determined about the regulated control of these critical hematopoietic changes and their likely relevance to hematopoietic stem cell-niche interactions in mammals.

We thank I. Ando, M. Crozatier, M. Haenlin, T. Kornberg and the various stock centers for antibodies and Drosophila strains; and T. Vargo-Gogola and the Indiana University Medical School, South Bend, for use of their Zeiss LSM710 laser confocal microscope. This work was supported by grants to R.A.S. from the National Institutes of Health (HL071540) and the Notre Dame Initiative in Adult Stem Cell Research. This research was facilitated through the use of the Notre Dame Integrated Imaging Facility. Deposited in PMC for release after 12 months.