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Hedgehog is required for murine yolk sac angiogenesis

¹ Department of Biology, Wesleyan University, CT 06459, USA
² Genetics Unit, The Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada
³ Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA

View larger version (61K): [in a new window]   Fig. 1. Cell types responding to hedgehog signaling in ES embryoid bodies and yolk sacs. (A) Day 13 R1 ES Ptch-lacZ embryoid body expressing ß-gal in the mesothelial layer surrounding blood islands (arrows) and beneath the visceral endoderm between blood islands. (B) Ptch-lacZ (ß-gal) and PECAM are expressed in distinct patterns in adjacent sections in day 13 embryoid bodies. (C) Double staining demonstrates overlapping expression for ß-gal and -SMA around blood islands (arrows) and between them in day 12 embryoid bodies. (D) 10.5 dpc Ptch-lacZ yolk sacs express ß-gal around blood vessels and in the mesothelial layer. (E) -SMA is also expressed around and between vessels in 10.5 dpc yolk sacs. Scale bars: 100 µm.

View larger version (30K): [in a new window]   Fig. 2. Generation of Ihh-deficient ES cells. A targeting construct designed to replace the first exon with a neoR cassette (A) was used to generate Ihh null cell lines (clone 14-61 and 14-63I (not shown)), identified using PCR (not shown) and Southern blot analysis (B). An NcoI digest yields an 8 kb endogenous allele and an 8.8 kb recombinant allele (see heterozygous clone14). Absence of Ihh mRNA expression and continued expression of Shh mRNA in Ihh null cell lines was demonstrated via RT-PCR analysis of embryoid body RNA (C) and loss of hedgehog response was detected by in situ hybridization analysis for Ptch in day 10 embryoid bodies (D).

View larger version (78K): [in a new window]   Fig. 3. Differentiation of Ihh deficient embryoid bodies. (A) Day 10 Ihh–/– embryoid bodies fail to cavitate compared with Ihh+/– embryoid bodies. Ihh–/– embryoid bodies also appear to have increased levels of parietal endoderm (arrows). (B) Day 13 Ihh+/– embryoid bodies contain blood cells (stained with benzidine) and blood islands (arrows), whereas Ihh–/– consistently fail to cavitate or form blood island structures. Scale bars: 100 µm.

View larger version (20K): [in a new window]   Fig. 4. Expression of vasculogenesis and hematopoiesis markers in Ihh–/– ES cell embryoid bodies. PECAM whole-mount immunocytochemistry in day 13 Ihh+/– embryoid bodies reveals expression in endothelial cells lining the interior of blood islands (A, arrows) and in core patches, whereas Ihh–/– embryoid bodies express PECAM in only core patches. Scale bar: 200 µm. (B) RT-PCR analysis examining expression of vasculogenesis and hematopoiesis markers. Ihh+/– embryoid bodies upregulate the markers Tal1, Flk1 and Flt1 during blood island development (days 10-13), whereas the loss of Ihh results in a dramatic reduction in mRNA levels for these genes. HPRT is shown as a loading control.

View larger version (66K): [in a new window]   Fig. 5. Morphology of Ihh–/– yolk sacs. (A) 10.5, 11.5 and 12.5 dpc Ihh mutant yolk sacs display vascular defects. Ihh–/– yolk sacs contain fewer and smaller vessels and undergo suboptimal vascular remodeling compared with Ihh+/– yolk sacs. Scale bar: 1mm. (B) Whole-mount PECAM staining in 11.5 dpc yolk sacs. Ihh+/– vessels appear uniform in diameter and well organized. Note the small vessels (arrow) and flattened morphology (arrowhead) in the Ihh–/– yolk sac compared with the similar junction in the Ihh +/– yolk sac. Scale bar: 500 µm.

View larger version (57K): [in a new window]   Fig. 6. Histological and immunocytochemical analysis of 11.5 dpc Ihh+/– and Ihh –/– yolk sacs. (A) Hematoxylin and Eosin stained sections illustrate the small diameter of blood vessels, as well as the flattened morphology, in mutant yolk sacs compared with heterozygotes (arrows). (B) PECAM stains endothelial cells more robustly in Ihh +/– yolk sacs than in Ihh mutants (first panel). -SMA stains vascular smooth muscle cells encircling the blood vessels in Ihh+/– yolk sacs, whereas it fails to do so in the mutants (second panel, note arrow on right side). Additional endothelial cell markers, CD34 and Flk1, also exhibit reduced expression levels in the Ihh mutant yolk sacs compared with Ihh+/– yolk sacs (third and fourth panels). Scale bars: 100 µm.

View larger version (64K): [in a new window]   Fig. 7. Smo–/– embryoid bodies resemble Ihh–/– embryoid bodies. PECAM is expressed in endothelial cells lining the interior of the blood island and in core patches in day 13 Ihh and Smo heterozygous embryoid bodies (A,C). Like Ihh–/– embryoid bodies, the majority of Smo–/– embryoid bodies fail to cavitate and do not express PECAM in cell-free pockets, only in core patches (B,D). Scale bar: 200 µm.

View larger version (78K): [in a new window]   Fig. 8. Smo mutant yolk sacs exhibit severe vascular defects. (A) Whole-mount immunocytochemistry for PECAM demonstrates the arrest in the primary vascular plexus stage and total absence of large vitelline vessels in the mutant yolk sacs (note proximal staining in upper right panel and enlargements below). Scale bars: 1mm. (B) Hematoxylin and Eosin stained sections show the vessels packed with hematopoietic cells in the mutant yolk sacs compared with the heterozygotes. In situ hybridization for Bh1 (C) identifies the clustered cells as erythrocytes. (D) PECAM staining is reduced in the Smo mutant yolk sacs compared with Smo+/– yolk sacs. Scale bars: 100 µm in B-D.