ABSTRACT
Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/metabolism , Epistasis, Genetic , Erythropoiesis/genetics , Evolution, Molecular , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic System/embryology , Hematopoietic System/metabolism , Models, Biological , Molecular Sequence Data , Zebrafish/embryology , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolismABSTRACT
Ocular coloboma is a developmental defect of the eye and is due to abnormal or incomplete closure of the optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, we identified a mutation in the ATP-binding cassette (ABC) transporter ABCB6 in a Chinese family affected by autosomal-dominant coloboma. The Leu811Val mutation was identified in seven affected members of the family and was absent in six unaffected members from three generations. A LOD score of 3.2 at θ = 0 was calculated for the mutation identified in this family. Sequence analysis was performed on the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma, and aniridia, and an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in the ethnically matched control populations. Immunostaining of transiently transfected, Myc-tagged ABCB6 in retinal pigment epithelial (RPE) cells showed that it localized to the endoplasmic reticulum and Golgi apparatus of RPE cells. RT-PCR of ABCB6 mRNA in human cell lines and tissue indicated that ABCB6 is expressed in the retinae and RPE cells. Using zebrafish, we show that abcb6 is expressed in the eye and CNS. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be corrected with coinjection of the wild-type, but not mutant, ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish are due to insufficient abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma.
