Charting ESCRT function reveals distinct and non-compensatory roles in blood progenitor maintenance and lineage choice in Drosophila

Tissue heterogeneity permits diverse biological outputs in response to systemic signals but requires context-dependent spatiotemporal regulation of a limited number of signaling circuits. In addition to their stereotypical roles of transport and cargo sorting, endocytic networks provide rapid, adaptable, and often reversible means of signaling. Aberrant function of the Endosomal Sorting Complex Required for Transport (ESCRT) components results in ubiquitinated cargo accumulation, uncontrolled signaling and neoplastic transformation. However, context-specific effects of ESCRT on developmental decisions are not resolved. By a comprehensive spatiotemporal profiling of ESCRT in Drosophila hematopoiesis in vivo, here we show that pleiotropic ESCRT components have distinct effects on blood progenitor maintenance, lineage choice and response to immune challenge. Of all 13 core ESCRT components tested, only Vps28 and Vp36 were required in all progenitors, whereas others maintained spatiotemporally defined progenitor subsets. ESCRT depletion also sensitized posterior progenitors that normally resist differentiation, to respond to immunogenic cues. Depletion of the critical Notch signaling regulator Vps25 did not promote progenitor differentiation at steady state but made younger progenitors highly sensitive to wasp infestation, resulting in robust lamellocyte differentiation. We identify key heterotypic roles for ESCRT in controlling Notch activation and thereby progenitor proliferation and differentiation. Further, we show that ESCRT ability to regulate Notch activation depends on progenitor age and position along the anterior-posterior axis. The phenotypic range and disparity in signaling upon depletion of components provides insight into how ESCRT may tailor developmental diversity. These mechanisms for subtle control of cell phenotype may be applicable in multiple contexts.

LSD1 inhibition sustains T cell invigoration with a durable response to PD-1 blockade

Exhausted CD8+ T cells are key targets of immune checkpoint blockade therapy and their ineffective reinvigoration limits the durable benefit in some cancer patients. Here, we demonstrate that histone demethylase LSD1 acts to enforce an epigenetic program in progenitor exhausted CD8+ T cells to antagonize the TCF1-mediated progenitor maintenance and to promote terminal differentiation. Consequently, genetic perturbation or small molecules targeting LSD1 increases the persistence of the progenitor exhausted CD8+ T cells, which provide a sustained source for the proliferative conversion to numerically larger terminally exhausted T cells with tumor-killing cytotoxicity, thereby leading to effective and durable responses to anti-PD1 therapy. Collectively, our findings provide important insights into epigenetic mechanisms that regulate T cell exhaustion and have important implications for durable immunotherapy.

Omics profiling identifies MAPK/ERK pathway as a gatekeeper of nephron progenitor metabolism

Nephron endowment is defined by fetal kidney growth and it critically dictates renal health in adults. Despite the advances in understanding the molecular regulation of nephron progenitor maintenance, propagation, and differentiation, the causes for low congenital nephron count and contribution of basic metabolism to nephron progenitor regulation remain poorly studied. Here we have analyzed the metabolic effects that depend on and are triggered by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, which is an essential intracellular cascade required for nephron progenitor maintenance. Our combined approach utilizing LC/MS-based metabolomics and transcriptional profiling of MAPK/ERK-deficient cells identified 18 out of total 46 metabolites (38 untargeted and 8 targeted) that were down-regulated. These represent glycolysis, gluconeogenesis, pentose phosphate, glycine, and proline pathways among others. We focused our functional characterization of identified metabolites on pyruvate and proline. Use of in vitro kidney cultures revealed dosage-specific functions for pyruvate in not only controlling ureteric bud branching but also determining progenitor and differentiated (tip-trunk) cell identities. Our in vivo characterization of Pycr1/2 double knockout kidneys revealed functional requirement for proline metabolism in nephron progenitor maintenance. In summary, our results demonstrate that MAPK/ERK cascade regulates energy and amino acid metabolism in developing kidney where these metabolic pathways specifically regulate progenitor preservation.

Alginate-Chitosan Microencapsulated Cells for Improving CD34+ Progenitor Maintenance and Expansion

Protocols for isolation, characterization, and transplantation of hematopoietic stem cells (HSCs) have been well established. However, difficulty in finding human leucocyte antigens (HLA)-matched donors and scarcity of HSCs are still the major obstacles of allogeneic transplantation. In this study, we developed a double-layered microcapsule to deliver paracrine factors from non-matched or low-matched HSCs to other cells. The umbilical cord blood-derived hematopoietic progenitor cells, identified as CD34+ cells, were entrapped in alginate polymer and further protected by chitosan coating. The microcapsules showed no toxicity for surrounding CD34+ cells. When CD34+ cells-loaded microcapsules were co-cultured with bare CD34+ cells that have been collected from unrelated donors, the microcapsules affected surrounding cells and increased the percentage of CD34+ cell population. This study is the first to report the potency of alginate-chitosan microcapsules containing non-HLA-matched cells for improving proliferation and progenitor maintenance of CD34+ cells.

Interplay Between Notch and YAP/TAZ Pathways in the Regulation of Cell Fate During Embryo Development

Cells in growing tissues receive both biochemical and physical cues from their microenvironment. Growing evidence has shown that mechanical signals are fundamental regulators of cell behavior. However, how physical properties of the microenvironment are transduced into critical cell behaviors, such as proliferation, progenitor maintenance, or differentiation during development, is still poorly understood. The transcriptional co-activators YAP/TAZ shuttle between the cytoplasm and the nucleus in response to multiple inputs and have emerged as important regulators of tissue growth and regeneration. YAP/TAZ sense and transduce physical cues, such as those from the extracellular matrix or the actomyosin cytoskeleton, to regulate gene expression, thus allowing them to function as gatekeepers of progenitor behavior in several developmental contexts. The Notch pathway is a key signaling pathway that controls binary cell fate decisions through cell–cell communication in a context-dependent manner. Recent reports now suggest that the crosstalk between these two pathways is critical for maintaining the balance between progenitor maintenance and cell differentiation in different tissues. How this crosstalk integrates with morphogenesis and changes in tissue architecture during development is still an open question. Here, we discuss how progenitor cell proliferation, specification, and differentiation are coordinated with morphogenesis to construct a functional organ. We will pay special attention to the interplay between YAP/TAZ and Notch signaling pathways in determining cell fate decisions and discuss whether this represents a general mechanism of regulating cell fate during development. We will focus on research carried out in vertebrate embryos that demonstrate the important roles of mechanical cues in stem cell biology and discuss future challenges.

Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program towards precocious differentiation, thereby bolstering the cellular arm of the immune response.

A Conserved Role for Asrij/OCIAD1 in Progenitor Differentiation and Lineage Specification Through Functional Interaction With the Regulators of Mitochondrial Dynamics

Mitochondria are highly dynamic organelles whose activity is an important determinant of blood stem and progenitor cell state. Mitochondrial morphology is maintained by continuous fission and fusion and affects stem cell proliferation, differentiation, and aging. However, the mechanism by which mitochondrial morphology and dynamics regulate cell differentiation and lineage choice remains incompletely understood. Asrij/OCIAD1 is a conserved protein that governs mitochondrial morphology, energy metabolism and human embryonic stem cell (hESC) differentiation. To investigate the in vivo relevance of these properties, we compared hESC phenotypes with those of Drosophila hematopoiesis, where Asrij is shown to regulate blood progenitor maintenance by conserved mechanisms. In concordance with hESC studies, we found that Drosophila Asrij also localizes to mitochondria of larval blood cells and its depletion from progenitors results in elongated mitochondria. Live imaging of asrij knockdown hemocytes and of OCIAD1 knockout hESCs showed reduced mitochondrial dynamics. Since key regulators of mitochondrial dynamics actively regulate mitochondrial morphology, we hypothesized that mitochondrial fission and fusion may control progenitor maintenance or differentiation in an Asrij-dependent manner. Knockdown of the fission regulator Drp1 in Drosophila lymph gland progenitors specifically suppressed crystal cell differentiation whereas depletion of the fusion regulator Marf (Drosophila Mitofusin) increased the same with concomitant upregulation of Notch signaling. These phenotypes were stronger in anterior progenitors and were exacerbated by Asrij depletion. Asrij is known to suppress Notch signaling and crystal cell differentiation. Our analysis reveals that synergistic interactions of Asrij with Drp1 and Marf have distinct impacts on lymph gland progenitor mitochondrial dynamics and crystal cell differentiation. Taken together, using invertebrate and mammalian model systems we demonstrate a conserved role for Asrij/OCIAD1 in linking mitochondrial dynamics and progenitor differentiation. Our study sets the stage for deciphering how regulators of mitochondrial dynamics may contribute to functional heterogeneity and lineage choice in vertebrate blood progenitors.

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF

ABSTRACT Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.

Relish plays a dynamic role in the niche to modulate Drosophila blood progenitor homeostasis in development and infection.

Immune challenges demand the gearing up of basal hematopoiesis to combat infection. Little is known about how during development, this switch is achieved to take care of the insult. Here, we show that the hematopoietic niche of the larval lymph gland of Drosophila senses immune challenge and reacts to it quickly through the nuclear factor-κB (NF-κB), Relish, a component of the immune deficiency (Imd) pathway. During development, Relish is triggered by ecdysone signaling in the hematopoietic niche to maintain the blood progenitors. Loss of Relish causes an alteration in the cytoskeletal architecture of the niche cells in a Jun Kinase dependent manner, resulting in the trapping of Hh implicated in progenitor maintenance. Notably, during infection, downregulation of Relish in the niche tilts the maintenance program towards precocious differentiation, thereby bolstering the cellular arm of the immune response.