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Assessment of hematopoietic failure due to RPL11 deficiency in a zebrafish model of Diamond–Blackfan anemia by deep sequencing

ABSTRACT: To comprehensively reflect the impact of RPL11 deficiency on the transcriptome of zebrafish embryos, we collected 40–50 RPL11-deficient and MO control zebrafish embryos at 48 hpf from separate experiments and constructed two mRNA-seq sequencing libraries in parallel. High-throughput sequencing was performed on the Hi-Seq2000 sequencing platform in parallel. The number of sequenced gene transcript reads was 35–40 million. We found that hemoglobin biosynthetic and hematological defects in RPL11-deficient zebrafish were related to dysregulation of iron metabolism-related genes, including tfa, tfr1b, alas2 and slc25a37, which are involved in heme and hemoglobin biosynthesis. In addition, we found reduced expression of the hematopoietic stem cells (HSC) marker c-myb and HSC transcription factor tal1 and hoxb4a in RPL11-deficient zebrafish embryos, indicating that the hematopoietic defects may be related to impaired HSC differentiation and proliferation. However, RPL11 deficiency did not affect the development of other blood cell lineages such as granulocytes and myelocytes.

ORGANISM(S): Danio rerio

PROVIDER: GSE51326 | GEO | 2014/01/21

SECONDARY ACCESSION(S): PRJNA222400

REPOSITORIES: GEO

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Project description:Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has informed our understanding of blood differentiation and leukemogenesis, but a similarly transformative analysis of the embryonic origins of hematopoiesis is lacking. To address this issue, we acquired gene expression profiles of developing HSC purified from over 2500 dissected murine embryos and adult mice, and applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs emerging from hemogenic endothelium, and definitive HSCs. We functionally validated several candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos, confirming changes in the expression of HSC markers runx1 and c-myb in the aorta-gonads-mesonephros (AGM), the site of definitive HSC specification. Moreover, we found that HSCs derived from differentiating embryonic stem cells in vitro (ESC-HSC) most closely resemble definitive HSC, yet lack a signature indicative of specification by Notch signaling, which likely accounts for their deficient lymphoid development. Our analysis and accompanying web resource will accelerate the characterization of regulators of HSC ontogeny, facilitate efforts to direct hematopoietic differentiation and cell fate conversion, and serve as a model to study the origins of other adult stem cells. Hematopoietic stem cells and their precursors were isolated from mouse embryos and distinct stages and sites during development, purified by fluorescence activated cell sorting, and gene expression profiled on Affymetrix arrays. We also profiled HSCs and their precurors derived by in vitro differentitation of embryonic stem cells [expression data from GSE16925 and GSE14012 was used for mESCs, available at http://hsc.hms.harvard.edu/].

Project description:Background: Severe combined immunodeficiency (SCID) is characterized by arrested T lymphocyte production and B lymphocyte dysfunction, resulting in life-threatening infections. Early diagnosis of SCID through population-based newborn screening (NBS) optimizes clinical management and outcomes, and also permits identification of previously unknown factors essential for human lymphocyte development. Methods: SCID was detected, prior to onset of infections, by NBS of T cell receptor excision circles, a biomarker for thymic output. Upon confirmation, the affected baby was treated by allogeneic hematopoietic cell transplantation (HCT). The genetic cause was sought by exome sequencing of the patient and parents, followed by functional analysis of a prioritized candidate gene using human hematopoietic stem cells (HSC) and zebrafish embryos. Results: An infant with leaky SCID, craniofacial and dermal abnormalities, and absent corpus callosum had his immune deficit fully corrected by HCT. Exome sequencing revealed a heterozygous, de novo, missense mutation pN441K in BCL11B. The mutant Bcl11b protein had dominant negative activity, abrogating the ability of wild type Bcl11b to bind DNA, arresting T cell lineage development and disrupting HSC migration, revealing a novel function of Bcl11b. The patientâs defects, recapitulated in Bcl11b-deficient zebrafish, were reversed by ectopic expression of intact, but not mutant, human BCL11B. Conclusions: Newborn screening facilitated treatment and identification of a novel etiology for human SCID. Coupling exome sequencing with candidate gene evaluation in human HSC and in zebrafish revealed that a constitutional BCL11B mutation causes human multisystem anomalies with SCID, while also revealing a novel, pre-thymic role for Bcl11b in hematopoietic progenitors. 3 samples were analyzed in duplicate, Sample 1 was human HSC transduced with GFP only lentivirus which served as controls, Sample 2 was human HSC transduced with lentivirus expressing FLAG-tagged WT BCL11B and GFP, Sample 3 was human HSC transduced with lentivirus expressing FLAG-tagged mutant BCL11B and GFP

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Assessment of hematopoietic failure due to RPL11 deficiency in a zebrafish model of Diamond–Blackfan anemia by deep sequencing

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