Live imaging of emerging hematopoietic stem cells and early thymus colonization

Abstract

We recently demonstrated in zebrafish the developmental migration of emerging hematopoietic stem cells (HSCs) that is thought to occur in mammalian embryos, from the aorta-gonad-mesonephros (AGM) area to the successive hematopoietic organs. CD41 is the earliest known molecular marker of nascent HSCs in mammalian development. In this study, we show that in CD41-green fluorescent protein (GFP) transgenic zebrafish embryos, the transgene is expressed by emerging HSCs in the AGM, allowing us for the first time to image their behavior and trace them in real time. We find that the zebrafish AGM contains no intraaortic cell clusters, so far considered a hallmark of HSC emergence. CD41-GFP low HSCs emerge in the subaortic mesenchyme and enter the circulation not through the dorsal aorta but through the axial vein, the peculiar structure of which facilitates their intravasation. The rise in CD41-gfp expression among cmyb ؉ HSC precursors is asynchronous and marks their competence to leave the AGM and immediately seed the caudal hematopoietic tissue (which has a hematopoietic function analogous to that of the mammalian fetal liver). Imaging the later migration of CD41-GFP ؉ precursors to the nascent thymus reveals that although some reach the thymus by extravasating from the nearest vein, most travel for hours through the mesenchyme from surprisingly diverse and remote sites of extravasation. (Blood. 2008;111: 1147-1156)

NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation

“…In addition, whereas CD41:GFP low lymphoid progenitors normally start colonizing the thymus rudiment from 54 h.p.f. onwards 18 , the naca morphant thymus rudiment showed no such colonization even at 84 h.p.f. (Fig.…”

“…3a,b). To follow their fate in live embryos, we turned to transgenic Tg[CD41:gfp] embryos, as definitive HSPCs are CD41:GFP low from as soon as they emerge from the VDA 18 (from here onwards, gfp will refer to the transgene, and, for example, CD41:GFP to the fluorescence of the GFP protein expressed from the CD41:gfp transgene). Consistently, naca MO1-injected Tg[CD41:gfp] embryos showed normal emergence of CD41:GFP low HSPCs from the VDA at 54 h.p.f.…”

“…Confocal fluorescence microscopy was performed on Leica SPE and SP8 setups as described previously 18 . For quantification of the mean nls-mCherry fluorescence intensity per cell type in double Tg [TCF:nls-mCherry; Fli1:gfp] embryos, a volume of interest (VOI) was defined in 10 cells of each population of interest (stromal, somite muscle and fin mesenchymal cells) per embryo, and for each VOI the mean fluorescence/ voxel was calculated using Imaris software (Bitplane).…”

Origine endothéliale des cellules souches hématopoïétiques

“…Les cellules entrent finalement dans la circulation par la veine, après qu'une partie des cellules se soient divisées de manière symétrique. Ce passage des cellules hématopoïétiques par la veine et non par l'artère pourrait s'expliquer par le fait que les jonctions entre les cellules endothéliales veineuses sont plus lâches qu'entre les cellules endothéliales artérielles, comme le visualise la microscopie électronique de la région [33]. L'utilisation de morpholinos bloquant l'expression de Runx1 a clairement montré qu'en l'absence de ce facteur, les tentatives d'émergence des cellules hématopoïétiques sont avortées, aboutissant à une apoptose massive des cellules en cours d'émergence [31].…”

Regulation of Stem Cells in the Zebra Fish Hematopoietic System

“…Soon after the formation of the ICM, erythromyeloid progenitor cells (lmo2-gata1 + ) appear in the posterior blood island (PBI) located in the tail region (Bertrand et al 2007), and a wave of definitive HSC (runx1 + , c-myb + , ikaros + , lmo2 + , scl + , and CD41 + ) production ensues in the aortagonad-mesonephros (AGM) region along the ventral wall of the dorsal aorta (Liao et al 1998;Thompson et al 1998;Willett et al 2001;Kalev-Zylinska et al 2002;Bertrand et al 2008;Kissa et al 2008). These AGM cells subsequently colonize the caudal hematopoietic tissue (CHT), an expansion of the PBI, as well as the thymus and kidney (Murayama et al 2006;Jin et al 2007;Kissa et al 2008).…”