Digital and Analog STAT5 Signaling in Erythropoiesis: A Dissertation

Erythropoietin (Epo) modulates red blood cell production (erythropoiesis) by binding to its receptor and activating STAT5, a Signal Transducer and Activator of Transcription (STAT) protein implicated in both basal and stress erythropoiesis. Epo concentration in serum changes over three orders of magnitude, as it regulates basal erythropoiesis and its acceleration during hypoxic stress. However, it is not known how STAT5 translates the changes in Epo concentration into the required erythropoietic rates. We addressed this question by studying STAT5 phosphorylation, at the single cell level, in developing erythroblasts. We divided erythroid progenitors in tissue into several flow-cytometric subsets and found that each of them exhibited distinct modes of Stat5 activation, based on their developmental stage. STAT5 activation is bistable in mature erythroblasts, resulting in a binary (or digital), low-intensity STAT5 phosphorylation signal (p-Stat5). In early erythroblasts, and in response to stress levels of Epo, the low intensity bistable p-Stat5 signal is superseded by a high-intensity graded, or analog, signal. The gradual shift from high-intensity graded signaling in early erythroblasts to low intensity binary signaling in mature erythroblasts is due to a decline in STAT5 expression with maturation. We were able to convert mature, digital transducing erythroblasts into analog transducers simply by expressing high levels of exogenous STAT5. We found that EpoR-HM mice, expressing a mutant EpoR that lacks STAT5 docking sites, generate the binary, but not the analog, STAT5 signal. Unlike Stat5-null mice, which die perinatally, the EpoR-HM mice are viable but deficient in their response to stress, demonstrating that while binary STAT5 signaling is sufficient to support basal erythropoiesis, analog signaling is required for the stress response. Bistable systems contain a positive loop, which is important for flipping the switch between the two stable ‘on’ or ‘off’ states. We show that bistable activation is the result of an autocatalytic loop in which active STAT5 promotes further STAT5 activation. The isolated STAT5 N-terminal domain, which is not required for STAT5 phosphorylation, enhanced autocatalysis, converting a high intensity graded signal into a high intensity binary response. The N-terminal domain is known to participate in a radical conformational reorientation of STAT5 dimers inherent in STAT5 activation. We propose that the N-terminal domains of active STAT5 dimers facilitate the conformational reorientation of inactive dimers, in a prion-like autocatalytic interaction that underlies bistability and binary signaling. Together, bistable STAT5 activation, combined with a graded response allow erythropoietic rate to faithfully reflect a wide Epo concentration range, while preventing aberrant signaling

Negative Autoregulation by FAS Mediates Robust Fetal Erythropoiesis

Tissue development is regulated by signaling networks that control developmental rate and determine ultimate tissue mass. Here we present a novel computational algorithm used to identify regulatory feedback and feedforward interactions between progenitors in developing erythroid tissue. The algorithm makes use of dynamic measurements of red cell progenitors between embryonic days 12 and 15 in the mouse. It selects for intercellular interactions that reproduce the erythroid developmental process and endow it with robustness to external perturbations. This analysis predicts that negative autoregulatory interactions arise between early erythroblasts of similar maturation stage. By studying embryos mutant for the death receptor FAS, or for its ligand, FASL, and by measuring the rate of FAS-mediated apoptosis in vivo, we show that FAS and FASL are pivotal negative regulators of fetal erythropoiesis, in the manner predicted by the computational model. We suggest that apoptosis in erythroid development mediates robust homeostasis regulating the number of red blood cells reaching maturity

Hormonal regulation of the humoral innate immune response in Drosophila melanogaster

Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2* cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2* cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno- suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway resets, allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using knock-in mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a stop-gap in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms

Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and graded modalities combine to generate high-fidelity Stat5 signaling over the entire basal and stress Epo range. They suggest that dynamic behavior may encode information during STAT signal transduction

Hormonal regulation of the humoral innate immune response in Drosophila melanogaster

Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2 cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2 cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno-suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment

Measurement of binary and graded signaling responses by flow cytometry.

<p>(A) Dose/response curves for graded and binary (switch-like) responses. The grey line shows an idealized switch response. An infinitesimally small change in stimulus such as Epo results in an increase of response from 0% to 100%. The black curve shows a graded response with n_H = 1. An 81-fold increase in stimulus is required to shift the response from 10% to 90% of maximum. The green curve shows a steep, switch-like or binary dose/response curve (n_H = 3). Only a 4-fold change in stimulus is required in order to generate a similar increase in response. (B) Contrasting graded and binary signaling responses: three hypothetical examples. In a graded signaling response (left panels), increasing Epo concentration results in a graded increase in p-Stat5 in individual cells, represented by increasingly darker shades of grey. Simulations of the corresponding flow-cytometric profiles show that increasing Epo concentration causes a gradual shift of the p-Stat5 fluorescence histogram to the right. A plot of the total p-Stat5 median fluorescence intensity (MFI) versus Epo concentration has Michaelian kinetics with a Hill coefficient (n_H) of 1 (lower left panel, please note a log scale was used for the <i>x</i>-axis). In binary signaling (middle and right panels), the p-Stat5 signal in individual cells can only assume two states, either “off” (white) or “on” (black), but intermediate states (shades of grey) are unstable. Two distinct cases of binary signaling are illustrated that differ in their threshold responses. In “variable threshold” (middle panels), the threshold at which Epo causes a cell to switch from “off” to “on” varies substantially between cells of the population. Consequently, increasing Epo concentration causes a gradual increase in the number of cells that are p-Stat5⁺ (“on”). The simulated flow-cytometric histograms at each Epo concentration (in color) are each the sum of two histograms, corresponding to cells that are “off” (light grey histograms) and cells that are “on” (black histograms). The median fluorescence of the “on” and “off” histograms each remain unaffected by Epo concentration, but as Epo increases, the number of cells in the “on” histogram, reflected by its height, increases, with a corresponding decrease in the height of the “off” histogram. Although individual cells have binary responses, there is a graded increase in the MFI of the colored histograms representing the whole population. Therefore, a plot of total p-Stat5 MFI versus Epo concentration shows a graded response (here, the Hill coefficient is n_H = 1). In the case of cells with binary responses and similar threshold (right panels), cells switch from “off” to “on” within a much narrower Epo concentration range. Consequently, the response of the whole population reflects the response of individual cells more closely. Flow-cytometric histograms representing the population tend to be in one of two principal positions, corresponding to the “on” or to the “off” states. A plot of total p-Stat5 MFI versus Epo concentration is steep, reflected by a high Hill coefficient (n_H>1).</p