A gene trap screen reveals the expression of the transcription factor Gfi1.1 in haemogenic endothelial cells of the Zebrafish embryo
Thambyrajah, Roshana Sutharshini (2012) A gene trap screen reveals the expression of the transcription factor Gfi1.1 in haemogenic endothelial cells of the Zebrafish embryo. PhD thesis, University of Nottingham.
In vertebrates, haematopoiesis occurs in two waves. The primitive wave gives rise to transient myeloid and erythroid cells whereas the definitive wave generates haematopoietic stem cells (HSCs), which maintain the blood system throughout life. These HSCs are able to self-renew and to give rise to progenitors that differentiate into mature cells of all blood lineages. Little is known about the cellular origin and molecular programming of HSCs. This knowledge is useful to generate HSCs in vitro from embryonic stem cells or induced pluripotent cells. In zebrafish, HSCs form in the intermediate cell mass (ICM), in the trunk of the embryo. Here, they develop dorsal to the primitive red blood cells and in close association with the ventral wall of the dorsal aorta (DA). Like their mammalian counterparts, they express the transcription factors runx-1 and c-myb. As in other vertebrates, zebrafish HSCs are thought to arise from the haemogenic endothelium in the ventral wall of the DA. A signalling cascade that involves the Hedgehog, Vascular endothelial growth factor (Vegf) and Notch signalling pathways is needed for arterial specification of the DA and for HSC formation. Short-term lineage tracing experiments showed that cells in the ventral wall of the DA first seed the tail mesenchyme (caudal haematopoietic tissue, CHT) through blood circulation before they seed the final sites of haematopoiesis, the thymus and kidney in the adult fish. Here, we conducted a tol2-transposon based gene trap vector screen with eGFP as the reporter gene with the aim to label nascent HSCs in vivo and to identify novel genes involved in haematopoiesis. We obtained 174 transgenic lines with tissue-specific eGFP expression in non-haematopoietic and haematopoietic tissues. We identified two lines with marker gene expression in haematopoietic cells. One of the transgenic lines, I-551:eGFP, showed reporter gene expression in primitive red blood cells and in endothelial cells in the ventral wall of the DA at 25 hours post fertilization (hpf). Using inverse PCR we identified the trapped gene in I-551:eGFP as gfi1.1, the homolog of the mouse Growth independence factor 1 (Gfi1), a transcriptional repressor expressed in HSCs.
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