Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2002 Feb 1;16(3):301-6.
doi: 10.1101/gad.959102.

The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages

Shireen Saleque  1 , Scott Cameron, Stuart H Orkin
Affiliations
Comparative Study

The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages

Shireen Saleque et al. Genes Dev. .

Abstract

Gfi-1 and Gfi-1b are novel proto-oncogenes identified by retroviral insertional mutagenesis. By gene targeting, we establish that Gfi-1b is required for the development of two related blood lineages, erythroid and megakaryocytic, in mice. Gfi-1b(-/-) embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b(-/-) embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. The fetal liver of mutant mice contains erythroid and megakaryocytic precursors arrested in their development. Myelopoiesis is normal. Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and megakaryocyte development.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Targeted disruption of the mouse Gfi-1b gene. (A) Partial restriction map of the mouse Gfi-1b locus (top), the targeting vector (middle), and the expected targeted loci with the floxed neoR cassette (bottom). The 130-bp probe extending from the SacI site to the end of the Gfi-1b coding sequence on exon 7 used to detect appropriate 3′ integration of the targeting vector on Southern blots is indicated (3′ probe). The positions of the primers used to determine the 5′ integration by PCR are also indicated by arrowheads (5′ primer and neo primer, respectively). The Gfi-1b coding exons are indicated as shaded boxes, and the noncoding ones by open boxes. The floxed neoR cassette is indicated by an open box (neoR) flanked by arrowheads (loxP sites), and the TK cassette is shown as a solid black box. The restriction enzyme sites indicated in the map are BamHI (B), EcoRI (E), HindIII (H), and SacI (S). The sizes of the BamHI fragment detected by the 3′ probe in the wild-type and the mutant allele with the inserted neoR cassette are 12 kb and 5 kb, respectively. (B) Southern blot analysis of G148- and gancyclovir-resistant ES cell clones with the 3′ probe. Positions of the wild-type and mutant alleles (with neoR) are indicated by open and solid arrows, respectively. (C) PCR amplification of selected clones shown in B with the 5′ and neo primers, respectively. The PCR product indicative of the homologous recombination is indicated (5′ PCR product).
Figure 2
Figure 2
Gfi-1b−/− ES cells fail to contribute to adult red cell hemoglobin in chimeric mice. Hemoglobin electrophoresis of peripheral blood from four Gfi-1b−/− chimeric mice (lanes 1–4), two Gfi-1b+/− chimeric mice (lanes 5,6), and a wild-type C57Bl/6 mouse.
Figure 3
Figure 3
Control and Gfi-1b mutant embryos and peripheral blood at different gestational ages. Control (a,e,i) and mutant (c,g,k) embryos at E10.5, E12.5, and E14.5 and May–Grunwald–Giemsa stains of their corresponding yolk sac blood (b, f, and j, and d, h, and l, respectively). Gfi-1b−/− embryos show aberrant primitive erythropoiesis characterized by abnormal cell morphology (d) and delayed cellular maturation (h,l). Embryos die by E15 (k) from a failure of fetal liver erythropoiesis, resulting in the complete absence of definitive enucleated erythrocytes (l).
Figure 4
Figure 4
Gfi-1b−/− fetal livers show arrested definitive erythropoiesis. (A) Flow cytometry of E12.5 fetal livers. Forward (FSC-H) and side scatter (SSC-H) profiles of control (a) and mutant fetal livers (c). FACS profiles of gated (b,d) fetal liver cells stained with antibodies to c-kit and ter119. The majority of cells (60%–70%) from control livers are ter119hi and c-kit− (b), showing normal erythroid maturation. Cells from Gfi-1b−/− livers are either ter119− or ter119lo and c-kit+ (d). (B) Fetal liver cells from control embryos produce CFU-Es (day 3, a) and BFU-Es (day 7, b) when cultured in vitro with epo and KL. Gfi-1b−/− cells proliferate in epo and KL (e) but cannot mature into BFU-Es (e) or CFU-Es (d). Cells from BFU-E colonies of control fetal livers stain positively for benzidine (brown/black cells, c), but those from Gfi-1b−/− liver colonies do not (f).
Figure 4
Figure 4
Gfi-1b−/− fetal livers show arrested definitive erythropoiesis. (A) Flow cytometry of E12.5 fetal livers. Forward (FSC-H) and side scatter (SSC-H) profiles of control (a) and mutant fetal livers (c). FACS profiles of gated (b,d) fetal liver cells stained with antibodies to c-kit and ter119. The majority of cells (60%–70%) from control livers are ter119hi and c-kit− (b), showing normal erythroid maturation. Cells from Gfi-1b−/− livers are either ter119− or ter119lo and c-kit+ (d). (B) Fetal liver cells from control embryos produce CFU-Es (day 3, a) and BFU-Es (day 7, b) when cultured in vitro with epo and KL. Gfi-1b−/− cells proliferate in epo and KL (e) but cannot mature into BFU-Es (e) or CFU-Es (d). Cells from BFU-E colonies of control fetal livers stain positively for benzidine (brown/black cells, c), but those from Gfi-1b−/− liver colonies do not (f).
Figure 5
Figure 5
Gfi-1b−/− fetal livers show arrested megakaryopoiesis. (A) Colonies (a,d) and cells (b,c,e,f) from control (a–c) and Gfi-1b−/− (d–f) fetal liver cells grown in thrombopoietin (tpo). When cultured in tpo, fetal liver cells from wild-type livers give colonies with large megakaryocytes (a), whereas Gfi-1b−/− cells proliferate in tpo but do not differentiate into large megakaryocytes (d). The Gfi-1b−/− cells also do not show any nuclear (multilobulation) or cytoplasmic (granulation) maturation upon May–Grunwald–Giemsa staining (b vs. e) and are negative for acetylcholine esterase staining (c vs. f). (B) Semiquantitative RT–PCR of control (wild-type) and Gfi-1b−/− fetal liver cells cultured in tpo. Gfi-1b−/− cells have far fewer transcripts encoding markers of mature megakaryocytes relative to controls (a–d, lanes 5–8 vs. 1–4), for example, von-Willebrand factor (vWF in a), the transcription factor NF-E2 (b), the c-MPL receptor (c), and the surface glycoprotein IIb (d).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cubadda Y, Heitzler P, Ray RP, Bourouis M, Ramain P, Gelbart W, Simpson P, Haenlin M. u-shaped encodes a zinc finger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila. Genes & Dev. 1997;11:3083–3095. - PMC - PubMed
    1. Fossett N, Tevosian SG, Gajewski K, Zhang Q, Orkin SH, Schulz RA. The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in Drosophila. Proc Natl Acad Sci. 2001;98:7342–7347. - PMC - PubMed
    1. Fuchs B, Wagner T, Rossel N, Antoine M, Beug H, Niessing J. Structure and erythroid cell-restricted expression of a chicken cDNA encoding a novel zinc finger protein of the Cys + His class. Gene. 1997;195:277–284. - PubMed
    1. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swat W, Wang J, et al. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell. 1998;95:891–902. - PubMed
    1. Gilks CB, Bear SE, Grimes HL, Tsichlis PN. Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein. Mol Cell Biol. 1993;13:1759–1768. - PMC - PubMed

Publication types