spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhu, J.
Right arrow Articles by Reichardt, L. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhu, J.
Right arrow Articles by Reichardt, L. F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

ß8 integrins are required for vascular morphogenesis in mouse embryos

Jiangwen Zhu, Karin Motejlek*, Denan Wang, Keling Zang, Andrea Schmidt and Louis F. Reichardt{dagger}

Howard Hughes Medical Institute and Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA
* Present address: Department of Pharmacology for Pharmacists Martinistrasse. 52, Institute of Pharmacology, University of Hamburg-UKE, 20251 Hamburg, Germany



View larger version (45K):

[in a new window]
  		Fig. 1. Generation of integrin ß8-deficient mice. (A) Schematic drawing of integrin ß8 genomic region encompassing Exons IV, V and VI in the wild type and mutant. Black boxes represent exons and white box represents the PGK-neor-cassette. Arrows indicate the transcriptional orientation of the cassette. 5' and 3' probes for Southern blot analyses are indicated. (B) Identification of ES clones containing a mutated ß8 allele with 5' and 3' probes by Southern blot analyses. The wild-type and mutant alleles are labeled. (C) PCR analysis of genotypes from a heterozygous intercross. Mutant and wild-type amplification products are indicated. (D) Northern blot analysis showing the absence of integrin ß8 transcripts in homozygous mutant mice. Comparable amounts of wild-type and mutant total RNA were loaded, as indicated by the presence of equal amounts of ß-actin RNA. RT-PCR analysis further verified that no functional transcript is expressed in the mutant (data not shown).

 


View larger version (77K):

[in a new window]
  		Fig. 2. Phenotypic defects in integrin ß8-deficient mice. (A,B) E10.5 yolk sacs. The vasculature is less prominent and often pale in mutant embryos (B) when compared with wild type (A, arrow). (C,D) Side views of E10.5 embryos. The class A mutant embryo exhibits a smaller body size (D) compared with a wild-type littermate (C). An example of enlarged pericardiac cavities is shown in D (*). (E,F) Side views of E12.5 embryos. Intracerebral hemorrhage in a class B mutant embryo is shown (F, arrow) when compared with its wild-type littermate (E). (G,H) Side views of P0 wild-type (G) and mutant (H) pups. Severe hemorrhage in the mutant head is obvious (H, arrow). (I,J) The P0 mutant brain (J) shows characteristics of hydrocephalus (arrowhead) and exhibits visible hemorrhage compared with a wild type (I). (K,L) A cleft palate is obvious in a mutant neonate (L, arrow), but is absent in a wild-type littermate (K). mc, metencephalon; tc, telencephalon; op, optic vesicle; ot, otic pit; ba, bronchial arches; he, heart; fl, forelimb; hl, hindlimb.

 


View larger version (118K):

[in a new window]
  		Fig. 3. Angiogenesis defects in class A integrin ß8-deficient mutants. (A,B) Hematoxylin and Eosin staining of transverse sections of placentas from an E10.5 wild type (A) and a mutant (B). While the chorionic plate (cp) and labyrinthine trophoblast layer (lt) are comparable in mutant and wild-type littermates, the labyrinthine layer (lbr) is reduced in the mutant. While the interdigitation of fetal blood vessels (A, arrows) and maternal blood vessels in wild-type embryos is elaborate, only a few fetal blood vessels have penetrated into the labyrinthine layer in the mutant (B, arrows). Inserts in A,B provide higher magnification photos of the vasculature. (C,D) Vasculature of E10.5 yolk sac in wild-type (C) and mutant (D) as depicted by X-gal staining of a Tie2:lacZ reporter gene in these mice. (E-G) Vascular patterns revealed by whole-mount staining of ß-galactosidase activity in E9.5 embryos expressing the Tie2:lacZ reporter gene (E,F) and whole-mount immunohistochemistry with anti-PECAM antibody in E10.5 embryos (G). An E9.5 mutant embryo (E) shows no obvious abnormalities in vascular pattern compared with a wild-type littermate (F) (the tails of the embryos in both E and F were used for genotyping). E10.5 mutant embryos (G, right) have similar vascular patterns as wild-type littermates (G, left) except for reduced vasculature development in the heart (G, arrow). (H,I) X-gal staining of transverse sections of E10.5 neural tubes in wild-type (H) and mutant embryos (I) with the Tie2:lacZ reporter gene reveals the vasculature (blue) and cell nuclei labeled with Nuclear Fast Red (pink). While perineural plexuses are present in both the wild type and the mutant (H,I, arrowheads), there is no apparent penetration of vessels into the neural tube in the mutant when compared with the wild-type embryo (H, arrows). Note that the floor plate in the mutant (I, *) is absent. cp, chorionic plate; lt, labyrinthine trophoblast; lbr, labyrinthine layer; iv, intersomitic vessel; pnp, perineural plexus; V, ventricle; NE, neuroepithelium. (J,K) Hematoxylin and Eosin staining of E10.5 yolk sac showing presence of endothelial cells (e), mesothelial cells (m), blood cells (b) and endoderm cells (n) in both wild type (J) and mutant (K). Scale bars: 100 µm in A,B,H,I (500 µm in insets); 150 µm in J,K.

 


View larger version (110K):

[in a new window]
  		Fig. 4. Integrin ß8 expression in E9.5 placenta tissue. (A-D) Sections of E9.5 placenta hybridized with integrin ß8 antisense (A,C,D) or sense (B) probes showing localization of integrin ß8 to most of placenta tissues, notably in the trophoblast giant cells (A), labyrinthine layer and spongiotrophoblast layer (C). In the yolk sac, integrin ß8 appears to be expressed specifically in the endoderm cells (D, arrow). sp, spongiotrophoblast layer; lb, labyrinthine layer. Scale bar: 200 µm in C; 50 µm in D.

 


View larger version (63K):

[in a new window]
  		Fig. 5. Abnormal cavitation and radial glial organization in the brains of class B integrin ß8-deficient mutants. (A-H) Hematoxylin and Eosin staining of transverse sections of E11.5 brain (A-D) and E14.5 brain (E-H), showing abnormal cavitation in integrin ß8-deficient mutant brains (B,D,F,H, arrows) compared with the wild-type littermates (A,C,E,G). Hemorrhage is visible in the ganglionic eminence (B, arrowhead) and diencephalon (D, arrowhead) of an E11.5 mutant brain and becomes much more severe in the E14.5 mutant brain (F, arrow; H, arrowhead). (I-N) Radial glial organization characterized by immunohistochemical staining using the RC2 antibody. Radial glial cells look grossly normal in an E10.5 mutant brain (J) compared with a wild-type littermate (I). However, they are apparently disorganized in the ganglionic eminence (L, arrow) and diencephalon (N, arrow) in the E12.5 mutant brain when compared with the same regions of E12.5 wild-type brains (K,M). GE, ganglionic eminence; DI, diencephalon. Scale bars: 100 µm in A-H; 50 µm in I-N.

 


View larger version (70K):

[in a new window]
  		Fig. 6. Abnormal brain capillary morphologies in class B integrin ß8-deficient mutants. (A-D) Paraffin wax embedded sections of E12.5 (A,B) and E14.5 (C,D) brains stained with anti-laminin antibodies that show the abnormal morphologies of capillary vessels in the integrin ß8-deficient mutant (B,D) when compared with a wild-type littermate (A,C). Discontinuous basement membranes are indicated by arrows (B). Aggregates of capillary vessels are visible (D, arrow). (E-F) Projected confocal images of E12.5 brain capillary vessels double labeled with anti-PECAM antibody (green) and anti-FN (red) antibodies. Capillary vessels in an integrin ß8-deficient mutant (F1-3) exhibit irregular distended morphologies and are often conjoined when compared with those in wild-type littermates (E1-3). The basement membrane is discontinuous and a blood cell is captured at a potential hemorrhage site (F2, arrow) in the mutant. Note that a blood cell is present clearly outside of the capillaries, indicating hemorrhage in a nearby location (F3, arrow). (G,H) Pericytes recognized with anti-desmin are present and recruited to the capillaries in the brains of E12.5 wild-type (G, arrow) and mutant embryos (H, arrow). Scale bars: 50 µm in A-D; 20 µm in E,F; 100 µm in G,H.

 


View larger version (183K):

[in a new window]
  		Fig. 7. Abnormal endothelial cell morphology in class B integrin ß8-deficient brains. (A-D) Electron micrographs of capillary structure in E12.5 wild-type (A) and integrin ß8-deficient mutant (B-D) brains. In contrast to the wild type (A), the endothelial cells in the mutant display abundant active membrane protrusions (B, arrows) and large empty spaces surrounding the capillaries (B,C, *). In addition, fenestrations (D, arrows) are often seen in the mutant endothelium. P, pericyte; EC, endothelial cell. Scale bars: 5 µm in A-C; 1 µm in D.

 


View larger version (116K):

[in a new window]
  		Fig. 8. Abnormal capillary vascular patterning and endothelial cell hyperplasia in class B integrin ß8-deficient brains. (A-D) Projected confocal images of brain capillary vessels stained with anti-PECAM in E10.5 (A,B) and E12.5 (C,D) embryos. Wild-type capillary vessels grow close to and couple near the ventricle (A, broken line marks boundary of ventricle, V). However, capillary vessels extend a shorter distance into the neuroepithelium and couple further away from the ventricle in the mutant (B). At E12.5, wild-type capillary vessels have branched and anastomosed to form an organized network (C); while in the mutant, capillary vessels show bulbous distentions and have not invaded regions of neuroepithelium immediately adjacent to the ventricle (D, arrows). (E) A brain capillary stained for isolectin BS (staining endothelial cell, green), DAPI (staining nuclei, blue) and BrdU (labeling proliferating cell, red, arrows). (F) The quantification of endothelial cell nuclei per vessel cross section in E11.5 wild-type and mutant brains. The percentage of BrdU-labeled endothelial cell nuclei in total endothelial cell nuclei scored is shown in the histogram (n=3). The error bars represent the s.e.m. Wild-type and mutant values are significantly different (P<0.005). Scale bars: 20 µm in A,B; 100 µm in C,D; 25 µm in E.

 


View larger version (71K):

[in a new window]
  		Fig. 9. Integrin ß8 is expressed in periventricular cells of the embryonic brain. (A,B) Whole-mount in situ hybridization analyses shows integrin ß8 mRNA expression in tissues surrounding the ventricles of E10.5 embryonic brain (A, antisense; B, sense). (C,D) Transverse sections of E12.5 brains hybridized with integrin ß8 antisense (C) or sense (D) probes showing localization of integrin ß8 to periventricular cells in the neuroepithelium. (E,F) Similar areas to those shown in C at E12.5 (C, box E and box F) are shown at a higher magnification in an E13.5 brain hybridized with an integrin ß8 antisense probe (E,F). Note the absence of integrin ß8 mRNA from the vascular cells of the brain (A) and inside of the brain area (C,E). Scale bar: 500 µm in C and D; 200 µm in E,F.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002