Regulation of aortic morphogenesis and VE-cadherin dynamics by VEGF.

In amniote vertebrates, the definitive dorsal aorta is formed by the fusion of two primordial aortic endothelial tubes. Formation of the definitive dorsal aorta requires extensive cellular migrations and rearrangements of the primordial tubes in order to generate a single vessel located at the embryonic ventral midline. This study examines the role of VEGF signaling in the generation of the definitive dorsal aorta. Through gain- and loss-of-function studies in vivo in the chick embryo, we document a requirement for VEGF signaling in growth and remodeling of the paired primordia. We find that regions of the aorta are differentially sensitive to levels of VEGF signaling, and present evidence that areas of low blood flow are more sensitive to the loss of VEGF signaling. We also find that VEGF signaling regulates the intracellular distribution between membrane and cytoplasm of the cell-cell adhesion molecule VE-cadherin in aortic endothelial cells in vivo. Together, these finding identify mechanisms that likely contribute to the dynamic behavior of endothelial cells during aorta morphogenesis.

Hedgehog Signaling Regulates Epithelial Morphogenesis to Position the Ventral Embryonic Midline.

Despite much progress toward understanding how epithelial morphogenesis is shaped by intra-epithelial processes including contractility, polarity, and adhesion, much less is known regarding how such cellular processes are coordinated by extra-epithelial signaling. During embryogenesis, the coelomic epithelia on the two sides of the chick embryo undergo symmetrical lengthening and thinning, converging medially to generate and position the dorsal mesentery (DM) in the embryonic midline. We find that Hedgehog signaling, acting through downstream effectors Sec5 (ExoC2), an exocyst complex component, and RhoU (Wrch-1), a small GTPase, regulates coelomic epithelium morphogenesis to guide DM midline positioning. These effects are accompanied by changes in epithelial cell-cell alignment and N-cadherin and laminin distribution, suggesting Hedgehog regulation of cell organization within the coelomic epithelium. These results indicate a role for Hedgehog signaling in regulating epithelial morphology and provide an example of how transcellular signaling can modulate specific cellular processes to shape tissue morphogenesis.

Disruption of the aortic wall by coelomic lining-derived mesenchymal cells accompanies the onset of aortic hematopoiesis.

In vertebrates, definitive hematopoietic stem cells (HSCs) first emerge in the ventral wall of the aorta in the Aorta-Gonad-Mesonephros (AGM) region of the embryo, where they differentiate from a specialized type of endothelium termed Hemogenic Endothelium (HE). While the transition from HE to hematopoietic tissue has received much experimental attention, much less is known regarding generation of HE itself. The current study investigates the emergence of the HE in the chick embryo aorta. Using the HE marker Runx1 as well as a new chicken-reactive antibody to the endothelial marker VE-Cadherin, we document the relationship between the emerging HE and surrounding tissues, particularly the coelomic epithelium (CE) and CE-derived sub-aortic mesenchyme. In addition, the fate of the CE cells was traced by electroporation of a GFP-expressing plasmid into the CE, followed by analysis using immunofluorescence and in situ hybridization. We make the novel observation that CE-derived mesenchyme transiently invades through the ventral wall of the aorta during the period of establishment of HE and just prior to the emergence of hematopoietic cell clusters in the ventral aortic wall. These observations emphasize a hitherto unappreciated dynamism in the aortic wall during the period of HE generation, and open the door to future studies regarding the role of invasive CE-derived cells during aortic hematopoiesis.

Nkx2.5 marks angioblasts that contribute to hemogenic endothelium of the endocardium and dorsal aorta.

Novel regenerative therapies may stem from deeper understanding of the mechanisms governing cardiovascular lineage diversification. Using enhancer mapping and live imaging in avian embryos, and genetic lineage tracing in mice, we investigated the spatio-temporal dynamics of cardiovascular progenitor populations. We show that expression of the cardiac transcription factor Nkx2.5 marks a mesodermal population outside of the cardiac crescent in the extraembryonic and lateral plate mesoderm, with characteristics of hemogenic angioblasts. Extra-cardiac Nkx2.5 lineage progenitors migrate into the embryo and contribute to clusters of CD41+/CD45+ and RUNX1+ cells in the endocardium, the aorta-gonad-mesonephros region of the dorsal aorta and liver. We also demonstrated that ectopic expression of Nkx2.5 in chick embryos activates the hemoangiogenic gene expression program. Taken together, we identified a hemogenic angioblast cell lineage characterized by transient Nkx2.5 expression that contributes to hemogenic endothelium and endocardium, suggesting a novel role for Nkx2.5 in hemoangiogenic lineage specification and diversification.

Establishment of the Visceral Embryonic Midline Is a Dynamic Process that Requires Bilaterally Symmetric BMP Signaling.

The vertebrate body plan contains both dorsal and ventral midline structures. While dorsal midline structures have been extensively studied, formation of ventral midline structures, and how they become aligned with the dorsal midline, is a fundamental aspect of vertebrate development that is poorly understood. This study uses the chick dorsal mesentery (DM) as a model for investigating the formation of ventral midline structures. We document formation of the DM by epithelial-to-mesenchymal transition (EMT) and medial ingression of the lateral plate coelomic lining and show that DM positioning is a fundamentally dynamic process regulated by relative levels of bone morphogenetic protein (BMP) signaling in the two sides of the ingressing lateral plate. Disruption of this process causes misalignment of the DM and disturbances during initial stages of lung morphogenesis. Since the dorsal midline is a source of BMP antagonists, these results suggest a mechanism for aligning the dorsal and ventral embryonic midlines.

Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development.

During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.

Wnt signaling orients the proximal-distal axis of chick kidney nephrons.

The nephron is the fundamental structural and functional unit of the kidney. Each mature nephron is patterned along a proximal-distal axis, with blood filtered at the proximal end and urine emerging from the distal end. In order to filter the blood and produce urine, specialized structures are formed at specific proximal-distal locations along the nephron, including the glomerulus at the proximal end, the tubule in the middle and the collecting duct at the distal end. The developmental processes that specify these different nephron segments are not fully understood. Wnt ligands, which are expressed in the nephric duct and later in the nascent nephron itself, are well-characterized inducers of nephrons, and are both required and sufficient for initiation of nephron formation from nephrogenic mesenchyme. Here, we present evidence that Wnt signaling also patterns the proximal-distal nephron axis. Using the chick mesonephros as a model system, a Wnt ligand was ectopically expressed in the coelomic lining, thereby introducing a source of Wnt signaling that is at right angles to the endogenous Wnt signal of the nephric duct. Under these conditions, the nephron axis was re-oriented, such that the glomerulus was always located at a position farthest from the Wnt sources. This re-orientation occurred within hours of exposure to ectopic Wnt signaling, and was accompanied initially by a repression of the early glomerular podocyte markers Wt1 and Pod1, followed by their re-emergence at a position distant from the Wnt signals. Activation of the Wnt signaling pathway in mesonephric explant cultures resulted in strong and specific repression of early and late glomerular markers. Finally, cytoplasmic ß-catenin, indicative of active canonical Wnt signaling, was found to be enriched in the distal as compared with the proximal region of the forming nephron. Together, these data indicate that Wnt signaling patterns the proximal-distal axis of the nephron, with glomeruli differentiating in regions of lowest Wnt signaling.