The glucocorticoid receptor elicited proliferative response in human erythropoiesis is BCL11A-dependent.

Prior evidence indicates that the erythroid cellular response to glucocorticoids (GC) has developmental specificity, namely, that developmentally more advanced cells that are undergoing or have undergone fetal to adult globin switching are more responsive to GC-induced expansion. To investigate the molecular underpinnings of this, we focused on the major developmental globin regulator BCL11A. We compared: a) levels of expression and nuclear content of BCL11A in adult erythroid cells upon GC stimulation; b) response to GC of CD34+ cells from patients with BCL11A microdeletions and reduced BCL11A expression, and; c) response to GC of two cellular models (HUDEP-2 and adult CD34+ cells) before and after reduction of BCL11A expression by shRNA. We observed that: a) GC-expanded erythroid cells from a large cohort of blood donors displayed amplified expression and nuclear accumulation of BCL11A; b) CD34+ cells from BCL11A microdeletion patients generated fewer erythroid cells when cultured with GC compared to their parents, while the erythroid expansion of the patients was similar to that of their parents in cultures without GC, and; c) adult CD34+ cells and HUDEP-2 cells with shRNA-depleted expression of BCL11A exhibit reduced expansion in response to GC. In addition, RNA-seq profiling of shRNA-BCL11A CD34+ cells cultured with and without GC was similar (very few differentially expressed genes), while GC-specific responses (differential expression of GILZ and of numerous additional genes) were observed only in controls cells with unperturbed BCL11A expression. These data indicate that BCL11A is an important participant of certain aspects of the stress pathway sustained by GC.

Statistics of maximum photon penetration depth in a two-layer diffusive medium.

We present numerical results for the probability density function f(z) and for the mean value of photon maximum penetration depth zmax> in a two-layer diffusive medium. Both time domain and continuous wave regime are considered with several combinations of the optical properties (absorption coefficient, reduced scattering coefficient) of the two layers, and with different geometrical configurations (source detector distance, thickness of the upper layer). Practical considerations on the design of time domain and continuous wave systems are derived. The methods and the results are of interest for many research fields such as biomedical optics and advanced microscopy.

Application limits of the scaling relations for Monte Carlo simulations in diffuse optics. Part 1: theory.

Monte Carlo (MC) is a powerful tool to study photon migration in scattering media, yet quite time-consuming to solve inverse problems. To speed up MC-simulations, scaling relations can be applied to an existing initial MC-simulation to generate a new data-set with different optical properties. We named this approach trajectory-based since it uses the knowledge of the detected photon trajectories of the initial MC-simulation, in opposition to the slower photon-based approach, where a novel MC-simulation is rerun with new optical properties. We investigated the convergence and applicability limits of the scaling relations, both related to the likelihood that the sample of trajectories considered is representative also for the new optical properties. For absorption, the scaling relation contains smoothly converging Lambert-Beer factors, whereas for scattering it is the product of two quickly diverging factors, whose ratio, for NIRS cases, can easily reach ten orders of magnitude. We investigated such instability by studying the probability-distribution for the number of scattering events in trajectories of given length. We propose a convergence test of the scattering scaling relation based on the minimum-maximum number of scattering events in recorded trajectories. We also studied the dependence of MC-simulations on optical properties, most critical in inverse problems, finding that scattering derivatives are ascribed to small deviations in the distribution of scattering events from a Poisson distribution. This paper, which can also serve as a tutorial, helps to understand the physics of the scaling relations with the causes of their limitations and devise new strategies to deal with them.

Two-layer reconstruction of Raman spectra in diffusive media based on an analytical model in the time domain.

We derive and validate an analytical model that describes the migration of Raman scattered photons in two-layer diffusive media, based on the diffusion equation in the time domain. The model is derived under a heuristic approximation that background optical properties are identical on the excitation and Raman emission wavelengths. Methods for the reconstruction of two-layer Raman spectra have been developed, tested in computer simulations and validated on tissue-mimicking phantom measurements data. Effects of different parameters were studied in simulations, showing that the thickness of the top layer and number of detected photon counts have the most significant impact on the reconstruction. The concept of quantitative, mathematically rigorous reconstruction using the proposed model was finally proven on experimental measurements, by successfully separating the spectra of silicone and calcium carbonate (calcite) layers, showing the potential for further development and eventual application in clinical diagnostics.

GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder with limited therapeutic options. Insufficient understanding of driver mutations and poor fidelity of currently available animal models has limited the development of effective therapies. Since GATA1 deficient megakaryocytes sustain myelofibrosis, we hypothesized that they may also induce fibrosis in lungs. We discovered that lungs from IPF patients and Gata1low mice contain numerous GATA1negative immune-poised megakaryocytes that, in mice, have defective RNA-seq profiling and increased TGF-ß1, CXCL1 and P-selectin content. With age, Gata1low mice develop fibrosis in lungs. Development of lung fibrosis in this model is prevented by P-selectin deletion and rescued by P-selectin, TGF-ß1 or CXCL1 inhibition. Mechanistically, P-selectin inhibition decreases TGF-ß1 and CXCL1 content and increases GATA1positive megakaryocytes while TGF-ß1 or CXCL1 inhibition decreased CXCL1 only. In conclusion, Gata1low mice are a novel genetic-driven model for IPF and provide a link between abnormal immune-megakaryocytes and lung fibrosis.

Single cell analysis of the localization of the hematopoietic stem cells within the bone marrow architecture identifies niche-specific proliferation dynamics.

Introduction: Hematopoietic stem cells (HSC) reside in the bone marrow (BM) in specialized niches which provide support for their self-replication and differentiation into the blood cells. Recently, numerous studies using sophisticated molecular and microscopic technology have provided snap-shots information on the identity of the BM niches in mice. In adults, HSC are localized around arterioles and sinusoids/venules whereas in juvenile mice they are in close to the osteoblasts. However, although it is well recognized that in mice the nature of the hematopoietic niche change with age or after exposure to inflammatory insults, much work remains to be done to identify changes occurring under these conditions. The dynamic changes occurring in niche/HSC interactions as HSC enter into cycle are also poorly defined. Methods: We exploit mice harboring the hCD34tTA/Tet-O-H2BGFP transgene to establish the feasibility to assess interactions of the HSC with their niche as they cycle. In this model, H2BGFP expression is driven by the TET trans-activator under the control of the human CD34 promoter which in mice is active only in the HSC. Since Doxycycline inhibits TET, HSC exposed to this drug no longer express H2BGFP and loose half of their label every division allowing establishing the dynamics of their first 1-3 divisions. To this aim, we first validated user-friendly confocal microscopy methods to determine HSC divisions by hemi-decrement changes in levels of GFP expression. We then tracked the interaction occurring in old mice between the HSC and their niche during the first HSC divisions. Results: We determined that in old mice, most of the HSC are located around vessels, both arterioles which sustain quiescence and self-replication, and venules/sinusoids, which sustain differentiation. After just 1 week of exposure to Doxycycline, great numbers of the HSC around the venules lost most of their GFP label, indicating that they had cycled. By contrast, the few HSC surrounding the arterioles retained maximal levels of GFP expression, indicating that they are either dormant or cycle at very low rates. Conclusion: These results reveal that in old mice, HSC cycle very dynamically and are biased toward interactions with the niche that instructs them to differentiate.

Inhibition of CXCR1/2 reduces the emperipolesis between neutrophils and megakaryocytes in the Gata1low model of myelofibrosis.

Emperipolesis between neutrophils and megakaryocytes was first identified by transmission electron microscopy. Although rare under steady-state conditions, its frequency greatly increases in myelofibrosis, the most severe of myeloproliferative neoplasms, in which it is believed to contribute to increasing the transforming growth factor (TGF)-ß microenvironmental bioavailability responsible for fibrosis. To date, the challenge of performing studies by transmission electron microscopy has hampered the study of factors that drive the pathological emperipolesis observed in myelofibrosis. We established a user-friendly confocal microscopy method that detects emperipolesis by staining with CD42b, specifically expressed on megakaryocytes, coupled with antibodies that recognize the neutrophils (Ly6b or neutrophil elastase antibody). With such an approach, we first confirmed that the bone marrow from patients with myelofibrosis and from Gata1low mice, a model of myelofibrosis, contains great numbers of neutrophils and megakaryocytes in emperipolesis. Both in patients and Gata1low mice, the emperipolesed megakaryocytes were surrounded by high numbers of neutrophils, suggesting that neutrophil chemotaxis precedes the actual emperipolesis event. Because neutrophil chemotaxis is driven by CXCL1, the murine equivalent of human interleukin 8 that is expressed at high levels by malignant megakaryocytes, we tested the hypothesis that neutrophil/megakaryocyte emperipolesis could be reduced by reparixin, an inhibitor of CXCR1/CXCR2. Indeed, the treatment greatly reduced both neutrophil chemotaxis and their emperipolesis with the megakaryocytes in treated mice. Because treatment with reparixin was previously reported to reduce both TGF-ß content and marrow fibrosis, these results identify neutrophil/megakaryocyte emperipolesis as the cellular interaction that links interleukin 8 to TGF-ß abnormalities in the pathobiology of marrow fibrosis.

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Fluence rate directly derived from photon pathlengths: a tool for Monte Carlo simulations in biomedical optics.

In biomedical optics, the mean fluence rate of photons, assessed in a sub-volume of a propagating medium, is classically obtained in Monte Carlo simulations by taking into account the power deposited by the absorbed photons in the sub-volume. In the present contribution, we propose and analytically demonstrate an alternative method based on the assessment of the mean pathlength traveled by all the photons inside the sub-volume. Few practical examples of its applications are given. This method has the advantage of improving, in many cases, the statistics and the convergence of the Monte Carlo simulations. Further, it also works when the absorption coefficient is nil and for a non-constant spatial distribution of the absorption coefficient inside the sub-volume. The proposed approach is a re-visitation of a well-known method applied in radiation and nuclear physics in the context of radiative transfer, where it can be derived in a more natural manner.

Patients with hypercortisolemic Cushing disease possess a distinct class of hematopoietic progenitor cells leading to erythrocytosis.

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Preclinical studies on the use of a P-selectin-blocking monoclonal antibody to halt progression of myelofibrosis in the Gata1low mouse model.

The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-ß (TGF-ß) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-ß and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients.

On the mean path length invariance property for random walks of animals in open environment.

Random walks are common in nature and are at the basis of many different phenomena that span from neutrons and light scattering to the behaviour of animals. Despite the evident differences among all these phenomena, theory predicts that they all share a common fascinating feature known as Invariance Property (IP). In a nutshell, IP means that the mean length of the total path of a random walker inside a closed domain is fixed by the geometry and size of the medium. Such a property has been demonstrated to hold not only in optics, but recently also in the field of biology, by studying the movement of bacteria. However, the range of validity of such a universal property, strictly linked to the fulfilment of equilibrium conditions and to the statistical distributions of the steps of the random walkers, is not trivial and needs to be studied in different contexts, such as in the case of biological entities occupied in random foraging in an open environment. Hence, in this paper the IP in a virtual medium inside an open environment has been studied by using actual movements of animals recorded in nature. In particular, we analysed the behaviour of a grazer mollusc, the chiton Acanthopleura granulata. The results depart from those predicted by the IP when the dimension of the medium increases. Such findings are framed in both the condition of nonequilibrium of the walkers, which is typical of animals in nature, and the characteristics of actual animal movements.

Monte Carlo simulations in anomalous radiative transfer: tutorial.

Anomalous radiative transfer (ART) theory represents a generalization of classical radiative transfer theory. The present tutorial aims to show how Monte Carlo (MC) codes describing the transport of photons in anomalous media can be implemented. We show that the heart of the method involves suitably describing, in a "non-classical" manner, photon steps starting from fixed light sources or from boundaries separating regions of the medium with different optical properties. To give a better sense of the importance of these particular photon step lengths, we also show numerically that the described approach is essential in preserving the invariance property for light propagation. An interesting byproduct of the MC method for ART is that it allows us to simplify the structure of "classical" MC codes, utilized, for example, in biomedical optics.

Special Section Guest Editorial: Introduction to the Special Section Celebrating 30 years of Open Source Monte Carlo Codes in Biomedical Optics.

The editorial introduces the JBO Special Section Celebrating 30 Years of Open Source Monte Carlo Codes in Biomedical Optics for Volume 27, Issue 8.

The CXCR1/CXCR2 Inhibitor Reparixin Alters the Development of Myelofibrosis in the Gata1 low Mice.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-ß, the megakaryocytes from the bone marrow of the Gata1 low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1 low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-ß1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1 low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-ß1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

Two-step verification method for Monte Carlo codes in biomedical optics applications.

SIGNIFICANCE: Code verification is an unavoidable step prior to using a Monte Carlo (MC) code. Indeed, in biomedical optics, a widespread verification procedure for MC codes is still missing. Analytical benchmarks that can be easily used for the verification of different MC routines offer an important resource. AIM: We aim to provide a two-step verification procedure for MC codes enabling the two main tasks of an MC simulator: (1) the generation of photons' trajectories and (2) the intersections of trajectories with boundaries separating the regions with different optical properties. The proposed method is purely based on elementary analytical benchmarks, therefore, the correctness of an MC code can be assessed with a one-sample t-test. APPROACH: The two-step verification is based on the following two analytical benchmarks: (1) the exact analytical formulas for the statistical moments of the spatial coordinates where the scattering events occur in an infinite medium and (2) the exact invariant solutions of the radiative transfer equation for radiance, fluence rate, and mean path length in media subjected to a Lambertian illumination. RESULTS: We carried out a wide set of comparisons between MC results and the two analytical benchmarks for a wide range of optical properties (from non-scattering to highly scattering media, with different types of scattering functions) in an infinite non-absorbing medium (step 1) and in a non-absorbing slab (step 2). The deviations between MC results and exact analytical values are usually within two standard errors (i.e., t-tests not rejected at a 5% level of significance). The comparisons show that the accuracy of the verification increases with the number of simulated trajectories so that, in principle, an arbitrary accuracy can be obtained. CONCLUSIONS: Given the simplicity of the verification method proposed, we envision that it can be widely used in the field of biomedical optics.

Direct Measurement of the Reduced Scattering Coefficient by a Calibrated Random Laser Sensor.

The research in optical sensors has been largely encouraged by the demand for low-cost and less or non-invasive new detection strategies. The invention of the random laser has opened a new frontier in optics, providing also the opportunity to explore new possibilities in the field of sensing, besides several different and peculiar phenomena. The main advantage in exploiting the physical principle of the random laser in optical sensors is due to the presence of the stimulated emission mechanism, which allows amplification and spectral modification of the signal. Here, we present a step forward in the exploitation of this optical phenomenon by a revisitation of a previous experimental setup, as well as the measurement method, in particular to mitigate the instability of the results due to shot-to-shot pump energy fluctuations. In particular, the main novelties of the setup are the use of optical fibers, a reference sensor, and a peristaltic pump. These improvements are devoted to: eliminating optical beam alignment issues; improving portability; mitigating the variation in pump energy and gain medium performances over time; realizing an easy and rapid change of the sensed medium. The results showed that such a setup can be considered a prototype for a portable device for directly measuring the scattering of liquid samples, without resorting to complicated numerical or analytic inversion procedures of the measured data, once the suitable calibration of the system is performed.

Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice.

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-ß1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

Verification method of Monte Carlo codes for transport processes with arbitrary accuracy.

In this work, we present a robust and powerful method for the verification, with arbitrary accuracy, of Monte Carlo codes for simulating random walks in complex media. Such random walks are typical of photon propagation in turbid media, scattering of particles, i.e., neutrons in a nuclear reactor or animal/humans' migration. Among the numerous applications, Monte Carlo method is also considered a gold standard for numerically "solving" the scalar radiative transport equation even in complex geometries and distributions of the optical properties. In this work, we apply the verification method to a Monte Carlo code which is a forward problem solver extensively used for typical applications in the field of tissue optics. The method is based on the well-known law of average path length invariance when the entrance of the entities/particles in a medium obeys to a simple cosine law, i.e., Lambertian entrance, and annihilation of particles inside the medium is absent. By using this law we achieve two important points: (1) the invariance of the average path length guarantees that the expected value is known regardless of the complexity of the medium; (2) the accuracy of a Monte Carlo code can be assessed by simple statistical tests. We will show that we can reach an arbitrary accuracy of the estimated average pathlength as the number of simulated trajectories increases. The method can be applied in complete generality versus the scattering and geometrical properties of the medium, as well as in presence of refractive index mismatches in the optical case. In particular, this verification method is reliable to detect inaccuracies in the treatment of boundaries of finite media. The results presented in this paper, obtained by a standard computer machine, show a verification of our Monte Carlo code up to the sixth decimal digit. We discuss how this method can provide a fundamental tool for the verification of Monte Carlo codes in the geometry of interest, without resorting to simpler geometries and uniform distribution of the scattering properties.

hGATA1 Under the Control of a µLCR/ß-Globin Promoter Rescues the Erythroid but Not the Megakaryocytic Phenotype Induced by the Gata1 low Mutation in Mice.

The phenotype of mice carrying the Gata1 low mutation that decreases expression of Gata1 in erythroid cells and megakaryocytes, includes anemia, thrombocytopenia, hematopoietic failure in bone marrow and development of extramedullary hematopoiesis in spleen. With age, these mice develop myelofibrosis, a disease sustained by alterations in stem/progenitor cells and megakaryocytes. This study analyzed the capacity of hGATA1 driven by a µLCR/ß-globin promoter to rescue the phenotype induced by the Gata1 low mutation in mice. Double hGATA1/Gata1 low/0 mice were viable at birth with hematocrits greater than those of their Gata1 low/0 littermates but platelet counts remained lower than normal. hGATA1 mRNA was expressed by progenitor and erythroid cells from double mutant mice but not by megakaryocytes analyzed in parallel. The erythroid cells from hGATA1/Gata1 low/0 mice expressed greater levels of GATA1 protein and of α- and ß-globin mRNA than cells from Gata1 low/0 littermates and a reduced number of them was in apoptosis. By contrast, hGATA1/Gata1 low/0 megakaryocytes expressed barely detectable levels of GATA1 and their expression of acetylcholinesterase, Von Willebrand factor and platelet factor 4 as well as their morphology remained altered. In comparison with Gata1 +/0 littermates, Gata1 low/0 mice contained significantly lower total and progenitor cell numbers in bone marrow while the number of these cells in spleen was greater than normal. The presence of hGATA1 greatly increased the total cell number in the bone marrow of Gata1 low/0 mice and, although did not affect the total cell number of the spleen which remained greater than normal, it reduced the frequency of progenitor cells in this organ. The ability of hGATA1 to rescue the hematopoietic functions of the bone marrow of the double mutants was confirmed by the observation that these mice survive well splenectomy and did not develop myelofibrosis with age. These results indicate that hGATA1 under the control of µLCR/ß-globin promoter is expressed in adult progenitors and erythroid cells but not in megakaryocytes rescuing the erythroid but not the megakaryocyte defect induced by the Gata1 low/0 mutation.