Kicking it up a Notch for the best in show: Scalloped leads Yorkie into the haematopoietic arena

Maintenance and differentiation of progenitor cells is essential for proper organ development and adaptation to environmental stress and injury. In Drosophila melanogaster, the haematopietic system serves as an ideal model for interrogating the function of signaling pathways required for progenitor maintenance and cell fate determination. Here we focus on the role of the Hippo pathway effectors Yorkie and Scalloped in mediating and facilitating Notch signaling-mediated lineage specification in the lymph gland, the primary center for haematopoiesis within the developing larva. We discuss the regulatory mechanisms which promote Notch activity during normal haematopoiesis and its modulation during immune challenge conditions. We provide additional evidence establishing the hierarchy of signaling events during crystal cell formation, highlighting the relationship between Yorkie, Scalloped and Lozenge, while expanding on the role of Yorkie in promoting hemocyte survival and the developmental regulation of Notch and its ligand, Serrate, within the lymph gland. Finally, we propose additional areas of exploration that may provide mechanistic insight into the environmental and non-cell autonomous regulation of cell fate in the blood system.     

Ngn3(+) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3^−/− mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.     

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila

The interconnected Insulin/IGF signaling (IlS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. Here, we addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. We show that, reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, we demonstrate that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, we show that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, our study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.     

Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate

Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination.     

Competition in Notch Signaling with Cis Enriches Cell Fate Decisions

Notch signaling is involved in cell fate choices during the embryonic development of Metazoa. Commonly, Notch signaling arises from the binding of the Notch receptor to its ligands in adjacent cells driving cell-to-cell communication. Yet, cell-autonomous control of Notch signaling through both ligand-dependent and ligand-independent mechanisms is known to occur as well. Examples include Notch signaling arising in the absence of ligand binding, and cis-inhibition of Notch signaling by titration of the Notch receptor upon binding to its ligands within a single cell. Increasing experimental evidences support that the binding of the Notch receptor with its ligands within a cell (cis-interactions) can also trigger a cell-autonomous Notch signal (cis-signaling), whose potential effects on cell fate decisions and patterning remain poorly understood. To address this question, herein we mathematically and computationally investigate the cell states arising from the combination of cis-signaling with additional Notch signaling sources, which are either cell-autonomous or involve cell-to-cell communication. Our study shows that cis-signaling can switch from driving cis-activation to effectively perform cis-inhibition and identifies under which conditions this switch occurs. This switch relies on the competition between Notch signaling sources, which share the same receptor but differ in their signaling efficiency. We propose that the role of cis-interactions and their signaling on fine-grained patterning and cell fate decisions is dependent on whether they drive cis-inhibition or cis-activation, which could be controlled during development. Specifically, cis-inhibition and not cis-activation facilitates patterning and enriches it by modulating the ratio of cells in the high-ligand expression state, by enabling additional periodic patterns like stripes and by allowing localized patterning highly sensitive to the precursor state and cell-autonomous bistability. Our study exemplifies the complexity of regulations when multiple signaling sources share the same receptor and provides the tools for their characterization.     

Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.     

Interaction of Notch and gp130 Signaling in the Maintenance of Neural Stem and Progenitor Cells

Notch and gp130 signaling are involved in the regulation of multiple cellular processes across various tissues during animal ontogenesis. In the developing nervous system, both signaling pathways intervene at many stages to determine cell fate—from the first neural lineage commitment and generation of neuronal precursors, to the terminal specification of cells as neurons and glia. In most cases, the effects of Notch and gp130 signaling in these processes are similar. The aim of the current review was to summarize the knowledge regarding the roles of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells during animal ontogenesis, from early embryo to adult. Recent data show a direct crosstalk between these signaling pathways that seems to be specific for a particular type of neural progenitors.     

Pleiotropic roles of Notch signaling in normal,malignant, and developmental hematopoiesis in the human

The Notch signaling pathway is evolutionarily conserved across species and plays an important role in regulating cell differentiation, proliferation, and survival. It has been implicated in several different hematopoietic processes including early hematopoietic development as well as adult hematological malignancies in humans. This review focuses on recent developments in understanding the role of Notch signaling in the human hematopoietic system with an emphasis on hematopoietic initiation from human pluripotent stem cells and regulation within the bone marrow. Based on recent insights, we summarize potential strategies for treatment of human hematological malignancies toward the concept of targeting Notch signaling for fate regulation.     

Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5⁺ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5⁺ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.     

Notch signaling coordinates the patterning of striatal compartments

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.     

Numb Independently Antagonizes Sanpodo Membrane Targeting and Notch Signaling in Drosophila Sensory Organ Precursor Cells

In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure–function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.