GATA-factor dependence of the multitype zinc-finger protein FOG-1 for its essential role in megakaryopoiesis

Abstract

The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1(KI) (GATA-1 is located on the X-chromosome) or GATA-2(KI/KI) mice, compound GATA-1(KI) GATA-2(KI/KI) mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1(-/-) mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.

Figure 1

Homologous recombination of targeting constructs and subsequent neo cassette excision in ES cells. (A) Amino acid sequence alignment of the amino-terminal zinc fingers of GATA-1 and GATA-2. Mutated positions in GATA-1 and GATA-2 N-terminal zinc fingers (N-f) are indicated by the red “V” and residue number. Identical sequences are shown in black, and nonidentical residues are shown in blue. (B) Schematic representation of V205G KI mutation gene-targeting strategy into the GATA-1 locus on the X-chromosome. Subsequent Cre-mediated excision of neomycin resistance gene (neo) cassette is shown below. Solid triangles represent loxP sites. Location of probe used for Southern analysis is indicated by solid bar (probe A). PCR primers are shown as horizontally opposed arrows. (C) Southern blot and PCR analyses depicting correctly recombined and Cre-excised GATA-1^KI loci. (D) Schematic representation of V296G KI targeting strategy in the GATA-2 locus and subsequent Cre-mediated excision of neo cassette. Locations of probes used for Southern analysis are indicated by solid bars (probes B and C). (E) Southern blot analysis depicting correctly recombined and Cre-excised GATA-2^KI loci.

Figure 2

May–Grunwald–Giemsa stains of cytospun erythrocytes collected from E10.5 yolk sacs (original magnification ×1,000).

Figure 3

In vitro megakaryocyte differentiation from E10.5 yolk sac. (A) Phase-contrast appearance and AChE stains of representative megakaryocyte colonies at day 7 of culture. Arrows indicate proplatelet processes. AchE-positive cells stain orange [original magnification, ×320 (phase-contrast); ×1,000 (AchE)]. (B) Quantitation of megakaryocyte colony formation. Colonies were counted on day 7 of culture and expressed as the mean number of megakaryocyte colonies (MK) per 1 × 10⁵ cells plated. Error bars represent the SEM. n = number of embryos analyzed. (C) RT-PCR analysis of the murine megakaryocytic markers AChE, von Willebrand factor (vWF), GPIbα and P-selectin, from yolk sac cells cultured for 7 days in liquid culture in the presence of recombinant Tpo. Hypoxanthine ribosyltransferase (HPRT) represents “housekeeping” control gene for normalization of cDNA content. Fold decrease of mRNA levels relative to wild type (WT) (after normalization to HPRT signal) is indicated below each panel. “−RT” indicates no reverse transcriptase was added to sample.

Figure 4

Myeloid colony formation from E10.5 yolk sacs. May–Grunwald–Giemsa stains of cytospun preparation from E10.5 yolk sac cells cultured in methylcellulose in the presence of erythropoietin, Tpo, and Kit ligand for 7 days.

Figure 5

Model of FOG-1–GATA factor interaction requirements during megakaryocyte development. During an early stage of megakaryocyte commitment, FOG-1 requires an interaction with either GATA-1 or GATA-2. During a later stage, FOG-1 specifically requires an interaction with GATA-1. GATA-2 also plays a role in proliferation of progenitor cells (indicated by looping arrow). F1, FOG-1; G1, GATA-1; G2, GATA-2.