Kalpana Makhijani

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future.

Arginine Methylation Initiates BMP-Induced Smad Signaling

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Of blood cells and the nervous system: Hematopoiesis in the Drosophila larva

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.

Rationally designed fluorogenic protease reporter visualizes spatiotemporal dynamics of apoptosis in vivo

Significance By harnessing the unique interactions between infrared fluorescent protein and its chromophore, we have designed an infrared fluorogenic protease reporter (iProtease). A fluorogenic protease reporter is ideal for imaging protease activity in vivo, whereas a FRET-based reporter is limited by poor signal and requirement of image processing. The iProtease scaffold may be used as a core module to design reporters of various proteases with specific activity. This technology will aide important applications, including monitoring protease activity in vivo, dissecting signaling pathways that regulate protease activity, and high-throughput screening of protease inhibitors for drug development and biological study. Our work shows that phytochrome-derived infrared fluorescent protein is a promising scaffold in engineering fluorogenic reporters for visualizing spatiotemporal dynamics of cell signaling in vivo. Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.

Drosophila twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of ∼36 kDa catalytic and ∼65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of PP2A role in Wg/Wnt pathway in different organisms are conflicting. We show that twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is a positive regulator of Wg/Wnt signaling. In tws- wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws- mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. Our results suggest that Tws is required for the stabilization of Armadillo/β-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits.

Rational design of a monomeric and photostable far-red fluorescent protein for fluorescence imaging in vivo.

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice.

A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. Here we report that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined.