Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.     

Developmental patterning deciphered in avian chimeras

I started my scientific carer by investigating the development of the digestive tract in the laboratory of a well-known embryologist, Etienne Wolff, then professor at the Collège de France. My animal model was the chick embryo. The investigations that I pursued on liver development together with serendipity, led me to devise a cell-marking technique based on the construction of chimeric embryos between two closely related species of birds, the Japanese quail ( Coturnix coturnix japonica ) and the chick ( Gallus gallus ).
The possibility to follow the migration and fate of the cells throughout development from early embryonic stages up to hatching and even after birth, was a breakthrough in developmental biology of higher vertebrates.
This article describes some of scientific achievements based on the use of this technique in my laboratory during the last 38 years.     

Origins of anteroposterior patterning and Hox gene regulation during chordate evolution

All chordates share a basic body plan and many common features of early development. Anteroposterior (AP) regions of the vertebrate neural tube are specified by a combinatorial pattern of Hox gene expression that is conserved in urochordates and cephalochordates. Another primitive feature of Hox gene regulation in all chordates is a sensitivity to retinoic acid during embryogenesis, and recent developmental genetic studies have demonstrated the essential role for retinoid signalling in vertebrates. Two AP regions develop within the chordate neural tube during gastrulation: an anterior 'forebrain-midbrain' region specified by Otx genes and a posterior 'hindbrain-spinal cord' region specified by Hox genes. A third, intermediate region corresponding to the midbrain or midbrain-hindbrain boundary develops at around the same time in vertebrates, and comparative data suggest that this was also present in the chordate ancestor. Within the anterior part of the Hox-expressing domain, however, vertebrates appear to have evolved unique roles for segmentation genes, such as Krox-20, in patterning the hindbrain. Genetic approaches in mammals and zebrafish, coupled with molecular phylogenetic studies in ascidians, amphioxus and lampreys, promise to reveal how the complex mechanisms that specify the vertebrate body plan may have arisen from a relatively simple set of ancestral developmental components.     

Contribution of BDNF-mediated inhibition in patterning avian skin innervation

Multiple factors are involved in the development and regulation of sensory innervation in skin. The findings we report here suggest that brain-derived neurotrophic factor (BDNF)-mediated inhibition may play an important role in determining the pattern of sensory innervation in avian skin. In birds, cutaneous innervation is restricted to dermis, where axons form a ring of innervation around the base of each feather. Here we show that both BDNF message and protein are more abundant in avian epidermis than dermis when innervation is being established; the BDNF in dermis is localized to feather buds. In vitro, BDNF caused growth cones of NGF-dependent dorsal root ganglion neurons to collapse. Similarly, outgrowth of neurites toward BDNF-secreting fibroblasts was inhibited. The inhibitory effects of BDNF appear to be mediated by the low-affinity p75 neurotrophin receptor, rather than a trk receptor. Thus, the distribution of BDNF in embryonic avian skin and the inhibitory effects of BDNF on cutaneous neurites in vitro suggest that BDNF may be important in restricting axons from entering the epidermis and the core of feather buds during development in vivo.     

Early arterial differentiation and patterning in the avian embryo model

Of the many models to study vascular biology the avian embryo remains an informative and powerful model system that has provided important insights into endothelial cell recruitment, assembly and remodeling during development of the circulatory system. This review highlights several discoveries in the avian system that show how arterial patterning is regulated using the model of dorsal aortae development along the embryo midline during gastrulation and neurulation. These discoveries were made possible through spatially and temporally controlled gain-of-function experiments that provided direct evidence that BMP signaling plays a pivotal role in vascular recruitment, patterning and remodeling and that Notch-signaling recruits vascular precursor cells to the dorsal aortae. Importantly, BMP ligands are broadly expressed throughout embryos but BMP signaling activation region is spatially defined by precisely regulated expression of BMP antagonists. These discoveries provide insight into how signaling, both positive and negative, regulate vascular patterning. This review also illustrates similarities of early arterial patterning along the embryonic midline in amniotes both avian and mammalians including human, evolutionarily specialized from non-amniotes such as fish and frog.     

Myocardialization of the cardiac outflow tract.

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally, (vi) ventricular myocardium did not produce factors capable of supporting myocardialization.     

Varix of the heart causing outflow tract obstruction.

We report a case about a neonate who died of severe subaortic stenosis due to a giant vascular dilation of the left ventricular outflow tract. We emphasize the fatal result of this benign lesion and make differential diagnosis with haemangiomas and valvular blood cysts.     

Left atrial endocardium and prostacyclin.

In patients with rheumatic mitral stenosis, intracardiac thrombi are found mostly, for reasons still unknown, in the left atrium. We compared the release of PGI2 from the endocardium of the left atrium with that of the right ventricle and from the endothelium of the pulmonary arteries. Endocardial endothelial cells (EECs) were isolated from right ventricles (RV) and left atrial appendages (LAA) of porcine hearts, and vascular endothelial cells (VECs) from pulmonary arteries (PA) were obtained from the same animals. Cultured EEC and PA-VEC monolayers were placed in a pressure loading apparatus and incubated for 30 min under various pressures. After incubation, the supernatants were sampled and the 6-keto-PGF1 alpha contents measured. PGI2 release from LAA-EEC was much less than from RV-EEC or from PA-VEC. Moreover, transmural pressure did not enhance PGI2 release from LAA-EEC, although it did from RV-EEC and PA-EEC in a pressure-dependent manner. These results may explain why the left atrium is a common site for intracardiac thrombus formation in patients with mitral valve disease.     

Acquired left coronary artery fistula to right ventricular outflow tract

A 59-year-old asymptomatic male was referred to our hospital for evaluation 44 years after surgical correction of a Fallot’s tetralogy. Transthoracic echocardiography showed a good surgical result with only a mild subvalvular pulmonary stenosis and mild pulmonary regurgitation. However, in the parasternal short axis a diastolic colour Doppler flow was seen in the right ventricular outflow tract with a maximum velocity over 4 m/s (figure 1). Pulmonary regurgitation seemed very unlikely because of the high velocity in the absence of elevated pulmonary artery pressure. Because a fistula was suspected, a multislice computer tomography scan was performed.     

Foodborne Salmonella ecology in the avian gastrointestinal tract

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.