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.

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.

Generation of the podocyte and tubular components of an amniote kidney: timing of specification and a role for Wnt signaling.

Kidneys remove unwanted substances from the body and regulate the internal body environment. These functions are carried out by specialized cells (podocytes) that act as a filtration barrier between the internal milieu and the outside world, and by a series of tubules and ducts that process the filtrate and convey it to the outside. In the kidneys of amniote vertebrates, the filtration (podocyte) and tubular functions are tightly integrated into functional units called nephrons. The specification of the podocyte and tubular components of amniote nephrons is currently not well understood. The present study investigates podocyte and tubule differentiation in the avian mesonephric kidney, and presents several findings that refine our understanding of the initial events of nephron formation. First, well before the first morphological or molecular signs of nephron formation, mesonephric mesenchyme can be separated on the basis of morphology and the expression of the transcription factor Pod1 into dorsal and ventral components, which can independently differentiate in culture along tubule and podocyte pathways, respectively. Second, canonical Wnt signals, which are found in the nephric duct adjacent to the dorsal mesonephric mesenchyme and later in portions of the differentiating nephron, strongly inhibit podocyte but not tubule differentiation, suggesting that Wnt signaling plays an important role in the segmentation of the mesonephric mesenchyme into tubular and glomerular segments. The results are discussed in terms of their broader implications for models of nephron segmentation.

A role for Vg1/Nodal signaling in specification of the intermediate mesoderm.

The intermediate mesoderm (IM) is the embryonic source of all kidney tissue in vertebrates. The factors that regulate the formation of the IM are not yet well understood. Through investigations in the chick embryo, the current study identifies and characterizes Vg1/Nodal signaling (henceforth referred to as 'Nodal-like signaling') as a novel regulator of IM formation. Excess Nodal-like signaling at gastrulation stages resulted in expansion of the IM at the expense of the adjacent paraxial mesoderm, whereas inhibition of Nodal-like signaling caused repression of IM gene expression. IM formation was sensitive to levels of the Nodal-like pathway co-receptor Cripto and was inhibited by a truncated form of the secreted molecule cerberus, which specifically blocks Nodal, indicating that the observed effects are specific to the Nodal-like branch of the TGFß signaling pathway. The IM-promoting effects of Nodal-like signaling were distinct from the known effects of this pathway on mesoderm formation and left-right patterning, a finding that can be attributed to specific time windows for the activities of these Nodal-like functions. Finally, a link was observed between Nodal-like and BMP signaling in the induction of IM. Activation of IM genes by Nodal-like signaling required an active BMP signaling pathway, and Nodal-like signals induced phosphorylation of Smad1/5/8, which is normally associated with activation of BMP signaling pathways. We postulate that Nodal-like signaling regulates IM formation by modulating the IM-inducing effects of BMP signaling.

Collective cell migration of the nephric duct requires FGF signaling.

BACKGROUND: During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood. RESULTS: A novel Gata3-enhancer-Gfp-based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path. CONCLUSIONS: FGF signaling acts as a "motor" that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157-167, 2015. © 2014 Wiley Periodicals, Inc.

Parallel waves of inductive signaling and mesenchyme maturation regulate differentiation of the chick mesonephros.

The mesonephros is a linear kidney that, in chicken embryos, stretches between the axial levels of the 15th to the 30th somites. Mesonephros differentiation proceeds from anterior to posterior and is dependent on signals from the nephric duct, which migrates from anterior to posterior through the mesonephric region. If migration of the nephric duct is blocked, markers of tubule differentiation, including Lhx1 and Wnt4, are not activated posterior to the blockade. However, activation and maintenance of the early mesonephric mesenchyme markers Osr1, Eya1 and Pax2 proceeds normally in an anterior-to-posterior wave, indicating that these genes are not dependent on inductive signals from the duct. The expression of Lhx1 and Wnt4 can be rescued in duct-blocked embryos by supplying a source of canonical Wnt signaling, although epithelial structures are not obtained, suggesting that the duct may express other tubule-inducing signals in addition to Wnts. In the absence of the nephric duct, anterior mesonephric mesenchyme adjacent to somites exhibits greater competence to initiate tubular differentiation in response to Wnt signaling than more posterior mesonephric mesenchyme adjacent to unsegmented paraxial mesoderm. It is proposed that mesonephric tubule differentiation is regulated by two independent parallel waves, one of inductive signaling from the nephric duct and the other of competence of the mesonephric mesenchyme to undergo tubular differentiation, both of which travel from anterior to posterior in parallel with the formation of new somites.

Analysis of nephric duct specification in the avian embryo.

Vertebrate kidney tissue exhibits variable morphology that in general increases in complexity when moving from anterior to posterior along the body axis. The nephric duct, a simple unbranched epithelial tube, is derived in the avian embryo from a rudiment located in the anterior intermediate mesoderm (IM) adjacent to somites 8 to 10. Using quail-chick chimeric embryos, the current study finds that competence to form nephric duct is fixed when IM precursor cells are still located in the primitive streak, significantly before the onset of duct differentiation. In the primitive streak, expression of the gene HoxB4 is associated with prospective duct IM, whereas expression of the more posterior Hox gene HoxA6 is associated with more posterior, non-duct-forming IM. Misexpression of HoxA6, but not of HoxB4, in prospective duct-forming regions of the IM resulted in repression of duct formation, suggesting a mechanism for the restriction of duct formation to the anterior-most IM. The results are discussed with respect to their implications for anterior-posterior patterning of kidney tissue and of mesoderm in general, and for the loss of duct-forming ability in more posterior regions of the IM that has occurred during vertebrate evolution.

A morphogenetic wave in the chick embryo lateral mesoderm generates mesenchymal-epithelial transition through a 3D-rosette intermediate.

Formation of epithelia through mesenchymal-epithelial transition (MET) is essential for embryonic development and for many physiological and pathological processes. This study investigates MET in vivo in the chick embryo lateral mesoderm, where a multilayered mesenchyme transforms into two parallel epithelial sheets that constitute the coelomic lining of the embryonic body cavity. Prior to MET initiation, mesenchymal cells exhibit non-polarized distribution of multiple polarity markers, albeit not aPKC. We identified an epithelializing wave that sweeps across the lateral mesoderm, the wavefront of which is characterized by the accumulation of basal fibronectin and a network of 3D rosettes composed of polarized, wedge-shaped cells surrounding a central focus of apical markers, now including aPKC. Initiation of the MET process is dependent on extracellular matrix-integrin signaling acting through focal adhesion kinase and talin, whereas progression through the rosette phase requires aPKC function. We present a stepwise model for MET, comprising polarization, 3D-rosette, and epithelialization stages.

An atypical basement membrane forms a midline barrier in left-right asymmetric gut development.

Correct intestinal morphogenesis depends on the early embryonic process of gut rotation, an evolutionarily conserved program in which a straight gut tube elongates and forms into its first loops. However, the gut tube requires guidance to loop in a reproducible manner. The dorsal mesentery (DM) connects the gut tube to the body and directs the lengthening gut into stereotypical loops via left-right (LR) asymmetric cellular and extracellular behavior. The LR asymmetry of the DM also governs blood and lymphatic vessel formation for the digestive tract, which is essential for prenatal organ development and postnatal vital functions including nutrient absorption. Although the genetic LR asymmetry of the DM has been extensively studied, a divider between the left and right DM has yet to be identified. Setting up LR asymmetry for the entire body requires a Lefty1+ midline barrier to separate the two sides of the embryo-without it, embryos have lethal or congenital LR patterning defects. Individual organs including the brain, heart, and gut also have LR asymmetry, and while the consequences of left and right signals mixing are severe or even lethal, organ-specific mechanisms for separating these signals are not well understood. Here, we uncover a midline structure composed of a transient double basement membrane, which separates the left and right halves of the embryonic chick DM during the establishment of intestinal and vascular asymmetries. Unlike other basement membranes of the DM, the midline is resistant to disruption by intercalation of Netrin4 (Ntn4). We propose that this atypical midline forms the boundary between left and right sides and functions as a barrier necessary to establish and protect organ asymmetry.

Promotion of avian endothelial cell differentiation by GATA transcription factors.

In the avian embryo, endothelial cells originate from several sources, including the lateral plate and somite mesoderm. In this study, we show that Gata transcription factors are expressed in the lateral plate and in vasculogenic regions of the avian somite and are able to promote a vascular endothelial fate when ectopically expressed in somite precursors. A fusion of GATA4 to the transcriptional activator VP16 promoted endothelium formation, indicating that GATA transcription factors promote vasculogenesis via activation of downstream targets, while a fusion of GATA4 to the transcriptional repressor engrailed repressed expression of Vascular Endothelial Growth Factor Receptor 2, a marker of endothelial precursors. These findings indicate a role for GATA transcription factors in the differentiation of the endothelium.

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.

Multimodality optical imaging of embryonic heart microstructure.

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

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.

Early molecular effects of ethanol during vertebrate embryogenesis.

Fetal alcohol spectrum disorder (FASD) is the combination of developmental, morphological, and neurological defects that result from exposing human embryos to ethanol (EtOH). Numerous embryonic structures are affected, leading to a complex viable phenotype affecting among others, the anterior/posterior axis, head, and eye formation. Recent studies have provided evidence suggesting that EtOH teratogenesis is mediated in part through a reduction in retinoic acid (RA) levels, targeting mainly the embryonic organizer (Spemann's organizer) and its subsequent functions. EtOH-treated Xenopus embryos were subjected to an analysis of gene expression patterns. Analysis of organizer-specific genes revealed a transient delay in the invagination of gsc- and chordin-positive cells that eventually reach their normal rostro-caudal position. Dorsal midline genes show defects along the rostro-caudal axis, lacking either their rostral (Xbra and Xnot2) or caudal (FoxA4b and Shh) expression domains. Head-specific markers like Otx2, en2, and Shh show abnormal expression patterns. Otx2 exhibits a reduction in expression levels, while en2 becomes restricted along the dorsal/ventral axis. During neurula stages, Shh becomes up-regulated in the rostral region and it is expressed in an abnormal pattern. These results and histological analysis suggest the existence of malformations in the brain region including a lack of the normal fore brain ventricle. An increase in the size of both the prechordal plate and the notochord was observed, while the spinal cord is narrower. The reduction in head and eye size was accompanied by changes in the eye markers, Pax6 and Tbx3. Our results provide evidence for the early molecular changes induced by EtOH exposure during embryogenesis, and may explain some of the structural changes that are part of the EtOH teratogenic phenotype also in FASD individuals.

Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels.

Fetal Alcohol Spectrum Disorder (FASD) is a set of developmental malformations caused by alcohol consumption during pregnancy. Fetal Alcohol Syndrome (FAS), the strongest manifestation of FASD, results in short stature, microcephally and facial dysmorphogenesis including microphthalmia. Using Xenopus embryos as a model developmental system, we show that ethanol exposure recapitulates many aspects of FAS, including a shortened rostro-caudal axis, microcephally and microphthalmia. Temporal analysis revealed that Xenopus embryos are most sensitive to ethanol exposure between late blastula and early/mid gastrula stages. This window of sensitivity overlaps with the formation and early function of the embryonic organizer, Spemann's organizer. Molecular analysis revealed that ethanol exposure of embryos induces changes in the domains and levels of organizer-specific gene expression, identifying Spemann's organizer as an early target of ethanol. Ethanol also induces a defect in convergent extension movements that delays gastrulation movements and may affect the overall length. We show that mechanistically, ethanol is antagonistic to retinol (Vitamin A) and retinal conversion to retinoic acid, and that the organizer is active in retinoic acid signaling during early gastrulation. The model suggests that FASD is induced in part by an ethanol-dependent reduction in retinoic acid levels that are necessary for the normal function of Spemann's organizer.

The competence of marginal zone cells to become Spemann's organizer is controlled by Xcad2.

The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation. Here, we show that the competence of marginal zone cells to respond to organizer-inducing signals is under temporal control, one of the regulators being the homeobox transcription factor Xcad2. Overexpression of Xcad2 restricts the temporal competence for axis induction, whereas partial loss of function expands this competence, supporting our suggestion. We propose that Xcad2 competes with putative axis-inducing signals within the marginal zone to prevent expression of organizer-specific genes. Elimination of endogenous Xcad2 allows for the activation of organizer genes beyond the normal competence window during early/mid-gastrulation. We conclude that Xcad2, through its early expression in the ventrolateral marginal zone, terminates the competence of this embryonic region to respond to organizer-inducing signals by preventing the activation of organizer-specific genes.