Zebrafish disease models in hematology: Highlights on biological and translational impact.

Zebrafish (Danio rerio) has proven to be a versatile and reliable in vivo experimental model to study human hematopoiesis and hematological malignancies. As vertebrates, zebrafish has significant anatomical and biological similarities to humans, including the hematopoietic system. The powerful genome editing and genome-wide forward genetic screening tools have generated models that recapitulate human malignant hematopoietic pathologies in zebrafish and unravel cellular mechanisms involved in these diseases. Moreover, the use of zebrafish models in large-scale chemical screens has allowed the identification of new molecular targets and the design of alternative therapies. In this review we summarize the recent achievements in hematological research that highlight the power of the zebrafish model for discovery of new therapeutic molecules. We believe that the model is ready to give an immediate translational impact into the clinic.

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.

Evi1 regulates Notch activation to induce zebrafish hematopoietic stem cell emergence.

During development, hematopoietic stem cells (HSCs) emerge from aortic endothelial cells (ECs) through an intermediate stage called hemogenic endothelium by a process known as endothelial-to-hematopoietic transition (EHT). While Notch signaling, including its upstream regulator Vegf, is known to regulate this process, the precise molecular control and temporal specificity of Notch activity remain unclear. Here, we identify the zebrafish transcriptional regulator evi1 as critically required for Notch-mediated EHT In vivo live imaging studies indicate that evi1 suppression impairs EC progression to hematopoietic fate and therefore HSC emergence. evi1 is expressed in ECs and induces these effects cell autonomously by activating Notch via pAKT Global or endothelial-specific induction of notch, vegf, or pAKT can restore endothelial Notch and HSC formations in evi1 morphants. Significantly, evi1 overexpression induces Notch independently of Vegf and rescues HSC numbers in embryos treated with a Vegf inhibitor. In sum, our results unravel evi1-pAKT as a novel molecular pathway that, in conjunction with the shh-vegf axis, is essential for activation of Notch signaling in VDA endothelial cells and their subsequent conversion to HSCs.

Endothelial-to-hematopoietic transition: Notch-ing vessels into blood.

During development, hematopoietic stem cells (HSCs) are formed in a temporally and spatially restricted manner, arising from specialized endothelial cells (ECs) in the ventral wall of the dorsal aorta within the evolutionary conserved aorta-gonad-mesonephros region. Our understanding of the processes regulating the birth of HSCs from ECs has been recently advanced by comprehensive molecular analyses of developing murine hematopoietic cell populations complemented by studies in the zebrafish model, with the latter offering unique advantages for genetic studies and direct in vivo visualization of HSC emergence. Here, we provide a concise review of the current knowledge and recent advances regarding the cellular origin and molecular regulation of HSC development, with particular focus on the process of endothelial-to-hematopoietic transition and its primary regulator, the Notch signaling pathway.

Peripheral blood WT1 expression predicts relapse in AML patients undergoing allogeneic stem cell transplantation.

To evaluate if WT1 expression may predict relapse after allo-SCT, we analyzed WT1 levels on peripheral blood (PB) and bone marrow (BM) before and after allo-SCT in 24 AML patients with WT1 overexpression at diagnosis. Five copies of WT1/ABL × 10(4) from PB were identified as the threshold value that correlated with relapse after allo-SCT. The same correlation was not identified when WT1 expression was assessed from bone marrow (BM). Eight out of 11 (73%) patients with a pre-allo-SCT PB-WT1 ≥ 5 and 4/13 (31%) patients with a pre-allo-SCT PB-WT1 < 5 relapsed, respectively (P = 0.04). The incidence of relapse was higher in patients with PB-WT1 ≥ 5 measured after allo-SCT, at the 3rd (56% versus 38%; P = 0.43) and at the 6th month (71% versus 20%; P = 0.03). Patients with pretransplant PB-WT1 < 5 had significantly better 2-year OS and LFS than patients with a PB-WT1 ≥ 5 (81% versus 0% and 63% versus 20%) (P = 0.02). Our data suggest the usefulness of WT1 monitoring from PB to predict the relapse in allotransplanted AML patients and to modulate the intensity of conditioning and/or the posttransplant immunosuppression in an attempt to reduce the posttransplant relapse risk.

Profile of toll-like receptors on peripheral blood cells in relation to acute graft-versus-host disease after allogeneic stem cell transplantation.

Toll-like receptors (TLRs) play a key role in the cross-talk between the innate and adaptive immune systems. Previous studies investigating associations between certain TLRs and acute graft-versus-host disease (aGVHD) have reported contrasting results, and no studies relating aGVHD to the expression and function of all human TLRs together have been published to date. We prospectively evaluated the expression of 9 TLRs on T lymphocytes and monocytes by flow cytometry in relation to aGVHD in 34 patients. Induction of TNF-α, IL-4, IFN-γ, and monocyte chemotactic protein 1 on TLR activation was assessed by ELISA on cell supernatants. Nineteen patients developed aGVHD, at a median time of 28 days (range, 20-50 days) after transplantation. A 2-step multivariate analysis was performed using principal component analysis and multifactor analysis of variance. The levels of TLR-5 expression on monocytes and T lymphocytes were positively correlated to aGVHD (P = .01), whereas levels of TLR-1 and -9 were negative predictors (P = .03 and .01, respectively). This profile of TLR-1, -5, and -9 can promote an overall immunostimulatory/proinflammatory response. If our findings are confirmed by further studies, this TLR profile could be a useful biomarker of aGVHD.