QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 September 2007
doi: 10.1242/dev.004432

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Astorga, J. Articles by Carlsson, P. Search for Related Content PubMed PubMed Citation Articles by Astorga, J. Articles by Carlsson, P. Social Bookmarking                         What's this?

Hedgehog induction of murine vasculogenesis is mediated by Foxf1 and Bmp4

Department of Cell and Molecular Biology, Göteborg University, Box 462, SE-405 30 Göteborg, Sweden.

View larger version (92K): [in this window] [in a new window]   Fig. 1. Defective vasculogenesis in Ihh-/- yolk sac is associated with reduced Foxf1 expression. (A-C) Comparison of yolk sac vasculature in E9.5 wild-type (wt) (A), Ihh-/- (B) and Foxf1-/- (C) mouse embryos. (D-G) Whole-mount in situ hybridization of E8.5 wt (D,F) and Ihh-/- (E,G) embryos with a Foxf1 probe. F and G are magnified views of the yolk sac surface of the embryos in D and E, respectively. Foxf1 expression in yolk sac blood islands is seen as diffuse blue speckles against the pale background of the out-of-focus embryo heads, and is much weaker in the Ihh mutant.

View larger version (83K): [in this window] [in a new window]   Fig. 2. Hedgehog signaling controls vasculogenesis and Foxf1 expression. (A-D) Morphology and histology of wt and Ihh-/-; Shh-/- E9.5 mouse embryos. (A) E9.5 wt embryo with well-developed yolk sac vasculature. The embryo has completed turning and reached the fetal position. (B) E9.5 Ihh-/-; Shh-/- embryo with an almost completely avascular yolk sac. The embryo is still in the primitive, unturned position and the amnion adheres to the inner surface of the yolk sac (arrowheads in B). (C) Section of a wt E9.5 yolk sac with large blood vessels containing embryonic erythrocytes. (D) Section of an Ihh-/-; Shh-/- E9.5 yolk sac. The yolk sac mesoderm consists of a thin lining of the inner surface of the yolk sac which in some areas adheres to the outer, mesodermal layer of the amnion (arrowhead). (E,F) Whole-mount in situ hybridization of E9.5 embryos with a Foxf1 probe. (E) Wt embryo showing high-level expression of Foxf1 in the lateral plate and extraembryonic mesoderm of allantois and yolk sac. (F) Lateral (top) and posterior-dorsal (bottom) views of an Ihh-/-; Shh-/- embryo. Foxf1 expression is absent in lateral mesoderm and yolk sac, but present in allantois and posterior primitive streak (arrowheads). al, allantois; am, amnion; lp, lateral plate mesoderm; ys, yolk sac; yse, yolk sac endoderm; ysm, yolk sac mesoderm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Defective vasculogenesis in Foxf1-/- yolk sac. (A-C) Whole-mount Pecam staining of E8.5 wt (A left, B) and Foxf1-/- (A right, C) mouse embryos. B and C are higher magnifications of parts of the yolk sacs of the embryos in A. A primitive vascular plexus of endothelial tubes is well developed in the wt, but absent from the mutant. (D-G) Pecam staining of paraffin sections of E9.5 wt (D,F) and Foxf1-/- (E,G) embryos. In the Foxf1 mutant, the yolk sac mesoderm is divided into a thin Pecam+ layer that lines the inner surface of the yolk sac endoderm, and a detached thick layer that only makes sporadic contact with the yolk sac endoderm (arrowhead in E). (H-J) Flk1 staining of cryosections of E8.5 wt (H) and Foxf1-/- (I,J) embryos. The separation of the yolk sac mesoderm has started in the mesometrial pole of the yolk sac (J), but not in the antimesometrial (I). al, allantois; am, amnion; post, posterior part of the embryo proper; yse, yolk sac endoderm; ysm, yolk sac mesoderm.

View larger version (102K): [in this window] [in a new window]   Fig. 4. The allantois undergoes normal vasculogenesis and maintains expression of Foxf1 and Bmp4 in the absence of hedgehog signaling. (A-D) Foxf1 whole-mount in situ hybridization. (A) E8.5 wt littermate of the Smo-/- mouse embryo in D, showing Foxf1 expression in lateral mesoderm, primitive streak and allantois. (B) Foxf1 expression in lateral mesoderm, yolk sac and allantois of an E8.5 wt embryo still in the primitive, unturned position (shown for comparison with the unturned Smo-/- embryo). (C,D) Smo-/- embryos have the same pattern of Foxf1 expression as Ihh-/-; Shh-/- (see Fig. 2), i.e. in the allantois and primitive streak, but not in the lateral mesoderm or yolk sac. To show the chorioallantoic fusion, the entire E9.5 conceptus in C was left intact during hybridization. After the staining, a hole was dissected in the yolk sac through which the allantois and chorio-allantoic connection can be seen. Foxf1 is expressed throughout the allantois, which emerges from the posterior end of the embryo and spreads out like a wide funnel over the chorionic surface (inset shows the entire yolk sac at low magnification). The E8.5 Smo-/- embryo in D has the yolk sac removed to show Foxf1 expression in the primitive streak and its absence in the lateral mesoderm. (E,F) Pecam immunostaining of allantoic explants grown in vitro. Explants of unfused allantoic buds (E8.25) were grown for 24 hours, after which the primitive vascular plexus was visualized by immunostaining for the endothelial marker Pecam. Explants from Smo-/- embryos (F) developed a vasculature indistinguishable from that of wt embryos (E). (G-J) X-Gal staining of Bmp4lacZ heterozygous E9.5 embryos homozygous for the wt (G,H) or null (I,J) alleles in the Smo locus. (G,I) Bmp4lacZ expression in primitive streak and allantois is independent of hedgehog signaling, whereas expression in lateral mesoderm is lost in Smo-/-. (H,J) Flat-mounted yolk sacs (top) and sections (bottom) showing Bmp4lacZ expression in mesodermal cells of blood vessels. The avascular phenotype of Smo-/- yolk sac corresponds to reduced Bmp4lacZ expression. al, allantois; am, amnion; lm, lateral mesoderm; pl, placenta; ps, primitive streak; ysm, yolk sac mesoderm.

View larger version (93K): [in this window] [in a new window]   Fig. 5. Foxf1 is required for Bmp4 expression in lateral and extraembryonic mesoderm. X-Gal staining was used to visualize expression from the Bmp4lacZ allele in E8.5 Foxf1+/+ (A-E) and Foxf1-/- (F-J) mouse embryos. (A) Intact wt conceptus with embryo that has just initiated turning. High Bmp4 expression can be seen in both the allantois and yolk sac. (B) Unturned wt embryo with yolk sac removed showing Bmp4 expression in the amnion (speckled pattern covering embryo), allantois, heart, and extending from the posterior primitive streak into the splanchnopleure and somatopleure. (C) Transverse section showing Bmp4 expression in yolk sac mesoderm (at the top of the image), throughout the allantois and in the amnion (bottom). (D) The inner (mesodermal) surface of the yolk sac shows Bmp4 expression associated with the developing vasculature. (E) Section showing Bmp4 expression in yolk sac mesoderm and amnion. (F,G) Lateral (F) and dorsal (G) views of an Foxf1-/- embryo showing lack of Bmp4 expression in the yolk sac and allantois. Only the primitive streak and heart retain high expression. (H) Section showing scattered and very weak Bmp4 expression in Foxf1-/- allantois. (I) The inner (mesodermal) surface of a Foxf1-/- yolk sac shows only traces of Bmp4 expression. (J) Section of Foxf1-/- yolk sac with characteristic separation between the endodermal and mesodermal layers. Occasional mesodermal cells express low levels of Bmp4. al, allantois; am, amnion; ps, primitive streak; ys, yolk sac; yse, yolk sac endoderm; ysm, yolk sac mesoderm.

View larger version (98K): [in this window] [in a new window]   Fig. 6. Bmp signaling is essential for blood vessel formation and is a mediator of the vasculogenic activity of Foxf1. Explants of E8.25 allantoic buds (A-F) and Smo-/- E9.5 yolk sacs (G,H) were cultured in vitro for 24 hours followed by staining with Pecam antibody to visualize the capillary network. (A-C) Allantoic explants from wt mouse embryos grown in the absence (A) or presence (B,C) of the Bmp antagonist noggin at 1 (B) or 10 (C) ng/ml. (D-F) Rescue of blood vessel formation in Foxf1-/- explants by exogenous Bmp4. (D) Normal vasculogenesis in Foxf1-/+ explant. (E) Foxf1-/- allantoic cells express Pecam, but fail to form a distinct vascular plexus. (F) Addition of 0.1 ng/ml Bmp4 to Foxf1-/- explants restored formation of the capillary network. (G,H) The yolk sac from an E9.5 Smo-/- embryo was dissected, cut in half and left- and right-hand sides spread on separate filters with (H) or without (G) Bmp4 (0.1 ng/ml) in the medium. After 24 hours in culture, explants were fixed and stained with Pecam antibody. Low-magnification images (top) show the entire explants and higher magnification views (bottom) show the formation of a vascular plexus in response to Bmp4.

View larger version (55K): [in this window] [in a new window]   Fig. 7. Bmp4 restricts allantoic smooth muscle cell differentiation. Immunofluorescence showing Flk1+ (red) and SMA+ (green) cells in wt (A) and Foxf1-/- (B,C) allantoic explants cultured for 24 hours with (C) or without (A,B) Bmp4 (0.1 ng/ml).

View larger version (24K): [in this window] [in a new window]   Fig. 8. Schematic summary of relationships between hedgehogs, Foxf1 and Bmp4 in different tissues. Expression of Bmp4 precedes that of Foxf1 in mesoderm formed in the primitive streak area during gastrulation; data from Xenopus (Tseng et al., 2004) indicate that Bmp4 activates transcription of Foxf1 at this stage. As the posterior mesoderm migrates out of the primitive streak and associates with the endoderm in the lateral plate and yolk sac, maintenance of Foxf1 expression depends on hedgehog ligands (Ihh and Shh) secreted by the endoderm. In the murine allantois, which lacks endoderm, Foxf1 expression remains high in the absence of hedgehog signaling. Maintenance of robust Bmp4 expression in the mesodermal cells, after exit from the primitive streak, requires Foxf1. Bmp4 induces the formation of endothelial tubes from mesodermal progenitors.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter