Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth.

In Drosophila, definitive haematopoiesis takes place in a specialized organ termed "lymph gland". It harbours multi-potent stem-like blood progenitor cells whose development controls overall growth of this haematopoietic tissue and formation of mature blood cells. With respect to its development, neurotransmitters have emerged as potent regulators of blood-progenitor cell development and function. In this study, we extend our understanding of neurotransmitters and show that progenitors are self-sufficient with regard to synthesizing dopamine, a well-established neurotransmitter. These cells also have modules for dopamine sensing through the receptor and transporter. We found that modulating expression of these components in progenitor cells affected lymph gland growth, which suggested growth-promoting function of dopamine in blood-progenitor cells. Cell-cycle analysis of developing lymph glands revealed an unexpected requirement for intracellular dopamine in moderating the progression of early progenitor cells from S to G2 phase of the cell cycle, while activation of dopamine receptor signalling later in development regulated their progression from G2 and entry into mitosis. The dual capacity in which dopamine operated, first intracellularly to coordinate S/G2 transition and later extracellularly in G2/M transition, was critical for the growth of the lymph gland. Overall, the data presented highlight a novel non-canonical use of dopamine in the myeloid system that reveals an uncharacterized function of intracellular dopamine in cell-cycle phasing with outcomes on haematopoietic growth and immunity as well.

Blood progenitor redox homeostasis through olfaction-derived systemic GABA in hematopoietic growth control in Drosophila.

The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. We show that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control.

Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts.

Progenitors of the thoracic tracheal system of adult Drosophila (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ataxia telangiectasia mutated and rad3-related kinase (ATR)-dependent phosphorylation of checkpoint kinase 1 (Chk1) that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018; Kizhedathu et al., 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an H2O2 generating dual oxidase. ROS quenching by overexpression of superoxide dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of H2O2 in minutes. The findings presented reveal that H2O2 activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

Metabolic control of cellular immune-competency by odors in Drosophila.

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. We find that Drosophila employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. We show that neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming.

Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila.

A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. Here, we explore the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. We report that Drosophila larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, our findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well.

Temporal specificity and heterogeneity of Drosophila immune cells.

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

Olfactory control of blood progenitor maintenance.

Drosophila hematopoietic progenitor maintenance involves both near neighbor and systemic interactions. This study shows that olfactory receptor neurons (ORNs) function upstream of a small set of neurosecretory cells that express GABA. Upon olfactory stimulation, GABA from these neurosecretory cells is secreted into the circulating hemolymph and binds to metabotropic GABAB receptors expressed on blood progenitors within the hematopoietic organ, the lymph gland. The resulting GABA signal causes high cytosolic Ca(2+), which is necessary and sufficient for progenitor maintenance. Thus, the activation of an odorant receptor is essential for blood progenitor maintenance, and consequently, larvae raised on minimal odor environments fail to sustain a pool of hematopoietic progenitors. This study links sensory perception and the effects of its deprivation on the integrity of the hematopoietic and innate immune systems in Drosophila. PAPERCLIP:

Direct sensing of systemic and nutritional signals by haematopoietic progenitors in Drosophila.

The Drosophila lymph gland is a haematopoietic organ in which progenitor cells, which are most akin to the common myeloid progenitor in mammals, proliferate and differentiate into three types of mature cell--plasmatocytes, crystal cells and lamellocytes--the functions of which are reminiscent of mammalian myeloid cells. During the first and early second instars of larval development, the lymph gland contains only progenitors, whereas in the third instar, a medial region of the primary lobe of the lymph gland called the medullary zone contains these progenitors, and maturing blood cells are found juxtaposed in a peripheral region designated the cortical zone. A third group of cells referred to as the posterior signalling centre functions as a haematopoietic niche. Similarly to mammalian myeloid cells, Drosophila blood cells respond to multiple stresses including hypoxia, infection and oxidative stress. However, how systemic signals are sensed by myeloid progenitors to regulate cell-fate determination has not been well described. Here, we show that the haematopoietic progenitors of Drosophila are direct targets of systemic (insulin) and nutritional (essential amino acid) signals, and that these systemic signals maintain the progenitors by promoting Wingless (WNT in mammals) signalling. We expect that this study will promote investigation of such possible direct signal sensing mechanisms by mammalian myeloid progenitors.

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance.

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.

Interaction between Notch and Hif-alpha in development and survival of Drosophila blood cells.

A blood cell type termed crystal cell in Drosophila functions in clotting and wound healing and requires Notch for specification and maintenance. We report that crystal cells express elevated levels of Sima protein orthologous to mammalian hypoxia-inducible factor-α (Hif-α) even under conditions of normal oxygen availability. In these platelet-like crystal cells, Sima activates full-length Notch receptor signaling via a noncanonical, ligand-independent mechanism that promotes hemocyte survival during both normal hematopoietic development and hypoxic stress. This interaction initiates in early endosomes, is independent of Hif-ß (Τangο in Drosophila), and does not activate hypoxia response targets. Studies in vertebrate myeloid cells have shown a similar up-regulation of Hif-α protein in well-oxygenated environments. This study provides a mechanistic paradigm for Hif-α/Notch interaction that may be conserved in mammals.

Identification of Drosophila genes modulating Janus kinase/signal transducer and activator of transcription signal transduction.

The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. We have used a dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed us to assemble a collection of genes whose products represent novel components and regulators of this important signal transduction cascade.

Opposing roles for Drosophila JAK/STAT signalling during cellular proliferation.

The JAK/STAT signalling pathway mediates both antiproliferative responses following interferon stimulation and cellular proliferation in response to cytokines such as interleukins and growth factors. Central to these responses are the seven vertebrate STAT molecules, misregulation of which is implicated in a variety of malignancies. We have investigated the proliferative role of the single Drosophila STAT92E, part of the evolutionarily conserved JAK/STAT cascade. During second instar larval wing disc development pathway activity is both necessary and sufficient to promote proliferation of this epithelial cell type. However by later stages, endogenous STAT92E is stimulated by a noncannonical mechanism to exert pronounced antiproliferative effects. Ectopic canonical activation is sufficient to further decrease proliferation and leads to the premature arrest of cells in the G2 phase of the cell cycle. The single STAT92E present in Drosophila therefore mediates both proproliferative functions analogous to vertebrate interleukin-stimulated STAT3 and antiproliferative functions analogous to interferon-stimulated STAT1. Pro- and antiproliferative roles therefore represent ancestral activities conserved through evolution and subsequently assigned to distinct molecules.

Ets-1 and VEGF expression correlates with tumor angiogenesis, lymph node metastasis, and patient survival in esophageal squamous cell carcinoma.

PURPOSE: Esophageal squamous cell carcinoma (ESCC) in the Indian population exhibits insidious symptomatology, late clinical presentation, aggressive behavior, and high propensity for metastasis. Ets-1, a transcription factor, is expressed in esophageal tumors and associated with poor prognosis. The aim of the present study was to determine the relationship between Ets-1 expression, tumor angiogenesis [vascular endothelial growth factor (VEGF) and microvessel density (MVD)] and the biological behavior of ESCCs. METHODS: In a prospective study the expression of Ets-1, VEGF, and PECAM-1 (CD-31) was determined in 55 ESCCs, by immunohistochemical analysis, correlated with clinicopathological parameters and outcome of the patients. RESULTS: Overexpression of Ets-1 and VEGF proteins was observed in 44/55 (80%) and 38/55 (69%) of ESCCs, respectively. VEGF immunopositivity was associated with lymph node metastasis ( P=0.002). Analysis of mRNA isoforms using RT-PCR revealed increased expression of VEGF 121 transcripts in ESCCs and MVD was correlated with de-differentiation status of the tumors ( P=0.049). Kaplan-Meier survival analysis showed significant correlation between poor disease-free survival and tumor stage ( P=0.02) and with nodal metastasis ( P=0.05). Concomitant expression of VEGF, Ets-1 proteins, and high MVD was correlated with poor disease-free survival ( P=0.004). CONCLUSION: Significant association of Ets-1 and VEGF proteins with tumor angiogenesis (MVD), lymph node invasion, and poor disease-free survival underscores their relevance regarding aggressive tumor behavior and highlights their potential utility as adverse prognostic factors in esophageal carcinomas.