Mary R. Hutson

A Caudal Proliferating Growth Center Contributes to Both Poles of the Forming Heart Tube

Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1. Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation.

The Neural Crest in Cardiac Congenital Anomalies

This review discusses the function of neural crest as they relate to cardiovascular defects. The cardiac neural crest cells are a subpopulation of cranial neural crest discovered nearly 30 years ago by ablation of premigratory neural crest. The cardiac neural crest cells are necessary for normal cardiovascular development. We begin with a description of the crest cells in normal development, including their function in remodeling the pharyngeal arch arteries, outflow tract septation, valvulogenesis, and development of the cardiac conduction system. The cells are also responsible for modulating signaling in the caudal pharynx, including the second heart field. Many of the molecular pathways that are known to influence specification, migration, patterning and final targeting of the cardiac neural crest cells are reviewed. The cardiac neural crest cells play a critical role in the pathogenesis of various human cardiocraniofacial syndromes such as DiGeorge, Velocardiofacial, CHARGE, Fetal Alcohol, Alagille, LEOPARD, and Noonan syndromes, as well as Retinoic Acid Embryopathy. The loss of neural crest cells or their dysfunction may not always directly cause abnormal cardiovascular development, but are involved secondarily because crest cells represent a major component in the complex tissue interactions in the head, pharynx and outflow tract. Thus many of the human syndromes linking defects in the heart, face and brain can be better understood when considered within the context of a single cardiocraniofacial developmental module with the neural crest being a key cell type that interconnects the regions.

Factors controlling cardiac neural crest cell migration

Cardiac neural crest cells originate as part of the postotic caudal rhombencephalic neural crest stream. Ectomesenchymal cells in this stream migrate to the circumpharyngeal ridge and then into the caudal pharyngeal arches where they condense to form first a sheath and then the smooth muscle tunics of the persisting pharyngeal arch arteries. A subset of the cells continue migrating into the cardiac outflow tract where they will condense to form the aorticopulmonary septum. Cell signaling, extracellular matrix and cell-cell contacts are all critical for the initial migration, pauses, continued migration, and condensation of these cells. This review elucidates what is currently known about these factors.

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.

Arterial pole progenitors interpret opposing FGF/BMP signals to proliferate or differentiate

During heart development, a subpopulation of cells in the heart field maintains cardiac potential over several days of development and forms the myocardium and smooth muscle of the arterial pole. Using clonal and explant culture experiments, we show that these cells are a stem cell population that can differentiate into myocardium, smooth muscle and endothelial cells. The multipotent stem cells proliferate or differentiate into different cardiovascular cell fates through activation or inhibition of FGF and BMP signaling pathways. BMP promoted myocardial differentiation but not proliferation. FGF signaling promoted proliferation and induced smooth muscle differentiation, but inhibited myocardial differentiation. Blocking the Ras/Erk intracellular pathway promoted myocardial differentiation, while the PLCγ and PI3K pathways regulated proliferation. In vivo, inhibition of both pathways resulted in predictable arterial pole defects. These studies suggest that myocardial differentiation of arterial pole progenitors requires BMP signaling combined with downregulation of the FGF/Ras/Erk pathway. The FGF pathway maintains the pool of proliferating stem cells and later promotes smooth muscle differentiation.

Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations

Point mutations in the calcium‐permeable TRPV4 ion channel have been identified as the cause of autosomal‐dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4V620I channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6‐fold) up‐regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up‐regulation (1.1‐fold). We generated a mouse model of theTRPV4V620I mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8‐fold). FST was significantly up‐regulated in primary chondrocytes transfected with 3 different dysplasia‐causing TRPV4 mutations (2‐ to 2.3‐fold), but was not affected by an arthropathy mutation (1.1‐fold). Furthermore, FST‐loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4‐fold in human chondrocytes from an individual natively expressing the TRPV4T89I mutation. Taken together, these data strongly support that up‐regulation of FST in chondrocytes by skeletal dysplasia‐inducing TRPV4 mutations contributes to disease pathogenesis.—Leddy, H. A., McNulty, A. L., Lee, S. H., Rothfusz, N. E., Gloss, B., Kirby, M. L., Hutson, M. R., Cohn, D. H., Guilak, F., Liedtke, W. Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations. FASEB J. 28, 2525–2537 (2014). www.fasebj.org

BMP signaling modulates hedgehog-induced secondary heart field proliferation

Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.

Loss of Wnt5a disrupts second heart field cell deployment and may contribute to OFT malformations in DiGeorge Syndrome

Outflow tract (OFT) malformation accounts for ∼30% of human congenital heart defects and manifests frequently in TBX1 haplo-insufficiency associated DiGeorge (22q11.2 deletion) syndrome. OFT myocardium originates from second heart field (SHF) progenitors in the pharyngeal and splanchnic mesoderm (SpM), but how these progenitors are deployed to the OFT is unclear. We find that SHF progenitors in the SpM gradually gain epithelial character and are deployed to the OFT as a cohesive sheet. Wnt5a, a non-canonical Wnt, is expressed specifically in the caudal SpM and may regulate oriented cell intercalation to incorporate SHF progenitors into an epithelial-like sheet, thereby generating the pushing force to deploy SHF cells rostrally into the OFT. Using enhancer trap and Cre transgenes, our lineage tracing experiments show that in Wnt5a null mice, SHF progenitors are trapped in the SpM and fail to be deployed to the OFT efficiently, resulting in a reduction in the inferior OFT myocardial wall and its derivative, subpulmonary myocardium. Concomitantly, the superior OFT and subaortic myocardium are expanded. Finally, in chick embryos, blocking the Wnt5a function in the caudal SpM perturbs polarized elongation of SHF progenitors, and compromises their deployment to the OFT. Collectively, our results highlight a critical role for Wnt5a in deploying SHF progenitors from the SpM to the OFT. Given that Wnt5a is a putative transcriptional target of Tbx1, and the similar reduction of subpulmonary myocardium in Tbx1 mutant mice, our results suggest that perturbing Wnt5a-mediated SHF deployment may be an important pathogenic mechanism contributing to OFT malformations in DiGeorge syndrome.

Evolutionary and developmental origins of the cardiac neural crest: Building a divided outflow tract

The cardiac neural crest cells (CNCCs) have played an important role in the evolution and development of the vertebrate cardiovascular system: from reinforcement of the developing aortic arch arteries early in vertebrate evolution, to later orchestration of aortic arch artery remodeling into the great arteries of the heart, and finally outflow tract septation in amniotes. A critical element necessary for the evolutionary advent of outflow tract septation was the co-evolution of the cardiac neural crest cells with the second heart field. This review highlights the major transitions in vertebrate circulatory evolution, explores the evolutionary developmental origins of the CNCCs from the third stream cranial neural crest, and explores candidate signaling pathways in CNCC and outflow tract evolution drawn from our knowledge of DiGeorge Syndrome.

One shall become two: Separation of the esophagus and trachea from the common foregut tube

The alimentary and respiratory organ systems arise from a common endodermal origin, the anterior foregut tube. Formation of the esophagus from the dorsal region and the trachea from the ventral region of the foregut primordium occurs by means of a poorly understood compartmentalization process. Disruption of this process can result in severe birth defects, such as esophageal atresia and tracheo‐esphageal fistula (EA/TEF), in which the lumina of the trachea and esophagus remain connected. Here we summarize the signaling networks known to be necessary for regulating dorsoventral patterning within the common foregut tube and cellular behaviors that may occur during normal foregut compartmentalization. We propose that dorsoventral patterning serves to establish a lateral region of the foregut tube that is capable of undergoing specialized cellular rearrangements, culminating in compartmentalization. We review established as well as new rodent models that may be useful in addressing this hypothesis. Finally, we discuss new experimental models that could help elucidate the mechanism behind foregut compartmentalization. An integrated approach to future foregut morphogenesis research will allow for a better understanding of this complex process. Developmental Dynamics 244:277–288, 2015.