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First published online September 30, 2004
doi: 10.1242/10.1242/dev.01393

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A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels

¹ Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT, 06519-1361, USA
² Department of Comparative Medicine, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT, 06519-1361, USA
³ Department of Pathology, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT, 06519-1361, USA
⁴ Yale Child Health Research Center, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT, 06519-1361, USA
⁵ Department of Pediatrics, Bridgeport Hospital, Bridgeport, CT 06610, USA
⁶ Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (117K): [in a new window]   Fig. 1. Hhex–/– mice have multiple cardiovascular abnormalities. (A,B) 13.5 embryos and (C,D) whole hearts. (A,B) Compared to Hhex+/+ embryos, Hhex–/– embryos have no livers (asterisks), forebrain truncations (arrowheads), and markedly dilated blood vessels (arrows in B). Hearts in Hhex–/– mice at E13.5 have a small right ventricle (arrows in C and D). (E-P) Transverse sections of E13.5 embryos. (E) Section of Hhex+/+ heart inferior to outflow tracts showing normal morphology. (F) Hhex–/– heart with hypoplastic RV (arrow) and abnormal accumulation of ECCs (box). (G) Higher power view of boxed region in F reveals abnormal accumulation of cells in the atrio-ventricular cushion (AVC) region that are mesenchymal in morphology and represent ECCs. (H,I) RV outflow tract. (H) The Hhex+/+ heart has a small region of ECCs just inferior to the pulmonic valve (asterisk). The arrows indicate the region of the RV outflow tract that is comprised of ECCs. (I) The Hhex–/– heart shows a marked increase in ECCs with subsequent narrowing of RV outflow tract. Arrows indicate the region of RVOT that is comprised of ECCs and the asterisk denotes the pulmonic valve. (J) A more caudal section of the same heart as in I showing the aortic valve (asterisk) communicating with RV, indicating the presence of DORV. (K,L) AV valve region showing abnormal accumulation of ECCs and dysplastic AV valves in Hhex–/– mice compared to wild-type mice. (M-Q) Ventricles of Hhex+/+ and Hhex–/– hearts. In the Hhex–/– heart, a VSD (double arrow) is present and the myocardium is abnormally thin (O). High power view of boxed areas in (M) and (O) is shown in (N) and (P) and highlights the abnormally thin compact myocardial layer in Hhex–/– hearts. The thickness of the compact layer of the myocardium is indicated. Arrowheads point to epicardium. (Q) High power image through the myocardium of a severely affected embryo in which it is difficult to identify the presence any compact layer. RV, right ventricle; Ao, aorta; TV, tricuspid valve; MV, mitral valve; ECC, endocardial cushion cells; DORV, double outlet right ventricle; CL, compact layer; TL, trabecular layer.

View larger version (51K): [in a new window]   Fig. 2. The epicardium is present in Hhex–/– mice. Cross-sections through the ventricular region of E11.5 hearts from (A) Hhex+/+ and (B) Hhex–/– mice. Immunohistochemistry for pan-cytokeratin was performed. Green staining and arrows indicates the cells that are cytokeratin-positive and therefore epicardial cells.

View larger version (76K): [in a new window]   Fig. 3. AV cushion apoptosis is decreased in Hhex–/– mice. ECC apoptosis is decreased by 75% in the absence of Hhex (representative panels shown in A and B) while proliferation is unchanged (representative panels shown in C and D). Composite data are shown in the graphs on the far right (n=3 animals for each genotype). Bars show means+s.e.m. n=3 for each phenotype. ECC, endocardial cushion cells; Atr, atrium.

View larger version (101K): [in a new window]   Fig. 4. Hhex–/– embryos have defective angiogenesis. (A,D) Low-power view of whole E11.5 Hhex+/+ and Hhex–/– embryos stained with Pecam antibody. Hhex–/– embryos have a striking profusion of small vessels and disorganization of large vasculature throughout the whole embryo. (B,E) High-power view of cranial vascular detected with PECAM antibody. Note the disorganization of large vessels and the multiple ectopic small vessels that are present. Visualization of various focal planes indicates that the punctate staining represents small vessels branching off the larger vessels that are not in the plane of focus. Arrows indicate the same cranial vessel in each embryo for comparison. (C,F) The intersomitic vessels are also enlarged and have abnormal small branches (arrowheads).

View larger version (140K): [in a new window]   Fig. 5. Vascular abnormalities in Hhex–/– mice at E13.5. (A,B) Transverse sections through the neck region of E13.5 embryos show massive dilation of the internal jugular vein in Hhex–/– embryo compared to wild-type embryo. A and B are shown at the same magnification. Other examples of abnormal vascular structures include enlarged intercostal vessels (arrows) (C,D) and massive vascular lake (arrow) in the region of the septum transversum (F) in place of normal liver tissue and vascular structures (E). CCA, common carotid artery; IJV, internal jugular vein; TYR, thyroid; IA, intercostal artery.

View larger version (120K): [in a new window]   Fig. 6. The defect in angiogenesis in Hhex–/– mice includes abnormal VSMC development. Whole-mount -SMA immunohistochemistry of E11.5 embryos. (A,D,G) Wild-type embryo. (B,E,H) Hhex–/– embryo with mild phenotype. (C,F,I) Hhex–/– with severe phenotype. In the mildly affected embryos, decreased -SMA staining is seen in the cranial vasculature (E) and the dorsal aorta (H). Arrows in (D) and (E) highlight the same vascular branches in both wild-type and Hhex–/– embryos, showing the absence of VSMCs in some vessels. Arrowheads in (G) and (H) indicate the VSMCs in the dorsal aorta. Note the abnormal pattern of -SMA staining in the Hhex–/– aorta. In severely affected embryos, there are no VSMCs present in the cranial vessels (F) and a dramatically decreased number are present in the dosal aorta (I), where the pattern of -SMA staining is decreased and very irregular.

View larger version (13K): [in a new window]   Fig. 7. Vegfa levels are increased in Hhex–/– hearts during cardiac morphogenesis. Vegfa levels were determined by ELISA at the gestational ages indicated. n=5 hearts for each age. Each value was determined in duplicate. Bars show means+s.e.m. Means were compared using Student's t-test.

View larger version (111K): [in a new window]   Fig. 8. The spatial expression pattern of Vegfa expression is not altered in Hhex–/– embryos. (A) and (B) Low-power views of sagittal sections of E9.5 embryos stained with Hoecsht (blue staining of cell nuclei). Higher-power views of the areas boxed in (A) and (B) are shown as indicated. Green staining represents presence of Vegfa. In the heart of both Hhex+/+ (C) and Hhex–/– (D) embryos, Vegfa is expressed in both the myocardium and endocardium. Vegfa is also highly expressed in the developing gut endoderm of both Hhex+/+ (E) and Hhex–/– (F) embryos.

View larger version (69K): [in a new window]   Fig. 9. E10.5 AVC explants from Hhex–/– hearts show increased EMT that is blocked by inhibiting Vegf signaling. (A) Top six panels show light microscopic images of E10.5 AVC explants from C57Bl/6J mice, wild-type mice from the Hhex+/– intercrosses, and Hhex–/– mice cultured for 72 hours showing increased numbers of transformed mesenchymal cells in the Hhex–/– explants. The bottom two panels are representative confocal images of endocardial cell outgrowths immunstained for -SMA. In the wild-type explant (left panel), the cells are more epitheliod in morphology with relatively few mesenchymal cells. In the Hhex–/– explants, most of the cells are spindle-shaped mesenchymal cells that exhibit little cell-cell contact. Thus, there is ongoing EMT of AVC explants in the absence of Hhex at E10.5, a gestational age when EMT is usually complete. (B) Confocal images of E10.5 AVC explants immunostained for -SMA in untreated wild-type explants (left panel), untreated Hhex–/– explants (middle panel), and in Hhex–/– explants treated with 25 µg/ml s-Flt (right panel). Below each panel is a corresponding z-plane image showing the distance cells have moved into the collagen gel, which is indicative of the invasive capacity of the cells. The broken line represents the top of the collagen gel. These panels show that wild-type cells at E10.5 are epithelioid in morphology, maintain close cell-cell contact, and do not invade the collagen gel. Hhex–/– cells undergo extensive transformation into spindle-shaped mesenchymal cells, maintain little cell-cell contact, and show extensive migration into the collage gel (arrows). Treatment of Hhex–/– explants with s-Flt reverts the cell morphology to the wild-type phenotype as evidenced by rounded cells with extensive cell-cell contact and no invasion of the collagen gel.

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