No abstract

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.

The p75NTR neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75NTR, many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75NTR (p75NTR-FX) allele made by flanking exons 4–6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75NTR/Wnt1-Cre mutants and conventional p75NTR null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75NTR in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75NTR mutants. This novel conditional p75NTR allele will offer new opportunities to investigate the role of p75NTR in specific tissues and cells.

Bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly in humans. Despite recent advances, the molecular basis of BAV development is poorly understood. Previously it has been shown that mutations in the Notch1 gene lead to BAV and valve calcification both in human and mice, and mice deficient in Gata5 or its downstream target Nos3 have been shown to display BAVs. Here we show that tissue-specific deletion of the gene encoding Activin Receptor Type I (Alk2 or Acvr1) in the cushion mesenchyme results in formation of aortic valve defects including BAV. These defects are largely due to a failure of normal development of the embryonic aortic valve leaflet precursor cushions in the outflow tract resulting in either a fused right- and non-coronary leaflet, or the presence of only a very small, rudimentary non-coronary leaflet. The surviving adult mutant mice display aortic stenosis with high frequency and occasional aortic valve insufficiency. The thickened aortic valve leaflets in such animals do not show changes in Bmp signaling activity, while Map kinase pathways are activated. Although dysfunction correlated with some pro-osteogenic differences in gene expression, neither calcification nor inflammation were detected in aortic valves of Alk2 mutants with stenosis. We conclude that signaling via Alk2 is required for appropriate aortic valve development in utero, and that defects in this process lead to indirect secondary complications later in life.

No abstract

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (‘persistent truncus arteriosus’ or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.

We have read with interest the recent r e v i e w~( I ,~,~) drawing the attention of a wider audience to the process of trabeculation during embryonic heart morphogenesis. Whilst welcoming this, we are concerned that some of what has been written could be confusing and misleading to a reader not familiar with embryonic hearts.Trabeculation is a morphogenetic process undergone by the luminal side of some parts of the myocardium (muscle wall) of the developing vertebrate heart. We think that the structure of trabeculated myocardium is not well described by 'finger-like projections' as is often used (e.g. as in refs 1,2 and 3). Trabeculated embryonic ventricular myocardium more resembles sheets or a sponge-like structure (e.g. as in refs 4 3 , with no free 'finger-tips'. Although some cross-sectional views can look consistent with finger-or villus-like outgrowths, more careful examination, scanning electron microscopy (e.g. as in refs 4 and 5), confocal microscopy or three-dimensional reconstruction techniques clearly demonstrate its real nature.A second point of confusion is the idea that trabeculation is confined to the ventricular myocardium. This is not the case. Atrial myocardium also becomes 'trabeculated', forming a leaf vein-like structure@) on the luminal side of the wall. This process commences slightly later than in the ventricles -too late to have occurred (or not) in the hearts of 'knockout' mice, in which ventricular trabeculation is severely compromised by the absence of neuregulin or some of its receptor (reviewed in refs 1 and 2).There are various hypotheses about functions of trabeculation: to provide sufficient pumping function during growth and allow for nutrition of embryonic myocardium before coronary vascularisation (reviewed by ref. 7), and to separate blood currents in the pre-septation heart@). Myocardial architecture, particularly in the ventricle, undergoes considerable changes during embryonic and fetal development. Some trabeculated myocardium remains both in the ventricles (including as papillary muscles supporting the atrioventricular valve leaflets) and in the atria (as pectinate muscles) of the mature heart@).Knowledge and understanding of the molecular mechanisms underlying development are becoming increasingly detailed. For the most rapid advances of this type to be made in morphogenesis, we would like to encourage a similar degree of attention with respect to knowledge, under-Fig. 1. Scanning electron micrograph of a transverse slice through an embryonic chick heart (day 5 of incubation, HH stage 27), showing the abundant, sponge-like trabecular meshwork in the basal part of the right ventricle (RV) and developing pectinate muscles in the left atrium (LA).standing and accurate communication of the morphology of the developing tissue itself.