Loss of GATA1 and gain of FLI1 expression during thrombocyte maturation

Abstract

In this paper, we characterized expression of GATA1 and FLI1 gene promoters in thrombocytes of zebrafish transgenic lines, G1-GM2 and TG(fli1:EGFP)y1 that carry transgenes of GATA1 and FLI1 gene promoters driving GFP. We found two discrete populations of thrombocytes verified by morphology, labeled with GFP in both G1-GM2 and TG(fli1:EGFP)y1 lines: (1) the more intensely labeled GFP+ thrombocyte, and (2) the less intensely labeled GFP+ thrombocytes. The more intensely labeled GFP+ thrombocyte in G1-GM2 line and the less intensely labeled GFP+ thrombocytes in the TG(fli1:EGFP)y1 line corresponded to young thrombocytes. These results showed that young thrombocytes have higher GATA1 promoter activity, while mature thrombocytes have more FLI1 gene promoter transcription. This finding suggests that there is a gradual loss of GATA1 and gain of FLI1 expression as the thrombocytes mature, and this overexpression of FLI1 may help maintain the thrombocyte lineage. Furthermore, the presence of transcriptional factors similar to those found in megakaryocytes raises the possibility that vertebrate thrombocytes may be the forerunners of mammalian megakaryocytes and, therefore, could serve as a model to study megakaryocyte maturation.

Figure 1

Identification of thrombocytes in TG(fli1:EGFP)y1 and G1-GM2 lines. Top and bottom panels, from left to right are images of thrombocytes, under bright field, fluorescence with excitation at 450–490nm (GFP fluorescence) and stained with Wright-Giemsa stain on blood smears obtained from TG(fli1:EGFP)y1 and G1-GM2 lines, respectively. Black arrows show thrombocytes.

Figure 2

Identification of young and mature thrombocytes in TG(fli1:EGFP)y1 and G1-GM2 lines. Top and bottom panels, from left to right show representative images of thrombocytes under bright field, fluorescence with excitation at 450–490nm and fluorescence with excitation at 510–560 nm on blood cells under a cover slip obtained from TG(fli1:EGFP)y1 and G1-GM2 lines respectively. Orange fluorescence is due to dual labeling of young thrombocyte by GFP and DiI.

Figure 3

ADP induced thrombocyte aggregates in TG(fli1:EGFP)y1 line. Left and right panels are images of thrombocyte cluster under bright field and fluorescence with excitation at 450–490nm (GFP fluorescence).

Figure 4

Thrombus growth in laser induced arterial thormbosis. Top and bottom panels, from left to right show growth of thrombus in arteries of two independent larvae with initiating young thrombocyte cluster (less intense thrombocytes) at the site of laser injury shown by arrow in TG(fli1:EGFP)y1 line. The mature more intense thrombocytes appear later. The rest of the area of green fluorescence is the blood vessels that are positive for GFP.

Figure 5

Analysis of young and mature thrombocytes by flow cytometry. Representative dot plots demonstrating presence of GFP and DiI positive cells in whole blood of TG(fli1:EGFP)y1 line. All cell populations were included in the gate. Top panels from left to right show unstained control and GFP+ thrombocytes. Bottom panels from left to right show DiI labeled thrombocytes (fluorescence was measured in FL2 channel shown on Y-axis) and GFP+ thrombocytes labeled with DiI.

Figure 6

Simultaneous amplification of GATA1, FLI1 and EF1-α mRNA by RT-PCR. A. Agarose gel showing the 410 bp, 299 bp and 220 bp RT-PCR amplified bands (shown by arrows) corresponding GATA1, FLI1 and EF1-α mRNA respectively in young and mature thrombocytes. 2-log DNA Ladder (New England Biolabs, Ipswich, MA) used as DNA size markers (Marker) are in left lane. B. Table showing the quantitation of the above bands. The numbers are the actual intensities measured by Quantity One software