Arterial dysgenesis and limb defects : Clinical and experimental examples

Acknowledgements This article is dedicated to Dr David S. Packard Jr. With thanks to Dr John DeSesso, Dr Lewis B. Holmes, Dr Mark Levinsohn, Dr David S. Packard Jr, Prof Lewis Wolpert for discussions on vascular disruption, particularly arterial dysgenesis and limb defects. We apologise to the many authors whose work we were unable to cite due to space limitations. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Peer reviewedPostprin

Engineering vascularised tissues in vitro

Tissue engineering aims at replacing or regenerating tissues lost due to diseases or traumas (Langer and Vacanti, 1993). However, mimicking in vitro the physiological complexity of vascularized tissue is a major obstacle, which possibly contributes to impaired healing in vivo. In higher organisms, native features including the vascular network, the lymphatic networks and interstitial flow promote both mass transport and organ development. Attempts to mimic those features in engineered tissues will lead to more clinically relevant cell-based therapies. Aside from current strategies promoting angiogenesis from the host, an alternative concept termed prevascularization is emerging. It aims at creating a biological vasculature inside an engineered tissue prior to implantation. This vasculature can rapidly anastamose with the host and enhances tissue survival and differentiation. Interestingly, growing evidence supports a role of the vasculature in regulating pattern formation and tissue differentiation. Thus, prevascularized tissues also benefit from an intrinsic contribution of their vascular system to their development. From those early attempts are emerging a body of principles and strategies to grow and maintain, in vitro, those self-assembled biological vascular networks. This could lead to the generation of engineered tissues of more physiologically relevant complexity and improved regenerative potential

Congenital anomalies from a physics perspective. The key role of "manufacturing" volatility

Genetic and environmental factors are traditionally seen as the sole causes of congenital anomalies. In this paper we introduce a third possible cause, namely random "manufacturing" discrepancies with respect to ``design'' values. A clear way to demonstrate the existence of this component is to ``shut'' the two others and to see whether or not there is remaining variability. Perfect clones raised under well controlled laboratory conditions fulfill the conditions for such a test. Carried out for four different species, the test reveals a variability remainder of the order of 10%-20% in terms of coefficient of variation. As an example, the CV of the volume of E.coli bacteria immediately after binary fission is of the order of 10%. In short, ``manufacturing'' discrepancies occur randomly, even when no harmful mutation or environmental factors are involved. Not surprisingly, there is a strong connection between congenital defects and infant mortality. In the wake of birth there is a gradual elimination of defective units and this screening accounts for the post-natal fall of infant mortality. Apart from this trend, post-natal death rates also have humps and peaks associated with various inabilities and defects.\qL In short, infant mortality rates convert the case-by-case and mostly qualitative problem of congenital malformations into a global quantitative effect which, so to say, summarizes and registers what goes wrong in the embryonic phase. Based on the natural assumption that for simple organisms (e.g. rotifers) the manufacturing processes are shorter than for more complex organisms (e.g. mammals), fewer congenital anomalies are expected. Somehow, this feature should be visible on the infant mortality rate. How this conjecture can be tested is outlined in our conclusion.Comment: 43 pages, 9 figure

THE EFFECTS OF POLYCHLORINATED BIPHEYNLS (PCBs) ON AVIAN CARDIAC DEVELOPMENT

Polychlorinated biphenyls (PCBs) are a class of synthetic organochlorines that are thermally stable, resistant to degradation, and persistent in the environment as a result of bioaccumulation and intermittent redistribution through trophic levels. These compounds were sold commercially as mixtures in the twentieth century and later banned due to their biological toxicity. There are 209 known PCB congeners, each with different toxicities and physical properties that cause a variety of adverse health effects. Moreover, the effects of PCB mixtures vary with exposure concentrations, PCB congener toxicity, and species sensitivity. However, limited information is available about the impact of PCBs on the development of the embryonic cardiovascular system. There is a major site of contamination along the upper Hudson River in New York; wildlife in that region have shown evidence of exposure to PCBs. The purpose of this research was to determine the impact of embryonic exposure to a PCB mixture and a single congener, both found in the upper Hudson River on the developing avian cardiovascular system. In study 1, tree swallow eggs (Tachycineta bicolor) were dosed with PCB 77 and incubated to hatch. Similarly, domestic chicken eggs (Gallus domesticus) were dosed with the PCB mixture at embryonic day zero and incubated to hatch in study 2. Eggs were monitored through incubation; other measures were taken at hatch along with tissue collection. Results showed that embryonic exposure to PCBs resulted in an absence of the ventricular wall compact layer and hypertrabeculation in tree swallow hatchlings in spite of no effect on survival. Embryonic exposure to a PCB mixture in domestic chickens resulted in compact layer absence as well as additional cardiomyopathies, including absence of the ventricular wall trabeculated layer, ventricular chamber dilation, abnormal heart wall and septal formations, and arrhythmias during embryonic development. In study 3, embryonic exposure to a PCB mixture was studied at Hamburger Hamilton stages 10, 16, and 20. Embryonic exposure to a PCB mixture resulted in abnormal proliferation of cardiomyocytes early in heart development. Dose-dependent mortality occurred in chicken embryos exposed to the PCB mixture. These results support other findings demonstrating PCB effects on the cardiovascular system. Further, these data showed dramatic adverse effects of the PCB mixture as well as a single congener found in the region of the upper Hudson River on the developing avian cardiac system

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids

International audienceThe amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution

Quantifying heart development

This thesis presents a series of papers on quantified heart development. It contains an atlas of human embryonic heart development, covering the first 8 weeks after conception. This atlas gives graphs of growth in size and volume of the various cardiac compartments. Such measures are still scarce in literature as illustrated in a review about ventricular wall development. The atlas also shows that by quantification of growth, new insights in developmental processes, such as sinus venosus incorporation can be gained. It, together with a series of ventricular wall growth curves covering foetal development, illustrates that a hypertrabeculated ventricle is the result of differential growth rather than a failure of compaction as has been presumed to underlie left ventricular non-compaction cardiomyopathy. Additionally, this thesis shows that trabecular myocardium is not necessarily weaker or ill-adapted to force generation compared to the compact wall as is assumed to be the case in aforementioned cardiomyopathy. Furthermore, quantification of atrioventricular canal growth on foetal ultrasounds lend support to the theory that aberrant atrioventricular canal development can lead to tricuspid valve agenesis. Finally, this thesis shows that there is a role for comparative anatomy, in a broader sense than just mouse and chicken, in understanding mammalian and human heart development by comparing a series of bird hearts from different species

Hypertension Programmed in Adult Hens by Isolated Effects of Developmental Hypoxia In Ovo.

In mammals, pregnancy complicated by chronic hypoxia can program hypertension in the adult offspring. However, mechanisms remain uncertain because the partial contributions of the challenge on the placenta, mother, and fetus are difficult to disentangle. Here, we used chronic hypoxia in the chicken embryo-an established model system that permits isolation of the direct effects of developmental hypoxia on the cardiovascular system of the offspring, independent of additional effects on the mother or the placenta. Fertilized chicken eggs were exposed to normoxia (N; 21% O2) or hypoxia (H; 13.5%-14% O2) from the start of incubation (day 0) until day 19 (hatching, ≈day 21). Following hatching, all birds were maintained under normoxic conditions until ≈6 months of adulthood. Hypoxic incubation increased hematocrit (+27%) in the chicken embryo and induced asymmetrical growth restriction (body weight, -8.6%; biparietal diameter/body weight ratio, +7.5%) in the hatchlings (all P<0.05). At adulthood (181±4 days), chickens from hypoxic incubations remained smaller (body weight, -7.5%) and showed reduced basal and stimulated in vivo NO bioavailability (pressor response to NG-nitro-L-arginine methyl ester, -43%; phenylephrine pressor response during NO blockade, -61%) with significant hypertension (mean arterial blood pressure, +18%), increased cardiac work (ejection fraction, +12%; fractional shortening, +25%; enhanced baroreflex gain, +456%), and left ventricular wall thickening (left ventricular wall volume, +36%; all P<0.05). Therefore, we show that chronic hypoxia can act directly on a developing embryo to program hypertension, cardiovascular dysfunction, and cardiac wall remodeling in adulthood in the absence of any maternal or placental effects.The British Heart Foundation The Wellcome Trus

Hypertension Programmed in Adult Hens by Isolated Effects of Developmental Hypoxia In Ovo.

In mammals, pregnancy complicated by chronic hypoxia can program hypertension in the adult offspring. However, mechanisms remain uncertain because the partial contributions of the challenge on the placenta, mother, and fetus are difficult to disentangle. Here, we used chronic hypoxia in the chicken embryo-an established model system that permits isolation of the direct effects of developmental hypoxia on the cardiovascular system of the offspring, independent of additional effects on the mother or the placenta. Fertilized chicken eggs were exposed to normoxia (N; 21% O2) or hypoxia (H; 13.5%-14% O2) from the start of incubation (day 0) until day 19 (hatching, ≈day 21). Following hatching, all birds were maintained under normoxic conditions until ≈6 months of adulthood. Hypoxic incubation increased hematocrit (+27%) in the chicken embryo and induced asymmetrical growth restriction (body weight, -8.6%; biparietal diameter/body weight ratio, +7.5%) in the hatchlings (all P<0.05). At adulthood (181±4 days), chickens from hypoxic incubations remained smaller (body weight, -7.5%) and showed reduced basal and stimulated in vivo NO bioavailability (pressor response to NG-nitro-L-arginine methyl ester, -43%; phenylephrine pressor response during NO blockade, -61%) with significant hypertension (mean arterial blood pressure, +18%), increased cardiac work (ejection fraction, +12%; fractional shortening, +25%; enhanced baroreflex gain, +456%), and left ventricular wall thickening (left ventricular wall volume, +36%; all P<0.05). Therefore, we show that chronic hypoxia can act directly on a developing embryo to program hypertension, cardiovascular dysfunction, and cardiac wall remodeling in adulthood in the absence of any maternal or placental effects.The British Heart Foundation The Wellcome Trus

Tissue-level Mechanisms Driving Cardiac Progenitor and Extracellular Matrix Movements during Early Vertebrate Heart Development

Vertebrate cardiogenesis involves heart progenitor cell movements from their initial lateral positions to the embryonic midline, where they assemble into a primitive heart. This early heart tube consists of an outer myocardium, a medial extracellular matrix (ECM), and an endocardial lining. Cardiac morphogenesis in avians and mammals is inseparable from development of the foregut, which provides molecular cues to regulate endocardial and myocardial differentiation from mesodermal progenitors. Concomitantly with the initiation of midline-directed cardiac progenitor movements, foregut endoderm undergoes dramatic folding and elongation. Following their initial assembly, the heart and foregut are transiently connected through a mesentery. Previous research focused on the molecular factors involved in guiding cardiac progenitors to the midline, yet cellular and tissue mechanisms coordinating these movements remain poorly understood. This work investigates movements of all three early heart constituents - the endocardial and myocardial progenitors, and surrounding ECM - in live quail embryos using a combination of time-lapse microscopy, chemical and mechanical perturbations, computational analysis and modeling. By visualizing the tissue environment for cell displacements, we distinguish the active (tissue-independent) movements from those cells undergo in a manner coordinated with the surrounding tissues. First, we analyzed the movements of endocardial progenitors and fluorescently-labeled ECM (fibronectin, fibrillin-2) fibrils. We found the bulk of midline-directed movement of pre-endocardial cells is coordinated with their surrounding ECM. Further, that ECM from extracardiac sources is transferred to and incorporated into the growing heart. By assessing the contributions of active cell motility to the observed midline endocardial displacements we found its role to be secondary to that of convective tissue movement within the anterior embryo. Second, we assessed myocardial progenitor movements relative to fibronectin ECM and endoderm. We discovered that observed antero-medial myocardial displacements are driven by a combination of: 1) medial tissue motion, and 2) anterior movement, accomplished via a coordinated deformation of myocardial progenitors, organized into a continuous epithelial sheet. Finally, we investigated the effects of VEGF overexposure on progenitor movements during early cardiogenesis. We found a dramatic VEGF-induced increase in cardiac inflow region size, which affected the coordinated movements/deformations displayed by myocardial progenitors, and resulted in heart tube elongation defects

Purification, characterization and molecular cloning of muscle paranemin

Paranemin is an incompletely characterized ~280 kilodalton protein previously identified and immunolocalized in embryonic chick skeletal muscle. Paranemin has been purified from the same tissue source, has the same molecular weight by SDS-PAGE, and has the same antibody localization at the Z-lines of adult avian cardiac muscle. The method developed for preparation of purified paranemin from embryonic (chick) skeletal muscle includes homogenization, centrifugation and gel filtration, hydroxyapatite, and DEAE-cellulose chromatography. By using this method, ~2 mg of purified paranemin was routinely obtained. Amino acid analysis revealed that paranemin has a high acidic to basic amino acid ratio, which agrees with the measured pI range of 4.1-4.5. When the purified protein was stained with a cationic carbocyanine dye, Stains-all, paranemin stained an intense blue, indicating it is a phosphoprotein and/or a glycoprotein. Further testing determined that paranemin is a glycoprotein. A monoclonal antibody (4D3) was made to use in one-and two-dimensional Western blots, which were used to identify paranemin throughout the purification procedure, and for immunofluorescence studies. Double-label confocal immunofluorescence showed colocalization of paranemin with desmin at the Z-lines of adult cardiac and skeletal muscle cells and at cardiac muscle intercalated disks;I determined the full-length cDNA sequence of paranemin by immunoscreening a [lambda]gt22 cDNA library from embryonic chick skeletal muscle with a monoclonal antibody specific for paranemin (4D3) and by hybridization screening. Northern blot analysis reveals a single transcript of 5.3 kb, which is much smaller than predicted from the size of paranemin (~280 kDa) by SDS-PAGE. The pI and molecular weight, predicted from the deduced amino acid sequence of paranemin, are 4.17 and 178,161 Daltons, respectively. I found that paranemin is a novel intermediate filament (IF) protein, which may be classified as a type VI IF protein. Paranemin contains the conserved IF rod domain (308 amino acids), which is 63.3% identical in amino acid sequence to the rod domain of tanabin and 45.5% identical to the rod domain of nestin. The partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, which overlap each other by 402 nucleotides, are almost identical to parts of the cDNA sequence of paranemin