FIGURE SUMMARY
Title

The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation

Authors
Liao, W., Bisgrove, B.W., Sawyer, H., Hug, B., Bell, B., Peters, K., Grunwald, D.J., and Stainier, D.Y.R.
Source
Full text @ Development

Localization of flk-1 transcripts in wild-type embryos. 7- somite stage (A-C), 20-somite stage (D,E), 24-somite stage (F,G), and 24 hpf (H,I). Embryos shown in A,D,F-H are lateral views with dorsal to the top and anterior to the left. All others are dorsal views with anterior to the left (B shows the head region, C shows the midtrunk region; F and G show the anterior and posterior trunk regions respectively, and I shows the head region). (A-C) flk-1-positive cells are found in discrete bilateral stripes both anteriorly and posteriorly. There is also a transverse ectodermal stripe of staining in the hindbrain region; this staining quickly becomes weaker (see D) and although blood vessels do form there at later stages, the fate of these flk-1-expressing cells is not entirely clear (see also Figs 4 and 5). (D,E) flk-1 expression appears to extend caudally from the head region as well as in both directions in the trunk region, until by the 20-somite stage there is a continuous band of flk-1-expressing cells from the anterior head region to the tailbud. Concurrently, flk-1- expressing cells in the mid- and posterior trunk regions converge medially. The site (arrowheads) where the paired lateral dorsal aortae fuse into a single medial aorta is the site of vascular breakdown in the gridlock mutation (Weinstein et al., 1995). At a slightly later stage, single cells (F, arrows) are starting to migrate into the intersomitic space in the upper trunk region to form the intersomitic vessels. The dorsal aorta (DA) and axial vein (AV) are clearly distinct in the trunk region at this stage, and further caudally, in the tail, there is a region of more intense staining (asterisk). [Similarly intense flk-1 expression is also observed in the tail region of early mouse embryos (Yamaguchi et al., 1993; Shalaby et al., 1995)]. By 24 hpf (H, I), the whole vasculature including the head vessels (I) is clearly outlined by staining for flk-1 transcript.

flk-1 is expressed only in cells of the lower trunk and tail regions in clo mutant embryos. flk-1 expression in wild-type and clo mutant embryos. Lateral view of 10-somite stage (A), 20-somite stage (B), 24 hpf (C) and 30 hpf embryos (D-G). (A-C) Wild-type above and clo mutant below. Until the 20-somite stage, the only flk-1 expression observed in mutant embryos is in the ectodermal region of the hindbrain (small arrows); there is also a small area of faint and hazy expression in the tailbud (A, B, arrowheads). At the 20-somite stage (B), staining is observed in the tail region (large arrow) (the tail begins at the level of the anus, which itself is located at the caudal end of the yolk extension). This staining is more noticeable by 24 hpf (C, large arrow) although it is very much weaker than in wild-type. By 30 hpf, the extent of flk-1 staining in mutant embryos (F,G) can be fully appreciated as compared to wild-type (D,E): only cells of the lower trunk and tail regions express flk-1 and this staining is much less intense than in wild-type.

Histological examination of wild-type and clo mutant embryos overstained with flk-1 (see Methods). Lateral view of the 36 hpf embryos used for histology (A,B), wild-type above and mutant below; transverse sections of the head, trunk and tail regions of wildtype (C,D,E) and clo mutants (F,G,H) respectively. Arrows in A indicate the A-P position of the sections shown. (A,B) flk-1 expression is seen in the lower trunk and tail regions of mutant embryos but is missing from the rest of the trunk. In the head region, flk-1 expression is observed in intracranial vessels in wild-type (C) but not in mutants (F); there is also staining on the dorsal aspect of the hindbrain region which is seen in both wild-type and mutant embryos (and can be observed at earlier stages (Fig. 2A-C)), as well as staining on the ventrolateral aspects of the caudal head region which appears on sections as hazy background on the outside of overstained embryos (data not included). (D,G) In the the trunk region, while vessels are clearly stained in wild-type embryos, no flk- 1 expression is seen in mutants and in fact, somitic tissue appears to occupy the space where the dorsal aorta and axial vein usually form (G, arrow). (E,H) In the tail region, flk-1-expressing cells are present in both wild-type and clo mutant embryos although at an apparently reduced number in the mutants. N, notochord.

tie is not expressed in clo mutant embryos. tie expression in wild-type and clo mutant embryos at 30 hpf. (A) Lateral view of wild-type above and clo mutant below. (B,C) Higher magnification view of the lower trunk and tail regions of wild-type (B) and clo mutant (C). At this stage, tie is strongly expressed throughout the wild-type vasculature. In clo mutants, tie expression is not detected at this or later stages.

GATA-1 is expressed in clo mutant embryos only in the region outlined by the flk-1-expressing cells. GATA-1 expression in wild-type and clo mutant embryos at 30 hpf. (A) Lateral view of 2 wild-type (top) and one clo mutant (bottom) embryos. (B) clo mutant embryos. (C,D) Higher magnification view of cells expressing GATA-1 (C) and flk-1 (D) in the lower trunk and tail regions of clo mutant embryos. GATA-1 expression is not detected at early stages in clo mutant embryos. Expression appears as flk-1 is being detected and only in the region lined by the flk-1-positive cells. Additional analysis reveals that although GATA-2 does not seem to be expressed in clo hematopoietic tissues, the GATA-1- expressing cells appear to mature normally as assessed by positive diaminofluorene staining used to detect heme (data not included).

Acknowledgments
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