Time-lapse imaging of chick cardiac precursor cells.

The chick embryo is easily accessible and has therefore been widely used in developmental biology studies. In particular, the early embryo can be removed from the egg and cultured, which allows real-time observations and imaging. Here, we describe ex vivo electroporation followed by long-term time-lapse microscopy, image capture, and processing. We have applied this approach to characterise the migration route of cardiac progenitor cells (CPCs) in live embryos. The heart is the first organ to function during vertebrate development and it is essential for the continued growth and survival of the embryo. In the chick, cardiac progenitors have been mapped to the anterior and mid-primitive streak at Hamburger-Hamilton stage 3. However, until recently it was not possible to observe cell migration trajectories directly. Furthermore, we used grafting of beads or cell pellets or electroporation of expression plasmids to show that Wnt3a acts as a repulsive signal to guide the movement of cardiac progenitors.

Cdx mutant axial progenitor cells are rescued by grafting to a wild type environment.

Cdx transcription factors are required for axial extension. Cdx genes are expressed in the posterior growth zone, a region that supplies new cells for axial elongation. Cdx2(+/-)Cdx4(-/-) (Cdx2/4) mutant embryos show abnormalities in axis elongation from E8.5, culminating in axial truncation at E10.5. These data raised the possibility that the long-term axial progenitors of Cdx mutants are intrinsically impaired in their ability to contribute to posterior growth. We investigated whether we could identify cell-autonomous defects of the axial progenitor cells by grafting mutant cells into a wild type growth zone environment. We compared the contribution of GFP labeled mutant and wild type progenitors grafted to unlabeled wild type recipients subsequently cultured over the period during which Cdx2/4 defects emerge. Descendants of grafted cells were scored for their contribution to differentiated tissues in the elongating axis and to the posterior growth zone. No difference between the contribution of descendants from wild type and mutant grafted progenitors was detected, indicating that rescue of the Cdx mutant progenitors by the wild type recipient growth zone is provided non-cell autonomously. Recently, we showed that premature axial termination of Cdx mutants can be partly rescued by stimulating canonical Wnt signaling in the posterior growth zone. Taken together with the data shown here, this suggests that Cdx genes function to maintain a signaling-dependent niche for the posterior axial progenitors.