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First published online 8 April 2004
doi: 10.1242/dev.01091

Development 131, 2195-2204 (2004)
Published by The Company of Biologists 2004
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Extra-embryonic vasculature development is regulated by the transcription factor HAND1

Yuka Morikawa1 and Peter Cserjesi1,2,*

1 Department of Cell Biology and Anatomy, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
2 Molecular and Human Genetics Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA



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  		Fig. 1. Targeting strategy and expression pattern of Hand1. (A) A region of the first exon of the Hand1 gene was replaced with the lacZ gene to monitor Hand1 expression. The coding region is shown as black shading and the 5' and 3' untranslated regions shown as gray shading. The targeting vector is shown in the middle. The lacZ gene was inserted in frame with the initiating methionine of the Hand1 gene and PGKneo gene was inserted in the opposite orientation to the transcription of Hand1. The MC1TK cassette was linked to the 3' untranslated region of Hand1 to provide negative selection with FIAU. (B-E) Hand1 expression is recapitulated by ß-gal expression during Hand1ß-gal/+ mouse development at 7.75 dpc (B), 10.5 dpc (C), 12.5 dpc (D) and 14.5 dpc (E). Embryos in D and E were cleared using benzyl alcohol and benzyl benzoate (1:2 ratio). ad, adrenal gland; h, heart; ht, heart tube; g, gut; lm, lateral mesoderm; m, mandible and tongue; scg, superior cervical ganglia; st, sympathetic trunk; t, thyroid; u, umbilical cord and gut.

 


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  		Fig. 2. Hand1-null embryos fail to develop an extra-embryonic vasculature. (A,C) A 9.5 dpc wild-type mouse embryo shows a well-developed vasculature within the yolk sac filled with red blood cells. (B,D,E) A 9.5 dpc Hand1ß-gal/ß-gal embryo appears to be devoid of a vasculature containing red blood cells within the yolk sac. Arrowheads indicate red blood cells collecting within the yolk sac. (C-E) ß-Gal staining. (F) Yolk sac section of 9.5 dpc wild-type mouse showing blood vessels between the endodermal and mesodermal layers (arrowheads). HAND1 expression is restricted to the mesodermal cells in the yolk sac (blue staining). (G) Yolk sac section of 9.5 dpc Hand1ß-gal/ß-gal yolk sac showing a disorganized mesodermal layer. a, allantois; em, extra-embryonic membrane; fg, foregut.

 


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  		Fig. 3. Distribution of endothelial cells in Hand1 mutant yolk sacs. Hand1 is expressed in the mesodermal layer of the yolk sac but not in differentiated endothelial cells. (A-F) Co-localization of PECAM protein and Hand1 expression. (G,H) Phase contrast images of A-F. Immunohistochemistry was performed with yolk sac sections of 8.5 dpc Hand1ß-gal/+ (A,C,E,G) and 9.5 dpc Hand1ß-gal/ß-gal (B,D,F,H). Anti-ß-gal antibody was used to visualize HAND1-expressing cells (A,B). Both yolk sacs expressed PECAM (C,D). end, extra-embryonic endoderm; mes, extra-embryonic mesoderm. Asterisks in Gindicate blood vessels. (I,J) Visualization of tissues expressing PECAM in yolk sacs. Whole-mount staining of 8.5 dpc Hand1+/+ (I), 9.5 dpc Hand1ß-gal/ß-gal (J) and 9.5 dpc Hand1+/+ littermate (K) yolk sacs with anti-PECAM antibody. Wild-type yolk sacs show integration into large vessels in both 8.5 dpc and 9.5 dpc (arrowheads). Hand1ß-gal/ß-gal yolk sac lacks integrated vasculature.

 


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  		Fig. 4. RT-PCR analysis of angiogenic genes in Hand1ß-gal/ß-gal and Hand1+/+ extra-embryonic membranes. cDNA was synthesized from RNA extracted from the individual extraembryonic membranes of 8.5 dpc Hand1+/+ embryos and 9.5 dpc Hand1ß-gal/ß-gal and 9.5 dpc Hand1+/+ littermates. Semi-quantitative RT-PCR was performed with synthesized cDNA. Expression of growth factor Vegf and its receptors are shown on the left (top). Expression of angiogenic growth factor Ang1 and its receptors are shown on the left (bottom). Expression of Hand1 was monitored to confirm our genotype analysis. Expression of related bHLH factor Hand2 was also examined. Vegf, Flt1, Flk1, Npr1, Ang1, Efnb2, Bmp4, Notch1, Notch4, Hey1 and Hand2 were upregulated in Hand1ß-gal/ß-gal extraembryonic membranes. Gapd was used to normalize each sample.

 


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  		Fig. 5. Hand1 expression in yolk sacs is not regulated by Hand2. The expression of Hand1 was examined in yolk sacs of Hand1ß-gal/+Hand2-/- mice by staining for ß-gal activity. Wild-type (A) and Hand2-null embryo (B) showed similar expression patterns of Hand1. Hand1 was broadly expressed in the mesoderm of Hand2 null yolk sacs (C). All embryos are to the same scale. ht, heart; lm, lateral mesoderm; ys, yolk sac.

 


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  		Fig. 6. Distribution of SMCs in yolk sacs. Hand1 is required for the SMC in yolk sacs to migrate from the mesoderm and surround the endothelial tubes of the developing vasculature. Sections of yolk sacs from 8.5 dpc Hand1ß-gal/+ (A,D,G,J) and 9.5 dpc Hand1ß-gal/ß-gal mice (B,C,E,F,H,I,K,L) were stained with anti-ß-gal antibody to visualize Hand1 expression (A-C) and anti-SMA antibody to visualize SMCs (D-F). SMCs were located in the peri-endothelial and peri-endodermal cells in Hand1ß-gal/+ yolk sac (D). In Hand1ß-gal/ß-gal yolk sacs, SMCs were rarely found in regions surrounding the developing vasculature (E). (G-I) Merged images of A-F. (J-L) Phase contrast images of A-I. Instead, SMCs were scattered in clusters in extra-embryonic mesoderm of Hand1ß-gal/ß-gal yolk sacs (F). end, extra-embryonic endoderm; mes, extra-embryonic mesoderm. Asterisk indicates blood vessel. C,F,I,L are images of Hand1ß-gal/ß-gal extra-embryonic mesoderm only.

 


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  		Fig. 7. The yolk sacs of Hand1ß-gal/ß-gal contain differentiated SMC and express genes required for SMC recruitment. Semi-quantitative RT-PCR was performed with cDNA from extra-embryonic membranes of 8.5 dpc Hand1+/+ embryos and 9.5 dpc Hand1ß-gal/ß-gal and 9.5 dpc Hand1+/+ littermates. (A) Analysis of SMC specific differentiation markers. Early and late markers of SMC differentiation were expressed in Hand1ß-gal/ß-gal extra-embryonic membranes. (B) Analysis of SMC migration/recruitment signaling genes. Expression of these genes was not significantly different between wild type and mutant.

 

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© The Company of Biologists Ltd 2004