2018 Group Project 4: Difference between revisions

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Development of the cardiovascular system begins with the formation of two endocardial tubes that merge together to form the tubular heart. These loop together and separate into the four chambers and paired arterial trunks form the adult heart. The tubular heart differentiates into the truncus arterioles, bulbus cordis, primitive ventricle, primitive atrium and the sinus venosus. The truncus arteriosus splits into the ascending aorta and pulmonary artery. The bulbus cordis forms part of the ventricles. The sinus venosus connects to the fetal circulation. Septa form within the atria and ventricles to separate the left and right sides of the heart.
Development of the cardiovascular system begins with the formation of two endocardial tubes that merge together to form the tubular heart. These loop together and separate into the four chambers and paired arterial trunks form the adult heart. The tubular heart differentiates into the truncus arterioles, bulbus cordis, primitive ventricle, primitive atrium and the sinus venosus. The truncus arteriosus splits into the ascending aorta and pulmonary artery. The bulbus cordis forms part of the ventricles. The sinus venosus connects to the fetal circulation. Septa form within the atria and ventricles to separate the left and right sides of the heart.
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== Cardiac Neural Crest Cells ==
== Cardiac Neural Crest Cells ==

Revision as of 10:45, 9 October 2018

Projects 2018: 1 Adrenal Medulla | 3 Melanocytes | 4 Cardiac | 5 Dorsal Root Ganglion

Project Pages are currently being updated (notice removed when completed)

Neural Crest and Cardiac Development

Introduction

The very first major system to develop its function within a vertebrate embryo is the cardiovascular system with the heart becoming active from the fourth week of development when the placenta is no longer able to sustain the requirements of the growing embryo by itself The four major embryonic regions that are involved in the process of vertebrate heart development are the primary heart field, secondary heart field, cardiac neural crest, and proepicardium. Each region have important contributions to the overall cardiac development, which occurs with complex and precise developmental timing and regulation. Neural crests are a population of multipotent cells which arises during embryonic development at the dorsal neural tube and were first identified by Wilhelm His as “Zwischenstrang,” the intermediate cord, in 1868, the year of Meiji Ishin, the westernizing revolution of Japan.[1]. Studies done in avian and fish embryos have shown that a specific subgroup of neural crest cells, known as cardiac neural crest cells are essential for the septation of the cardiac outflow track as well as the development of aortic arch artery. Since then, the studies have allowed for the classification of neural crest associated human cardiac defects such as DiGeorge syndrome.

Development of the Cardiovascular System

Development of the heart in the fetus and partitioning of the heart into four chambers

Development of the cardiovascular system begins with the formation of two endocardial tubes that merge together to form the tubular heart. These loop together and separate into the four chambers and paired arterial trunks form the adult heart. The tubular heart differentiates into the truncus arterioles, bulbus cordis, primitive ventricle, primitive atrium and the sinus venosus. The truncus arteriosus splits into the ascending aorta and pulmonary artery. The bulbus cordis forms part of the ventricles. The sinus venosus connects to the fetal circulation. Septa form within the atria and ventricles to separate the left and right sides of the heart.

Week 2 - 3 *Bilateral cardiogenic areas form
Week 3 - 4
  • Mesoderm splitting
  • Folding brings heart tubes into the ventral midline
  • Heart tube fusion
  • Heart tube begins to beat
Week 4 - 5
  • Heart looping
  • Neural crest migration starts
  • Dorsal and ventral endocardial cushions fused
  • Foramen premium closed, septum secundum begins to develop
Week 5-6
  • Deep, muscular interventricular septum
  • Bulbar ridges and trabeculations evident
Week 7
  • Aortic and pulmonary trunks cleave
  • Valves developed

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Cardiac Neural Crest Cells

Neural crest cells are a population of multipotent cells which arises during embryonic development at the dorsal neural tube. Neural crest cells originate from the dorsal-most region of the neural tube. These cells are capable of migrating and differentiating throughout the body to give rise to many different cell types. These cells, which originate from the ectoderm in a region lateral to the neural plate in the neural fold, give rise to neurons, glia, melanocytes, chondrocytes, smooth muscle cells, odontoblasts and neuroendocrine cells, among others[2]. Cardiac neural crest cells (CNCCs) are a subpopulation of the cranial neural crest cells and migrate ventrally from the dorsal neural tube PubmedParser error: Invalid PMID, please check. (PMID: [1]). CNCCs will then proceed and fall in place into third, fourth and the sixth caudal pharyngeal arches as they develop during their migration to the cardiac outflow tract. They will form condensed mesenchymal cells of the aorticopulmonary septation complex and also differentiate into cardiac ganglia[3].NCCs are necessary for aortic arch artery remodeling and outflow tract septation (OFT). [4]


Cardiac neural Cells can develop into:

  • Melanocytes near the heart region
  • neurons associated with cardiac innervation
  • cartilage
  • connective tissue (they form the connective tissue wall of the large arteries from the heart, as well as the septum between the branches in the heart)
  • provide signals required for the maintenance and differentiation of the other cell layers in the pharyngeal apparatus [4]

Early Development

Induction

Initially, NCCs are morphologically similar to other neuroepithelial cells and cannot be differentiated from them. With contact-mediated inductive signals from the surface ectoderm and underlying mesoderm through a process known as Induction where progenitor cells begin to differentiate PubmedParser error: Invalid PMID, please check. (PMID: [2]). Progenitor cells are found in the epiblast around Henson's node and are brought into the neural folds where signalling molecules will induce the progenitor cells to turn into CNCCs PubmedParser error: Invalid PMID, please check. (PMID: [3]). While key signalling regulators of neural crest cell formation such as bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) have been identified in species such as fish and avians, there is currently no evidence that suggests the same factors play a role in mammalian neural crest cell induction, thus more studies have to be carried out to identify the signaling pathway for mammalian neural crest cell formation PubmedParser error: Invalid PMID, please check. (PMID: [4]). Studies have also shown that if BMP levels are too high or low, the progenitor cells will not be able to migrate, thus an intermediate level of BMP is ideal for the induction process.PubmedParser error: Invalid PMID, please check. (PMID: [5]) As for the other signaling cascades involved, little information is known.

Migration From Neural Crest to Circumpharyngeal Ridge

After the Induction process, cranial neural crest cells undergo an epithelial-to-mesenchymal transition and emigrate from the neural tube to the circumpharyngeal ridge which is an arc-shaped ridge structure that is found dorsal to developing caudal pharyngeal arches PubmedParser error: Invalid PMID, please check. (PMID: [6]). In higher vertebrates, cranial neural crest cells will migrate in three clusters (cranial, middle and caudal) and eventually develop cranial nerve ganglia at even-numbered rhombomeres proximally and populate pharyngeal arches distally. The caudal stream comprises most of the CNCCs.

There are multiple signaling factors which control the migration of CNCCs.

  • Snail2 inhibits the expression of cadherins and studies on avians and fish have shown that the presence of Snail2 helps to facilitate cell migration PubmedParser error: Invalid PMID, please check. (PMID: [7]). However, the expression of Snail does not seem to be needed for the neural crest induction in mammals PubmedParser error: Invalid PMID, please check. (PMID: [8]).
  • RhoA/B, a GTPase protein, regulates and remodels the actin cytoskeleton of the cells to alter the planar cell polarity to allow neural crest migration PubmedParser error: Invalid PMID, please check. (PMID: [9]).
  • CNCC expresses integrin receptors and MMP-2 to allow them to migrate on fibronectin in extracellular matrix which is believed to provide a permissive environment to allow the migration of crest cells to the circumpharyngeal ridge PubmedParser error: Invalid PMID, please check. (PMID: [10]).

Formation of Pharyngeal Arches and Cardiac Outflow Tract

The Cardiac Outflow Tract (OFT) is a transient embryonic structure located at the arterial pole of the heart that functions initially as a conduit for the blood flowing from the right ventricle into the aortic sac.[5].

CNCCs will initially pause their migration at the circumpharyngeal ridge as their destined pharyngeal arches have not been developed. The pericardial cavity will regress caudally, allowing pharyngeal pouches to indent the body wall and delineate the pharyngeal arches from the cranial to the caudal direction and generate arches (3, 4 and 6). As the arches develop, they will be populated by cardiac neural crest cells which migrates from the circumpharyngeal ridge.

CNCCs express different factors that target the cells to the pharyngeal arches. Slit cells can target cells to migrate to arch 3. FGF-8 targets for arch 4. EphA targets for arch 6. Rac1 and Sdf1 are both expressed in the cells, causing them to condensate around the arch arteries. Semaphorin is expressed and causes the cells to migrate further to the cardiac outflow tract. Notch and BMP are then expressed condensing the cells, forming the semilunar valve and aorticopulmonary septum.

  • The 3rd arch is dedicated in the formation of the carotid system. It forms the left and right common carotid arteries which will sprout into internal and external carotid arteries by angiogenesis PubmedParser error: Invalid PMID, please check. (PMID: [11]).
  • The 4th arch gives rise to the definitive aortic arch along with the pulmonary artery.
  • The 6th arch initially develops into the pulmonary trunk that emerges from the right ventricles, but this structure will be remodeled asymmetrically and gives rise to the ductus arteriosus which is a crucial embryonic structure that connects the pulmonary artery with the descending aorta for blood circulation in the fetus. This shunt allows blood from the right ventricle to bypass the lungs because the fetal blood is oxygenated through the placenta. At birth, as the lungs start their function, the ductus arteriosus closes allowing circulation through to the lungs to oxygenate the blood that subsequently reaches the systemic circulation of the newborn PubmedParser error: Invalid PMID, please check. (PMID: [12]).


Ectomesenchyme that is derived from CNCCs in pharyngeal arches 3, 4 and 6 are critical for the repatterning of the bilaterally symmetrical pharyngeal arch arteries to form the asymmetric great arteries of the thorax.

Migration of cardiac neural crest cells from the neuroectoderm into the outflow tract cushions induces the formation of the aortopulmonary (AP) septum, which divides the common outflow tract at the cardiac to vascular border into an aortic and pulmonary orifice and more proximally intracardiac into a right and left ventricular outflow tract. [6]

Later Development

Outflow Septation

After migrating into the pharynx, some CNCCs will remain in the pharyngeal arches while the rest would continue and migrate into cardiac outflow cushions which converges to separate blood flow from the embryonic left and right ventricles PubmedParser error: Invalid PMID, please check. (PMID: [13]). Cushion formation and septation rely on the interaction of 3 distinct cell types, cardiac neural crest cells (NCCs), second heart field-derived (SHF-derived) cells, and endothelial cells (ECs) [5] . Currently, not much is known about which factors are responsible for attracting neural crest cells into the outflow tract cushions. Within the cushions, the CNCCs will condense and form the aorticopulmonary septation complex which is essentially two centrally placed columns and divides the common arterial outflow into the aorta and pulmonary trunks. Studies have shown that the TGFbeta/BMP signaling family is involved in this condensation processPubmedParser error: Invalid PMID, please check. (PMID: [14]). The cushions will be populated with three main types of mesenchymal cells, depending on their proximal-distal location in the outflow tract PubmedParser error: Invalid PMID, please check. (PMID: [15]).

There are three main components that are responsible for forming the septa in the outflow tract:

  1. Conus Septum
  2. truncus Septum
  3. Aorto-Pulmonary Septum

Failure of proper OFT septation during embryogenesis will result in inappropriate mixing of oxygenated and deoxygenated blood at birth and this may cause an unfavourable clinical prognosis.

Valvulogenesis

The majority of heart defects in live births arise from disruption of cardiac outflow tract development. The OFT is an embryonic structure that gives rise to the ascending aortic and pulmonary arteries as well as their respective tricuspid aortic (AV) and pulmonary (PV) valves.[7].The cNCC also contribute to the aortic and pulmonary valves, thereby connecting the heart to the vascular system. OFT endothelial cells that have undergone endoMT are thought to give rise to the bulk of the semilunar valves, which form within the aorta and pulmonary artery, to prevent the backflow of blood into the ventricles. In addition, cardiac NCCs also colonize the semilunar valves, where they mainly contribute to the two leaflets adjacent to the aorticopulmonary septum. Cells of the NCC have also been found to contribute to the atrioventricular valves, consisting of the bicuspid (mitral) valve and tricuspid valve, which is located between the upper atria and the lower ventricles.

Atrial and Ventricular Separation

The heart first transforms from the embryo into parts such as the atrial and ventricular chambers, arterial trunks and great veins. The first part of cardiac development involves the separation into phases of formation of the primary myocardial tube, looping of the tube, additional parts for future topography compartments, and assembly of the components into arterial trunks and cardiac chambers subsequently. [8]


The initial heart formation will involve the separation into four cardiac chambers.

involves more than simple formation of partitions between them.

The significance of morphologic distinctions of this type should become evident as we discuss the development of the various structures that, eventually, separate the atrial and ventricular chambers within the definitive heart. These are the atrial septum, the atrioventricular septum, and the ventricular septum. We will start, however, with a brief consideration of another area often described as a septum,

that subsequent to the formation of the two arterial trunks, there was disappearance of the cushions that initially divided them. Thus, in the definitive heart, the proximal parts of the aorta and pulmonary trunk, along with the sinuses of the arterial roots and the subpulmonary infundibulum, possess their own discrete walls, separated by extra-cardiac space.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1767797/

<<not edited>>

Formation of the Cardiac Ganglia

Cardiac ganglia are made entirely from cardiac crest cells.

"Virtually nothing is known about the factors that control their separation from the cardiac crest forming the aorticopulmonary septum or their condensation as ganglia. However, cardiac crest cells also participate in the formation of the nodose ganglion. This is the distal sensory ganglion of the vagus nerve. The nodose ganglion is formed from neurons derived from the nodose placode located dorsal to pharyngeal arches 4/6. Cells migrate from this placode to coalesce with cardiac crest to form the nodose ganglion. Condensation of this ganglion depends on N-cadherin and signaling by Slit/Robo signaling. In cranial crest Slit1/Robo signaling in conjunction with N-cadherin is important for coalescence of crest cells and placode-derived neurons into ganglia. N-cadherin and Robo2 are expressed by placodal neurons and Slit1 is on neural crest cells. If either N-cadherin or Robo2 is knocked down, the ganglia do not coalesce properly.115"

PubmedParser error: Invalid PMID, please check. (PMID: [16])

Signaling Molecules

  1. Wnt: extracellular growth factors that activate intracellular signaling pathways. The decrease of B-catenin results in a reduction in the proliferation of cardiac neural crest cells.
  2. Notch: a transmembrane protein whose signaling is required for differentiation of CNCCs to vascular smooth muscle cells and for proliferation of cardiac myocytes.
  3. BMP (bone morphogenetic proteins): they are required for neural crest cell migration into the cardiac cushions (=precursors to heart valves and septa) and for differentiation of neural crest cells to smooth muscle cells of the aortic arch arteries.
  4. FGF8(fibroblast growth factor 8): Transcription factors that are essential for regulating the addition of secondary heart field cells into the cardiac outflow tract.
  5. GATA: Transcription factors which play a critical role in cell lineage differentiation restriction during cardiac development.
  • Meis2 PubmedParser error: Invalid PMID, please check. (PMID: [17])

Developmental Time Course

Week 3-4 Day 22-28 Neural crest migration starts
Week 5-6 Day 32-37 Cardiac neural crest migrates through the aortic arches and enters the outflow tract of the heart
Week 9 Day 57+ Outflow tract and ventricular septation complete

Human Congenital Heart Diseases associated with Neural Crest Cells

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. [9]



not yet edited----

Cardiac neural crest ablation experiments demonstrated that upon removal of the pre-migratory cardiac neural crest cardiovascular abnormalities are induced.

that the quantity rather than the quality of neural crest cells is important in OFT septation.

Furthermore, cardiac malformations associated with partial ablation of the cardiac neural crest, show normal formation of the aorticopulmonary septum and as a result an aorta and a pulmonary trunk. However, the aorta and pulmonary trunk are malaligned with respect to the ventricles. One might argue that the neural crest-derived cells are not only crucial in the regulation of septation but also in the alignment of the great arteries with respect to the ventricles. On the other hand, one might argue that the malalignment is due to an indirect effect of neural crest ablation. https://academic.oup.com/cardiovascres/article/47/2/212/363634


End of unedited part----


Conotruncal Heart Malformations

OFT remodeling is a process whereby the embryonic outflowtract undergoes a series of developmental transitions that involves extra cardiac cell recruitment and transformations that when disrupted, can result in Persistent Truncus Arteriosus [7] : if the cardiac neural crest is removed before it begins to migrate, the conotruncal septa completely fails to develop, and blood leaves both the ventricles through what is termed a persistent truncus arteriosus, a rare congenital heart anomaly in humans. (Martinson) Failure of outflow tract septation may also be responsible for other forms of congenital heart disease, including transposition of the great vessels, high ventricular septal defects, and tetralogy of Fallot (Martinson).

This is a defect on the NKX2 gene/locus

DiGeorge Syndrome

DiGeorge syndrome (DGS), aka Velocardiofacial Syndrome, is a congenital condition which affects the development of many tissues that are patterned by or derived from NCCs. People suffering from DGS display symptoms such as craniofacial defects, aplasia or hypoplasia of the thymus and parathyroid glands, mental disorders and cardiovascular defects. [1]. DGS results primarily due to defective development of cranial and cardiac NCCs which invades the first four pharyngeal arches that contribute to the development of the lower jaw, neck and cardiac structures. Studies have shown in chicks that the ablation of the pharyngeal NCCs population produce cardiocraniofacial anomalies which can be found in DGS patients. (how to cite this?: http://dev.biologists.org/content/143/4/582)

DGS

  • Caused by a chromosomal 22q11.2 deletion.
  • a hemizygous deletion within chromosome band 22q11.2 has been found in 25% of DGS patients.
  • Characterized by interrupted aortic arch type B, outflow tract malformations that include xxx

[9]


(to edit) The DiGeorge syndrome consists of a PTA, type B interrupted aortic arch, absent or hypoplastic thymus, craniofacial dysmorphology and cognitive or behavioral disorders. (ref) It can also include absent or hypoplastic parathyroid and thyroid glands.

https://www.ncbi.nlm.nih.gov/pubmed/9514586 https://www.ncbi.nlm.nih.gov/pubmed/3728313

A variant of the DiGeorge phenotype, called Sprintzen or Velocardiofacial syndrome, also includes cleft palate. https://www.ncbi.nlm.nih.gov/pubmed/272242

C.H.A.R.G.E Syndrome

CHARGE is an acronym for a collection of symptoms including, heart defects, retarded growth, and development, genital hypoplasia, ear anomalies, and deafness.

  • Coloboma
  • Heart anomaly
  • Atresia of choanae
  • Retardation of physical and mental development
  • Genital hypoplasia
  • Ear anomalies and/or deafness

PubmedParser error: Invalid PMID, please check. (PMID: [18])

CHARGE syndrome is a sporadic, autosomal dominant malformation disorder diagnosed in 1/8,500-1/10,000 live births [10]. In addition, malformations of the foregut, kidneys, limbs, lung, and liver have been described in infants with CHARGE syndrome. The gene most commonly affected in patients with CHARGE syndrome is CHD7, which encodes a DNA binding protein involved in chromatin remodeling [10].Mutations in the chromatin helicase DNA-binding protein 7(CHD7) gene are causative of CHARGE syndrome and the loss-of-function.

The link between the chromatin-remodeling protein CHD7 with cardiac NCC-associated defects suggests that epigenetic regulation is important for genes controlling NCC function.


--> https://pdfs.semanticscholar.org/42cc/ee7fbb545ea6752e1c126cc2769e8e33e7b7.pdf



"Neural crest cells contribute to these deformations and abnormalities of the tissues. How is because NC development involves many convoluted steps such as specification, delamination, migration, induction, and differentiation [10]. These processes are controlled by regulatory gene networks. If certain genes are disrupted affecting the neural crest cells then a variety of human diseases arise categorized as neurocristopathies." -> necessary? or can throw?

Models and Research

Animal Models

  • Main animal models are chicken, fish, and mice.

Chick embryo models: (to edit)

the chick embryo using quail-chick chimeras to study neural crest migration and derivatives as well as using ablation of premigratory neural crest cells to study their function. These studies show that cardiac neural crest cells are absolutely required to form the aorticopulmonary septum dividing the cardiac arterial pole into systemic and pulmonary circulations. They support the normal development and patterning of derivatives of the caudal pharyngeal arches and pouches, including the great arteries and the thymus, thyroid and parathyroids.

https://www.ncbi.nlm.nih.gov/pubmed/17224285


These studies show that after the cardiac neural crest cells migrate into pharyngeal arches 3, 4 and 6, a subset of the cells continue migrating into the cardiac outflow cushions.

<<insert image>>

https://www.ncbi.nlm.nih.gov/pubmed/3568286


Mouse models:

Research

  • Can cardiac neural crest cells be used to repair human heart tissue? They are basically neural crest stem cells. In 2005, Tomita transplanted neural crest cells from mammal hearts to the neural crest of chick embryos --> find more research for this
  • What is the contribution of the cardiac NCCs to the myocardium and conduction system of the heart.

Glossary

Number | A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z

References

Nakanishi T, Markwald RR, Baldwin HS, Keller BB, Srivastava D, Yamagishi H, Miyagawa-Tomita S, Arima Y & Kurihara H. (2016). The “Cardiac Neural Crest” Concept Revisited. , , . PMID: 29787146 DOI. Odelin G, Faure E, Coulpier F, Di Bonito M, Bajolle F, Studer M, Avierinos JF, Charnay P, Topilko P & Zaffran S. (2018). Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve. Development , 145, . PMID: 29158447 DOI. Pauli S, Bajpai R & Borchers A. (2017). CHARGEd with neural crest defects. Am J Med Genet C Semin Med Genet , 175, 478-486. PMID: 29082625 DOI.

  1. 1.0 1.1 Nakanishi T, Markwald RR, Baldwin HS, Keller BB, Srivastava D, Yamagishi H, Miyagawa-Tomita S, Arima Y & Kurihara H. (2016). The “Cardiac Neural Crest” Concept Revisited. , , . PMID: 29787146 DOI.
  2. Vega-Lopez GA, Cerrizuela S, Tribulo C & Aybar MJ. (2018). Neurocristopathies: New insights 150 years after the neural crest discovery. Dev. Biol. , , . PMID: 29802835 DOI.
  3. Taneyhill LA & Schiffmacher AT. (2013). Cadherin dynamics during neural crest cell ontogeny. Prog Mol Biol Transl Sci , 116, 291-315. PMID: 23481200 DOI.
  4. 4.0 4.1 Odelin G, Faure E, Coulpier F, Di Bonito M, Bajolle F, Studer M, Avierinos JF, Charnay P, Topilko P & Zaffran S. (2018). Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve. Development , 145, . PMID: 29158447 DOI.
  5. 5.0 5.1 Plein A, Calmont A, Fantin A, Denti L, Anderson NA, Scambler PJ & Ruhrberg C. (2015). Neural crest-derived SEMA3C activates endothelial NRP1 for cardiac outflow tract septation. J. Clin. Invest. , 125, 2661-76. PMID: 26053665 DOI.
  6. Peterson JC, Chughtai M, Wisse LJ, Gittenberger-de Groot AC, Feng Q, Goumans MTH, VanMunsteren JC, Jongbloed MRM & DeRuiter MC. (2018). Nos3 mutation leads to abnormal neural crest cell and second heart field lineage patterning in bicuspid aortic valve formation. Dis Model Mech , , . PMID: 30242109 DOI.
  7. 7.0 7.1 Mifflin JJ, Dupuis LE, Alcala NE, Russell LG & Kern CB. (2018). Intercalated cushion cells within the cardiac outflow tract are derived from the myocardial troponin T type 2 (Tnnt2) Cre lineage. Dev. Dyn. , 247, 1005-1017. PMID: 29920846 DOI.
  8. Moorman A, Webb S, Brown NA, Lamers W & Anderson RH. (2003). Development of the heart: (1) formation of the cardiac chambers and arterial trunks. Heart , 89, 806-14. PMID: 12807866
  9. 9.0 9.1 Keyte A & Hutson MR. (2012). The neural crest in cardiac congenital anomalies. Differentiation , 84, 25-40. PMID: 22595346 DOI.
  10. 10.0 10.1 10.2 Cite error: Invalid <ref> tag; no text was provided for refs named PMID29082625