Generation of transgenic zebrafish with 2 populations of RFP- and GFP-labeled thrombocytes: analysis of their lipids

Abstract

Zebrafish thrombocytes are similar to mammalian platelets. Mammals have young platelets (also called reticulated platelets) and mature platelets. Likewise, zebrafish have 2 populations of thrombocytes; one is DiI-C18 (DiI)+ (DP), and the other is DiI− (DN). However, the mechanism of selective thrombocyte labeling by DiI is unknown. Furthermore, there is no transgenic zebrafish line where DP and DN thrombocytes are differentially labeled with fluorescent proteins. In this study, we found that Glo fish, in which the myosin light chain 2 promoter drives the rfp gene, have a population of thrombocytes that are red fluorescent protein (RFP) labeled. We also generated transgenic GloFli fish in which DP and DN thrombocytes are labeled with RFP and green fluorescent protein (GFP), respectively. Single-cell lipid analysis showed a twofold increase in phosphatidylethanolamine (PE) and a twofold decrease in phosphatidylcholine (PC) in RFP+ thrombocytes compared with GFP+ thrombocytes, suggesting that lipid composition may be important for DiI differential labeling. Therefore, we tested liposomes prepared with different ratios of PC and PE and observed that liposomes prepared with higher amounts of PE favor DiI labeling, whereas the PC concentration had a modest effect. In liposomes prepared using only PE or PC, increased concentrations of PE resulted in increased DiI binding. These results suggest that because RFP+ thrombocytes have higher PE concentrations, DiI may bind to them efficiently, thus explaining the selective labeling of thrombocytes by DiI. This work also provides GloFli fish that should be useful in understanding the mechanism of thrombocyte maturation.

Analysis of transcribed sequences from young and mature zebrafish thrombocytes

“…We used the GloFli adult zebrafish, where young thrombocytes were labeled with RFP, and mature thrombocytes were labeled with more intense GFP. This adult zebrafish line, GloFli, was previously characterized in our laboratory [ 4 ]. We collected 2 μL of blood from a single fish and subjected this to flow cytometry for separating RFP+ and GFP+ cells.…”

“…The comparative analysis of the GFP+ and RFP+ thrombocytes showed, more highly expressed genes in GFP+ thrombocytes compared to RFP+ thrombocytes, as confirmed by the heat map studies, probably because during maturation more number of genes are turned on while less number of genes are silenced. In our previous work on young and mature thrombocytes, we have shown that there was an increase in the expression of a Fli1 in mature thrombocytes [ 1 , 4 ]. These results are consistent with the heatmap results suggesting that maturation of thrombocytes involves an increase in thrombocyte gene expression [ 4 ].…”

“…In our previous work on young and mature thrombocytes, we have shown that there was an increase in the expression of a Fli1 in mature thrombocytes [ 1 , 4 ]. These results are consistent with the heatmap results suggesting that maturation of thrombocytes involves an increase in thrombocyte gene expression [ 4 ]. The number of genes analyzed using excel spreadsheet analysis tools is larger than the ones obtained by PANTHER analysis and does not match because there are several genes that are not recognized by the PANTHER program.…”

“…Therefore, we concluded that less intense GFP+ thrombocytes are young thrombocytes. Surprisingly, these less intense GFP+ thrombocytes are also positive for RFP driven by the mlc2 promoter [ 4 ]. Thus, the RFP+ thrombocytes are young, and the rest of the more intense GFP+ thrombocytes are shown to be mature thrombocytes, probably in different stages of maturation.…”

“…For example, the young thrombocytes, when they mature, gain more Fli1 with the concomitant loss of GATA-1 transcription factor [ 9 ]. We have also shown that there is a decrease in phosphatidylethanolamine (PE) and an increase in phosphatidylcholine (PC) in mature thrombocytes compared to young thrombocytes [ 4 ]. Interestingly, amphibian thrombocytes under in vitro conditions showed polyploidy-like features [ 10 ].…”