In this paper we characterized transgenic lines of zebrafish, which express GFP driven by two different gene promoters GATA1 and FLI1 for the promoter expression in thrombocytes. The fact that in both G1-GM2 and TG(fli1:EGFP)y1 lines thrombocytes were GFP+ suggests that GATA1 and FLI1 gene promoters are active in thrombocytes. In the G1-GM2 line we have shown the more intense GFP+ thrombocytes are young thrombocytes and that the mature thrombocytes do not express GFP very well. Sometimes it was difficult to observe any GFP fluorescence in certain thrombocytes. Thus, from these results it is evident that the maturation of thrombocytes appears to coincide with the loss of activity of the GATA1 gene promoter, and suggests loss of GATA1. In contrast, in the TG(fli1:EGFP)y1 line the young thrombocytes maintain low levels of GFP expression, whereas the mature thrombocytes have high levels of GFP expression suggesting that there is enhancement of FLI1 gene promoter expression resulting in increased expression of FLI1. These transgene expression results are consistent with RT-PCR results in which we have observed reduced levels of endogenous GATA1 mRNA and increased levels of FLI1 mRNA in mature thrombocytes and vice versa in young thrombocytes. These findings are especially interesting due to the fact that GATA1, along with FLI1, has been shown to synergistically activate genes in megakaryocytes. Thus, since both GATA1 and FLI1 are present in young thrombocytes, even though FLI1 is at low levels, this will still confer thrombocyte-specific gene expression. However, as the thrombocytes mature, the loss of GATA1 may be compensated by the over expression of FLI1. It is not yet clear whether such altered expression of these factors will result in alterations in gene expression between young and mature thrombocytes. Nevertheless, the current finding that thrombocyte maturation involves transcription factors that are involved in controlling megakaryocytes is important because understanding regulation in zebrafish thrombocyte maturation might provide insight into the gene control in megakaryocyte maturation. In addition, the observation of selective labeling of thrombocytes in TG(fli1:EGFP)y1, similar to the recently developed transgenic line where thrombocytes are labeled by GFP using GPIIb gene promoter, provides additional transgenic line for studies on thrombocyte biology.
The current work has demonstrated that young thrombocytes first appear at the site of injury. Also, the results showed clustering of young thrombocytes is followed by clustering of mature thrombocytes. These results are similar to the earlier findings using the dye labeling methods. The current results have confirmed previous findings and offer significance due to the fact that previous studies used dye labeling methods which begged the question of whether the observed thrombus formation could be due to dye effects despite the documentation that DiI labeling did not alter thrombocyte function. The current study eliminates such concerns and provides additional proof for the earlier observations.
The present work also establishes that the size of the thrombocyte is increased when a young thrombocyte matures. It is interesting to note in this context that in platelet maturation it has been claimed that the young platelets are in fact larger than mature platelets. Thus, fish thrombocyte departs from the mammalian platelet in this regard. However, it resembles the megakaryocyte maturation with respect to increases in size although the polyploidy does not appear to exist in fish thrombocytes.
In light of the above finding, as well as the previous studies, it is important to address the role of thrombocytes. Are they platelet equivalents or megakaryocyte forerunners? On one side, even though nucleated, they are found in circulation, aggregate in response to platelet agonists, and play a central role in arterial thrombus formation. Thus, physiologically, with respect to hemostatic function, one could acknowledge that they are equivalent to platelets. However, by the fact the thrombocytes are nucleated and express transcription factors that are present in megakaryocytes, in transcriptional sense they have similarities to megakaryocytes. Moreover, the young thrombocytes appear to increase in size similar to increases in megakaryocytes, although at present, the mechanism for the increases in size of thrombocytes is not known. Furthermore, thrombocytes are produced in kidney marrow in fish but they are synthesized in bone marrow in birds. Since they are roughly half the size of the mammalian red cells, they could exit the marrow easily. However, during evolution, at the time of avian and mammalian radiation, a mutation probably created polyploidization of thrombocytes, resulting in a mammalian megakaryocyte that is so large it would explain entrapment within marrow. Considering the role required for hemostasis, apoptosis of the megakaryocyte results in the release of platelets into the blood stream in mammals. As platelets are megakaryocyte vesicles, megakaryocytes should have functions that platelets possess. In fact, Shattil and coworkers have used megakaryocytes to understand platelet functions11
. It makes teleological sense that initially thrombocytes, the forerunners of megakaryocytes, were performing the hemostatic function and evolved to reside in the bone marrow of mammals releasing their vesicles as platelets. Based on transcriptional machinery of thrombocytes reported here, and the previous reports claiming that thrombocytes are platelet equivalents, thrombocytes provide a model system to study both megakaryocyte and platelet functions.
In summary, we have provided evidence that there is a gradual loss of GATA1 and gain of FLI1 expression during maturation of thrombocytes therefore, making thrombocytes a novel model for studying certain aspects of megakaryocyte maturation in consideration that the thrombocytes may be the forerunners of the megakaryocytes. In addition, the finding that the circulating thrombocytes are selectively labeled in TG(fli1:EGFP)y1 fish will provide a tool in understanding thrombocyte development and differentiation in zebrafish.