Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

The PAX-SIX-EYA-DACH network modulates GATA-FOG function in fly hematopoiesis and human erythropoiesis.

The GATA and PAX-SIX-EYA-DACH transcriptional networks (PSEDNs) are essential for proper development across taxa. Here, we demonstrate novel PSEDN roles in vivo in Drosophila hematopoiesis and in human erythropoiesis in vitro Using Drosophila genetics, we show that PSEDN members function with GATA to block lamellocyte differentiation and maintain the prohemocyte pool. Overexpression of human SIX1 stimulated erythroid differentiation of human erythroleukemia TF1 cells and primary hematopoietic stem-progenitor cells. Conversely, SIX1 knockout impaired erythropoiesis in both cell types. SIX1 stimulation of erythropoiesis required GATA1, as SIX1 overexpression failed to drive erythroid phenotypes and gene expression patterns in GATA1 knockout cells. SIX1 can associate with GATA1 and stimulate GATA1-mediated gene transcription, suggesting that SIX1-GATA1 physical interactions contribute to the observed functional interactions. In addition, both fly and human SIX proteins regulated GATA protein levels. Collectively, our findings demonstrate that SIX proteins enhance GATA function at multiple levels, and reveal evolutionarily conserved cooperation between the GATA and PSEDN networks that may regulate developmental processes beyond hematopoiesis.

Hedgehog signaling from the Posterior Signaling Center maintains U-shaped expression and a prohemocyte population in Drosophila.

Hematopoietic progenitor choice between multipotency and differentiation is tightly regulated by intrinsic factors and extrinsic signals from the surrounding microenvironment. The Drosophila melanogaster hematopoietic lymph gland has emerged as a powerful tool to investigate mechanisms that regulate hematopoietic progenitor choice in vivo. The lymph gland contains progenitor cells, which share key characteristics with mammalian hematopoietic progenitors such as quiescence, multipotency and niche-dependence. The lymph gland is zonally arranged, with progenitors located in medullary zone, differentiating cells in the cortical zone, and the stem cell niche or Posterior Signaling Center (PSC) residing at the base of the medullary zone (MZ). This arrangement facilitates investigations into how signaling from the microenvironment controls progenitor choice. The Drosophila Friend of GATA transcriptional regulator, U-shaped, is a conserved hematopoietic regulator. To identify additional novel intrinsic and extrinsic regulators that interface with U-shaped to control hematopoiesis, we conducted an in vivo screen for factors that genetically interact with u-shaped. Smoothened, a downstream effector of Hedgehog signaling, was one of the factors identified in the screen. Here we report our studies that characterized the relationship between Smoothened and U-shaped. We showed that the PSC and Hedgehog signaling are required for U-shaped expression and that U-shaped is an important intrinsic progenitor regulator. These observations identify a potential link between the progenitor regulatory machinery and extrinsic signals from the PSC. Furthermore, we showed that both Hedgehog signaling and the PSC are required to maintain a subpopulation of progenitors. This led to a delineation of PSC-dependent versus PSC-independent progenitors and provided further evidence that the MZ progenitor population is heterogeneous. Overall, we have identified a connection between a conserved hematopoietic master regulator and a putative stem cell niche, which adds to our understanding of how signals from the microenvironment regulate progenitor multipotency.

Signal transduction pathways, intrinsic regulators, and the control of cell fate choice.

BACKGROUND: Information regarding changes in organismal status is transmitted to the stem cell regulatory machinery by a limited number of signal transduction pathways. Consequently, these pathways derive their functional specificity through interactions with stem cell intrinsic master regulators, notably transcription factors. Identifying the molecular underpinnings of these interactions is critical to understanding stem cell function. SCOPE OF REVIEW: This review focuses on studies in Drosophila that identify the gene regulatory basis for interactions between three different signal transduction pathways and an intrinsic master transcriptional regulator in the context of hematopoietic stem-like cell fate choice. Specifically, the interface between the GATA:FOG regulatory complex and the JAK/STAT, BMP, and Hedgehog pathways is examined. MAJOR CONCLUSIONS: The GATA:FOG complex coordinates information transmitted by at least three different signal transduction pathways as a means to control stem-like cell fate choice. This illustrates emerging principles concerning regulation of stem cell function and describes a gene regulatory link between changes in organismal status and stem cell response. GENERAL SIGNIFICANCE: The Drosophila model system offers a powerful approach to identify the molecular basis of how stem cells receive, interpret, and then respond to changes in organismal status. This article is part of a Special Issue entitled: Biochemistry of Stem Cells.

Odd-skipped maintains prohemocyte potency and blocks blood cell development in Drosophila.

Studies using Drosophila have contributed significantly to our understanding of regulatory mechanisms that control stem cell fate choice. The Drosophila blood cell progenitor or prohemocyte shares important characteristics with mammalian hematopoietic stem cells, including quiescence, niche dependence, and the capacity to form all three fly blood cell types. This report extends our understanding of prohemocyte fate choice by showing that the zinc-finger protein Odd-skipped promotes multipotency and blocks differentiation. Odd-skipped was expressed in prohemocytes and downregulated in terminally differentiated plasmatocytes. Furthermore, Odd-skipped maintained the prohemocyte population and blocked differentiation of plasmatocytes and lamellocytes but not crystal cells. A previous study showed that Odd-skipped expression is downregulated by Decapentaplegic signaling. This report provides a functional basis for this regulator/target pair by suggesting that Decapentaplegic signaling limits Odd-skipped expression to promote prohemocyte differentiation. Overall, these studies are the basis for a gene regulatory model of prohemocyte cell fate choice.

Salmonella pathogenesis reveals that BMP signaling regulates blood cell homeostasis and immune responses in Drosophila.

Intercellular signaling by bone morphogenetic proteins (BMPs) regulates developmental decisions in virtually all animals. Here, we report that Decapentaplegic (Dpp; a Drosophila BMP family member) plays a role in blood cell homeostasis and immune responses by regulating a transcription factor cascade. The cascade begins with Dpp repression of Zfh1, continues with Zfh1 activation of Serpent (Srp; a GATA factor), and terminates with Srp activation of U-shaped (Ush) in hematopoietic cells. Hyperactivation of Zfh1, Srp, and Ush in dpp mutants leads to hyperplasia of plasmatocytes. Salmonella challenge revealed that in dpp mutants the misregulation of this cascade also prevents the generation of lamellocytes. These findings support the hypothesis that Ush participates in a switch between plasmatocyte and lamellocyte fate in a common precursor and further suggests a mechanism for how all blood cell types can arise from a single progenitor. These results also demonstrate that combining Drosophila and Salmonella genetics can provide novel opportunities for advancing our knowledge of hematopoiesis and innate immunity.

The GATA factor Serpent cross-regulates lozenge and u-shaped expression during Drosophila blood cell development.

The Drosophila GATA factor Serpent interacts with the RUNX factor Lozenge to activate the crystal cell program, whereas SerpentNC binds the Friend of GATA protein U-shaped to limit crystal cell production. Here, we identified a lozenge minimal hematopoietic cis-regulatory module and showed that lozenge-lacZ reporter-gene expression was autoregulated by Serpent and Lozenge. We also showed that upregulation of u-shaped was delayed until after lozenge activation, consistent with our previous results that showed u-shaped expression in the crystal cell lineage is dependent on both Serpent and Lozenge. Together, these observations describe a feed forward regulatory motif, which controls the temporal expression of u-shaped. Finally, we showed that lozenge reporter-gene activity increased in a u-shaped mutant background and that forced expression of SerpentNC with U-shaped blocked lozenge- and u-shaped-lacZ reporter-gene activity. This is the first demonstration of GATA:FOG regulation of Runx and Fog gene expression. Moreover, these results identify components of a Serpent cross-regulatory sub-circuit that can modulate lozenge expression. Based on the sub-circuit design and the combinatorial control of crystal cell production, we present a model for the specification of a dynamic bi-potential regulatory state that contributes to the selection between a Lozenge-positive and Lozenge-negative state.

Exposure to Concentrated Ambient PM2.5 Shortens Lifespan and Induces Inflammation-Associated Signaling and Oxidative Stress in Drosophila.

Exposure to ambient PM 2.5 is associated with human premature mortality. However, it has not yet been toxicologically replicated, likely due to the lack of suitable animal models. Drosophila is frequently used in longevity research due to many incomparable merits. The present study aims to validate Drosophila models for PM 2.5 toxicity study through characterizing their biological responses to exposure to concentrated ambient PM 2.5 (CAP). The survivorship curve demonstrated that exposure to CAP markedly reduced lifespan of Drosophila. This antilongevity effect of CAP exposure was observed in both male and female Drosophila, and by comparison, the male was more sensitive [50% survivals: 20 and 48 days, CAP- and filtered air (FA)-exposed males, respectively; 21 and 40 days, CAP- and FA-exposed females, respectively]. Similar to its putative pathogenesis in humans, CAP exposure-induced premature mortality in Drosophila was also coincided with activation of pro-inflammatory signaling pathways including Jak, Jnk, and Nf-κb and increased systemic oxidative stress. Furthermore, like in humans and mammals, exposure to CAP significantly increased whole-body and circulating glucose levels and increased mRNA expression of Ilp2 and Ilp5 , indicating that CAP exposure induces dysregulated insulin signaling in Drosophila. Similar to effects on humans exposure to CAP leads to premature mortality likely through induction of inflammation-associated signaling, oxidative stress, and metabolic abnormality in Drosophila, strongly supporting that it can be a useful model organism for PM 2.5 toxicity study.

The Friend of GATA Transcriptional Co-Regulator, U-Shaped, Is a Downstream Antagonist of Dorsal-Driven Prohemocyte Differentiation in Drosophila.

Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

Hemocyte development during Drosophila embryogenesis.

The model genetic organism Drosophila melanogaster has a rudimentary hematopoietic system with two embryonic blood cell types, crystal cells, and plasmatocytes. These distinct lineages provide the animal with an innate immune response and a means to remove apoptotic cells. Genetic analyses of Drosophila hematopoiesis have identified specific genes that function in blood cell formation. Complimentary deoxyribonucleic acid and antibody probes for these hematopoietic factors serve as important reagents to follow hemocyte lineage commitment and differentiation during embryogenesis. This is possible through methods described in this chapter on messenger ribonucleic acid localization by in situ hybridization and protein localization by immunohistochemical staining within hemocyte precursors and mature blood cells.

Antioxidants maintain E-cadherin levels to limit Drosophila prohemocyte differentiation.

Mitochondrial reactive oxygen species (ROS) regulate a variety of biological processes by networking with signal transduction pathways to maintain homeostasis and support adaptation to stress. In this capacity, ROS have been shown to promote the differentiation of progenitor cells, including mammalian embryonic and hematopoietic stem cells and Drosophila hematopoietic progenitors (prohemocytes). However, many questions remain about how ROS alter the regulatory machinery to promote progenitor differentiation. Here, we provide evidence for the hypothesis that ROS reduce E-cadherin levels to promote Drosophila prohemocyte differentiation. Specifically, we show that knockdown of the antioxidants, Superoxide dismutatase 2 and Catalase reduce E-cadherin protein levels prior to the loss of Odd-skipped-expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, two established targets of ROS, Enhancer of Polycomb and FOS, control the level of E-cadherin protein expression. Finally, we show that knockdown of either Superoxide dismutatase 2 or Catalase leads to an increase in the E-cadherin repressor, Serpent. As a result, antioxidants and targets of ROS can control E-cadherin protein levels, and over-expression of E-cadherin can ameliorate the prohemocyte response to oxidative stress. Collectively, these data strongly suggest that ROS promote differentiation by reducing E-cadherin levels. In mammalian systems, ROS promote embryonic stem cell differentiation, whereas E-cadherin blocks differentiation. However, it is not known if elevated ROS reduce E-cadherin to promote embryonic stem cell differentiation. Thus, our findings may have identified an important mechanism by which ROS promote stem/progenitor cell differentiation.

Drosophila E-cadherin functions in hematopoietic progenitors to maintain multipotency and block differentiation.

A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology.

Upregulation of the Drosophila Friend of GATA gene U-shaped by JAK/STAT signaling maintains lymph gland prohemocyte potency.

Studies using Drosophila melanogaster have contributed significantly to our understanding of the interaction between stem cells and their protective microenvironments or stem cell niches. During lymph gland hematopoiesis, the Drosophila posterior signaling center functions as a stem cell niche to maintain prohemocyte multipotency through Hedgehog and JAK/STAT signaling. In this study, we provide evidence that the Friend of GATA protein U-shaped is an important regulator of lymph gland prohemocyte potency and differentiation. U-shaped expression was determined to be upregulated in third-instar lymph gland prohemocytes and downregulated in a subpopulation of differentiating blood cells. Genetic analyses indicated that U-shaped maintains the prohemocyte population by blocking differentiation. In addition, activated STAT directly regulated ush expression as evidenced by results from loss- and gain-of-function studies and from analyses of the u-shaped hematopoietic cis-regulatory module. Collectively, these findings identify U-shaped as a downstream effector of the posterior signaling center, establishing a novel link between the stem cell niche and the intrinsic regulation of potency and differentiation. Given the functional conservation of Friend of GATA proteins and the role that GATA factors play during cell fate choice, these factors may regulate essential functions of vertebrate hematopoietic stem cells, including processing signals from the stem cell niche.

U-shaped protein domains required for repression of cardiac gene expression in Drosophila.

U-shaped is a zinc finger protein that functions predominantly as a negative transcriptional regulator of cell fate determination during Drosophila development. In the early stages of dorsal vessel formation, the protein acts to control cardioblast specification, working as a negative attenuator of the cardiogenic GATA factor Pannier. Pannier and the homeodomain protein Tinman normally work together to specify heart cells and activate cardioblast gene expression. One target of this positive regulation is a heart enhancer of the D-mef2 gene and U-shaped has been shown to antagonize enhancer activation by Pannier and Tinman. We have mapped protein domains of U-shaped required for its repression of cardioblast gene expression. Such studies showed GATA factor interacting zinc fingers of U-shaped are required for enhancer repression, as well as three small motifs that are likely needed for co-factor binding and/or protein modification. These analyses have also allowed for the definition of a 253 amino acid interval of U-shaped that is essential for its nuclear localization. Together, these findings provide molecular insights into the function of U-shaped as a negative regulator of heart development in Drosophila.

Regulation of Drosophila friend of GATA gene, u-shaped, during hematopoiesis: a direct role for serpent and lozenge.

Friend of GATA proteins interact with GATA factors to regulate development in a variety of tissues. We analyzed cis- and trans-regulation of the Drosophila gene, u-shaped, to better understand the transcriptional control of this important gene family during hematopoiesis. Using overlapping genomic fragments driving tissue-specific reporter-gene (lacZ) expression, we identified two minimal hematopoietic enhancers within the 7.4 kb region upstream of the transcription start site. One enhancer was active in all classes of hemocytes, whereas the other was active in hemocyte precursors and plasmatocytes only. The GATA factor, Serpent, directly regulated the activity of both enhancers. However, activity in the crystal cell lineage not only required Serpent but also the RUNX homologue, Lozenge. This is the first demonstration of GATA and RUNX direct regulation of Friend of GATA gene expression and provides additional evidence for the combinatorial control of crystal cell lineage commitment by Serpent, Lozenge, and U-shaped. In addition, we analyzed cis-regulation of ush expression in the lymph gland and identified similarities and differences between regulatory strategies used during embryonic and lymph gland hematopoiesis. The results of these studies provide information to analyze further the regulation of this conserved gene family and its role during hematopoietic lineage commitment.

Combinatorial interactions of serpent, lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis.

The GATA factor Serpent (Srp) is required for hemocyte precursor formation during Drosophila hematopoiesis. These blood cell progenitors give rise to two distinct lineages within the developing embryo. Lozenge, a Runx protein homologue, and Glial cells missing-1 and -2 are essential for crystal cell and plasmatocyte production, respectively. In contrast U-shaped, a Friend of GATA class factor, antagonizes crystal cell formation. Here we show that Srp, Lozenge, and U-shaped interact in different combinations to regulate crystal cell lineage commitment. Coexpression of Srp and Lozenge synergistically activated the crystal cell program in both embryonic and larval stages. Furthermore, expression of Lozenge and SrpNC, a Srp isoform with N- and C-terminal zinc fingers, inhibited u-shaped expression, indicating that crystal cell activation coincided with the down-regulation of this repressor-encoding gene. In contrast, whereas U-shaped and SrpNC together blocked crystal cell production, coexpression of U-shaped with noninteracting Srp proteins failed to prevent overproduction of this hemocyte population. Such results indicated that U-shaped and SrpNC must interact to block crystal cell production. Taken together, these studies show that the specialized SrpNC isoform plays a pivotal role during crystal cell lineage commitment, acting as an activator or repressor depending on the availability of specific transcriptional coregulators. These findings provide definitive proof of the combinatorial regulation of hematopoiesis in Drosophila and an in vivo demonstration of GATA and Runx functional interaction in a blood cell commitment program.