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.

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.

Fgfr2 is required for the expansion of the early adrenocortical primordium.

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from intermediate mesoderm of the anterior urogenital ridge. Previous work has pinpointed a minor role of the FGFR2IIIb isoform in expansion and differentiation of the fetal adrenal cortex in mice but did not address the complete role of FGFR2 and FGFR1 signaling in adrenocortical development. Here, we show that a Tbx18(cre) line mediates specific recombination in the coelomic epithelium of the anterior urogenital ridge which gives rise by a delamination process to the adrenocortical primordium. Mice with conditional (Tbx18(cre)-mediated) deletion of all isoforms of Fgfr2 exhibited severely hypoplastic adrenal glands around birth. Cortical cells were dramatically reduced in number but showed steroidogenic differentiation and zonation. Neuroendocrine chromaffin cells were also reduced and formed a cell cluster adjacent to but not encapsulated by steroidogenic cells. Analysis of earlier time points revealed that the adrenocortical primordium was established in the intermediate mesoderm at E10.5 but that it failed to expand at subsequent stages. Our further experiments show that FGFR2 signaling acts as early as E11.5 to prevent apoptosis and enhance proliferation in adrenocortical progenitor cells. FGFR1 signaling does not contribute to early adrenocortical development. Our work suggests that FGFR2IIIb and IIIc isoforms largely act redundantly to promote expansion of the adrenocortical primordium.

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.

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.

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.

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.

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.

Dynamic positional fate map of the primary heart-forming region.

Here we show the temporal-spatial orchestration of early heart morphogenesis at cellular level resolution, in vivo, and reconcile conflicting positional fate mapping data regarding the primary heart-forming field(s). We determined the positional fates of precardiac cells using a precision electroporation approach in combination with wide-field time-lapse microscopy in the quail embryo, a warm-blooded vertebrate (HH Stages 4 through 10). Contrary to previous studies, the results demonstrate the existence of a "continuous" circle-shaped heart field that spans the midline, appearing at HH Stage 4, which then expands to form a wide arc of progenitors at HH Stages 5-7. Our time-resolved image data show that a subset of these cardiac progenitor cells do not overlap with the expression of common cardiogenic factors, Nkx-2.5 and Bmp-2, until HH Stage 10, when a tubular heart has formed, calling into question when cardiac fate is specified and by which key factors. Sub-groups and anatomical bands (cohorts) of heart precursor cells dramatically change their relative positions in a process largely driven by endodermal folding and other large-scale tissue deformations. Thus, our novel dynamic positional fate maps resolve the origin of cardiac progenitor cells in amniotes. The data also establish the concept that tissue motion contributes significantly to cellular position fate - i.e., much of the cellular displacement that occurs during assembly of a midline heart tube (HH Stage 9) is NOT due to "migration" (autonomous motility), a commonly held belief. Computational analysis of our time-resolved data lays the foundation for more precise analyses of how cardiac gene regulatory networks correlate with early heart tissue morphogenesis in birds and mammals.

Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.

Despite recent advances in delineating the mechanisms involved in cardiogenesis, cellular lineage specification remains incompletely understood. To explore the relationship between developmental fate and potential, we isolated a cardiac-specific Nkx2.5(+) cell population from the developing mouse embryo. The majority of these cells differentiated into cardiomyocytes and conduction system cells. Some, surprisingly, adopted a smooth muscle fate. To address the clonal origin of these lineages, we isolated Nkx2.5(+) cells from in vitro differentiated murine embryonic stem cells and found approximately 28% of these cells expressed c-kit. These c-kit(+) cells possessed the capacity for long-term in vitro expansion and differentiation into both cardiomyocytes and smooth muscle cells from a single cell. We confirmed these findings by isolating c-kit(+)Nkx2.5(+) cells from mouse embryos and demonstrated their capacity for bipotential differentiation in vivo. Taken together, these results support the existence of a common precursor for cardiovascular lineages in the mammalian heart.

Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells.

Formation of kidney tissue requires the generation of kidney precursor cells and their subsequent differentiation into nephrons, the functional filtration unit of the kidney. Here we report that the gene odd-skipped related 1 (Odd1) plays an important role in both these processes. Odd1 is the earliest known marker of the intermediate mesoderm, the precursor to all kidney tissue. It is localized to mesenchymal precursors within the mesonephric and metanephric kidney and is subsequently downregulated upon tubule differentiation. Mice lacking Odd1 do not form metanephric mesenchyme, and do not express several other factors required for metanephric kidney formation, including Eya1, Six2, Pax2, Sall1 and Gdnf. In transient ectopic expression experiments in the chick embryo, Odd1 can promote expression of the mesonephric precursor markers Pax2 and Lim1. Finally, persistent expression of Odd1 in chick mesonephric precursor cells inhibits differentiation of these precursors into kidney tubules. These data indicate that Odd1 plays an important role in establishing kidney precursor cells, and in regulating their differentiation into kidney tubular tissue.

Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cell-autonomous and translation-dependent manner.

The intermediate mesoderm lies between the somites and the lateral plate and is the source of all kidney tissue in the developing vertebrate embryo. While bone morphogenetic protein (Bmp) signaling is known to regulate mesodermal cell type determination along the medio-lateral axis, its role in intermediate mesoderm formation has not been well characterized. The current study finds that low and high levels of Bmp ligand are both necessary and sufficient to activate intermediate and lateral mesodermal gene expression, respectively, both in vivo and in vitro. Dose-dependent activation of intermediate and lateral mesodermal genes by Bmp signaling is cell-autonomous, as demonstrated by electroporation of the avian embryo with constitutively active Bmp receptors driven by promoters of varying strengths. In explant cultures, Bmp activation of Odd-skipped related 1 (Odd-1), the earliest known gene expressed in the intermediate mesoderm, is blocked by cyclohexamide, indicating that the activation of Odd-1 by Bmp signaling is translation-dependent. The data from this study are integrated with that of other studies to generate a model for the role of Bmp signaling in trunk mesodermal patterning in which low levels of Bmp activate intermediate mesoderm gene expression by inhibition of repressors present in medial mesoderm, whereas high levels of Bmp repress both medial and intermediate mesoderm gene expression and activate lateral plate genes.

Cell fate specification along the anterior-posterior axis of the intermediate mesoderm.

The vertebrate intermediate mesoderm (IM) is highly patterned along the anterior-posterior (A-P) axis. In the chick embryo, the kidney tissue, which is a derivative of the IM, is generated only from IM located posterior to the sixth somite axial level, which also marks the border between cranial and trunk segments. The cellular and molecular mechanisms that govern the formation of the anterior border of the kidney morphogenetic field are currently unknown. In this study, we asked whether specific A-P patterning information is conveyed by the movement of cells through the primitive streak (PS) at different time points that consequently affects the expression of kidney genes, or by the environment that these cells encounter during their migration to the IM. In this study, we show that kidney-inductive signals are present along the whole axis, including anterior non-kidney-generating regions. These inductive signals are generated by tissues that are located medial to the anterior IM. We also demonstrate that cells that migrate through the PS of early embryonic stages (Hamburger and Hamilton stage 3-4 and earlier), which will give rise to anterior nonkidney IM, are competent to respond to these inductive factors. This prospective anterior IM tissue loses its competence to respond to kidney inducing signals during its migration from the PS to its final location in the anterior IM. We present here a model in which changes in cell competence determine the formation of the anterior border of kidney gene expression and discuss the possible evolutionary implications of this developmental mechanism.