2018 Group Project 1
- 1 The Contribution of Neural Crest Cells to the Adrenal Medulla
- 1.1 Introduction
- 1.2 History
- 1.3 Developmental time course
- 1.4 Embryonic origins
- 1.5 Developmental/adult function
- 1.6 Molecular mechanisms/factors/genes
- 1.7 Abnormalities/abnormal development
- 1.8 Animal models
- 1.9 Current research (labs)
- 1.10 Glossary
- 1.11 Reference list
The Contribution of Neural Crest Cells to the Adrenal Medulla
|Projects 2018: 1 Adrenal Medulla | 3 Melanocytes | 4 Cardiac | 5 Dorsal Root Ganglion|
The neural crest of an embryo differentiates and migrates to form various components of the body. One of these tissues is the medulla of the adrenal gland which is involved in the production of adrenalin and noradrenalin. This project will comprehensively outline the history of neural crest research, the embryonic developmental time course, tissue and organ structure, molecular mechanisms, abnormalities, animal models and current research.
Describing the neural crest
The "neural crest" is a structure in the embryo and is the junction between the neural and epidermal ectoderm. It is exclusive to vertebrates and forms a range of structures. The neural crest contributes a large number of cells of varying structure and function that directly or indirectly contribute to the development of tissues and organs within the body . In 1868, the neural crest cells were first identified in a chick embryo as a collection of cells known as "Zwischenstrang" by Professor Wilhelm His . Wilhelm His was among the first to provide an explanation for the mechanics governing the developing embryo as well as the physiology and the organs that the differing germinal regions would eventually give rise to. In 1878, the term neural ridge was used to describe this collection of cells but the term neural crest was later coined by Arthur Milnes Marshall in 1879 in a paper he worked on disseminating the knowledge from his research on the development of cranial nerves in chicken embryos. .
1890s-1950s The neural crest is the point of origin for spinal/cranial ganglia and neurons. This theory received little criticism and was not contested much by other researchers prior to the 1890s. This is owing to the fact that the neural crest is quite closely related to the neural tube and thus this relationship makes sense. This discovery was made by His and Marshall, however, they found this out independently of one another.In the 1890s, Julia Platt, claimed that odontoblasts as well as the cartilages forming the facial and pharyngeal arch skeletons came from the ectoderm. This created much debate and was contested by many.
This is because this theory completely contradicted the germ-layer theory, whereby skeletal tissues originate at the mesoderm and not the ectoderm. The controversies this finding created brought about a slowing down of research, as demonstrated by a gap of almost 40 years between the theory that skeletal tissues originating from the neural crest. Today, it is known that the neural crest does indeed play a role in the development of the skeleton in vertebrates, particularly, the cranial neural crest. Although these studies were occurring, the focus was still primarily on researching pigment cells and ganglia of the spine up until the 1950s, when Sven Hörstadius' major work on the neural crest was published, titled "The Neural Crest: Its properties and derivatives in the light of experimental research". This work focused on experimental data tracing the development of the cartilaginous skeleton derived from the neural crest .
1960s - 1970s In the 1960s, the migration patterns of neural crest cells were studied in avian embryos (birds). This was a shift from studying the embryos of amphibians. This was inspired by studies conducted in the 1960s on neural crest cells migrating from the brain and spinal cord, known as trunk and cranial neural crest cells . The cranial neural crest eventually becomes the bones, cartilage and connective tissues forming the foundation of the face, ears and teeth. The trunk neural crest cells can either become melanocytes or can migrate into sclerotomes which are later contributors to the cartilage of the spine and the dorsal root ganglia . The cells in the trunk neural crest that migrate vertically become the sympathetic ganglia, the nerves proximal to the aorta and the adrenal medulla . In the 1970s many detailed maps signifying the pathways of many of the neural crest cells appeared as well as an understanding that the environment in which the neural crest cells are found plays a major role in their final development as well as the occurrences of abnormalities in the fetus .
1980s - 21st century
From the 1980s-1990s, the causative agent of organ transformation from one to another (homeotic patterning), as well as the Hox genes which regulate the axis of most symmetrical animal embryos were founded and studied. The neural crest, which previously was divided into the cranial and trunk regions, was now further divided into the vagal and sacral neural crest, the cardiac neural crest and the cephalic neural crest . The vagal & sacral neural crest gives rise to the parasympathetic ganglia found in the gut. Without these ganglia, peristalsis, is not possible . The cardiac neural crest contributes to the valves, septa and major blood vessels of the heart, as well as connective tissues and cartilage. This is important for the structure of the heart as well as its overall function, enabling it to move blood around the body efficiently as well as keep pulmonary and aortic circulation separated .
Today, knowledge of the neural crest cells, their migration patterns, modes of differentiation, derivatives and apoptosis (cell death) are being studied extensively. This is being done on a comparative basis between different organisms, to add further evidence for evolution . The neural crest is definitely forging a "crest" between the fields of developmental and evolutionary biology, as it is a structure shared exclusively by all vertebrates. Invertebrates do not derive their specialised cells from the neural crest as they do not have one, rather, they derive these from the endomesoderm .
Since its discovery by His, the neural crest field has made great progress, from studies performed on the structure, to analysis of the molecules and whether certain aspects of the neural crest are specific to a single species of organism, or to all vertebrates collectively. Future work on the cranial neural crest needs to be done as well as identifying and categorising the networks responsible for the proliferation and death of cells, as well as how the cells move and migrate, as well as the gene regulatory networks (GRNs) that regulate the synthesis of proteins. Advances in technology will undeniably lead us to more answers on the subject .
Developmental time course
The adrenal gland has two distinct sections, the cortex and the medulla, both with different embryonic origins. The cortex accounts for 90% of the adrenal glands and arises from intermediate mesoderm. The medulla only accounts for 10% of the adult adrenal glands, however it has neural crest embryonic origin. The medulla consists of chromaffinoblast cells which are migrant neuroblast cells from the neural crest, therefore the adrenal medulla is a component of the sympathetic nervous system. Neural crest cells replicate in week 7 and begin to differentiate in week 8, the cells then migrate to the developing adrenal gland in week 9 of development. The neural crest cells infiltrate the cortex and the chromaffin cells are scattered islands in the cortex. Throughout development the cells will become more compact in the centre of the gland, during this period of development the adrenal gland is growing rapidly. The gland is abnormally large until the second trimester and the medulla is much thicker prenatally.
The adrenal gland is made up of the adrenal cortex and adrenal medulla, which both have different embryonic origins. The adrenal medulla arises from the neural crest tissue, found near the level of the coeliac plexus & sympathetic ganglion at somites 18-24 . Specifically, this neural crest tissue comes from the trunk neural crest cells (see above figure to determine the approximate location in an embryo) . The trunk neural crest coordinates the development of the endocrine system, secretory cells, the peripheral nervous system and, to some degree, skeletal development, as well as innervation of the intestine . The trunk neural cells arise caudally, undergo an epithelial to mesenchymal transition and follow three migratory pathways .
- A dorsolateral pathway (between ectoderm and somites), these become melanocytes, travel up to the point of the dermis and become a major part of the hair follicles and skin . - A ventrolateral pathway (through the somites) - A ventromedial pathway (between neural tube and posterior schlerotome).
Migration of neural crest cells from the sympathetic ganglion is what leads to the development of the adrenal medulla. In week 8 of development, immature cells called neuroblasts migrate through to the inner portion of the medulla, where they become the adrenal medulla . Chromaffin cells are neuroendocrine cells and are found in the sympathetic ganglia and make up the majority of the adrenal medulla. They synthesise and release a range of hormones in the body and arise from the temporary neural crest. The term chromaffin was inspired by the staining capacity of these cells when they came into contact with chrome salts. They make, store, export and use catecholamines, responsible for making noradrenaline and originate at the caudal region of somites 18-24 of the neural crest, also known as the adrenomedullary region .
For cells to leave the neural crest and begin their migration, certain conditions need to be met, that is, the cells need to be loosely connected, their tight junctions becoming relaxed, initiated by "Slug protein" and a loss of N-Cadherin which also leads to the loosening of these junctions. The surrounding matrix as well as chemotactic and stem cell factors of the neural tube are then what dictates how the neural crest cells move, some proteins stopping their migration to that area and others encouraging the movement . After splitting apart, the first neural crest cells move ventrally and pass into the spaces between the somites and then, through the anterior portions of each segment until they arrive at the para-aortic sites, where they become the cells of the sympathetic ganglia and the adrenal medulla . BMP-4 (bone morphogenetic protein 4) then acts on these neural crest cells, preventing them from becoming cells that have a neural function and causing them to become chromaffin cells at the adrenal medulla. Glucocorticoids then play a role in maintenance of chromaffin cells in postnatal life, this was only found recently, as previously it was thought that glucocorticoids were responsible entirely for the differentiation of neural crest cells into chromaffin, or adrenomedullary cells .
Normal Structure and Function of the Adrenal Medulla
The cells of the adrenal medulla are derived from the neural crest as opposed to the mesodermal origins of the cortex. The medulla contains secretory cells, rich vasculature with a high surface area and presynaptic sympathetitic ganglion cells. These cells secrete epinephrine and norepinephrine in response to various substances such as acetylcholine.
There are multiple cell types of in the adult adrenal medulla:
- Chromaffin cells
- Epinephrine cells
- Norepinephrine cells
- Small granule-containing cells (SGCs)
These cells also produce various other peptides such as substance P and neurotensin. The adrenal medulla also contains presynaptic sympathetic ganglion cells. 
These cells have unique neuroendocrine characteristics as a result of their neural-crest origin. They receive preganglionic innervation by thoracic splanchnic nerves, and could almost be considered a specialised sympathetic ganglion. With postganglgionic bodies, cells of the medulla are densely clustered around their vasculature for the secretion of hormones into the bloodstream to moderate the concentration of epinephrine and norepinephrine.
The tissue and organ structure of the adrenal medulla should be understood with regard to its specialised function within the sympathetic nervous system.
The medullary region of the adrenal glands is tasked with the endocrine secretions of adrenaline and nor-adrenaline in response to environmental stressors that are signalled for by the sympathetic nervous system. That is, the secretion of fight-or-flight response hormones in order to restrict vasculature to the trunk and increase vascular activity in the peripheral musculature.
The adrenal glands are supplied by several branches of the great vessels in the abdominal cavity. The secreted catecholamines in the medulla are directly able to pass to the blood stream this way.
These adrenal glands and their contents are retroperitoneal in the adult and varied in shape, with the left often being semilunar and right being pyramidal. Nervous supply of the adrenal glands is achieved by contributions from the splanchnic nerves of the celiac plexus. 
Role of the Adrenal Medulla
Understanding the biochemistry of catecholamines is necessary to see how the adrenal medulla is designed in the stages of embryology and how it is suited for life in the neonate and adult.
The epinephrine and norepinephrine in the neonate and adult work by primarily changing the osmoregulatory state of vasculature in organs. With the exclusion of their neuromodulatory and neurotransmitter functions, epinephrine and norepinephrine are mainly tasked with the control of sympathetic and parasympathetic supplies.
Norepinephrine is a constrictor of peripheral vasculature by antagonising the action of surface receptors expressed on the endothelium of blood vessels, specifically Alpha-1 and Alpha-2 receptors, such that vascular resistance increases.
Epinephrine is both a vasoconstrictor and vasodilator, depending on what receptors it attaches to. As a non-selective adrenergic agonist, it acts on Alpha-1, Alpha-2, Beta-1, Beta-2 and Beta-3 receptors that are found throughout the body's tissues, yielding many different physiological responses. 
Genes and Transcription Factors Involved with the Adrenal Medulla's Development
The 'story' of the Adrenal Medulla's neural crest tissue begins at the process of neurulation, the formation of the neural tube from the folding neural plate, which is comprised of developing ectoderm which is opposite to the primitive streak. It is here that several key chemical players are at work. The classic interpretation of Sonic Hedgehog (Shh) and Wnt signalling pathways as the only mediators here, has been shown to be incorrect. Research has uncovered other signallers such as:
- Bone-Morphogenic-Protein-4 (BMP4) for the regional expression of nervous system distinctions in the growing neural plate. This maintains two important embryonic trancription factors known as Paired-Box (PAX) Genes 3 and 7 which can allow for expression of neural crest markers HNK-1, SRY-Box (SOX) 8, 9 and 10 as the neural tube closes and the neural crest delaminates from the folding. 
- Slug via Wnt signalling, this allows for the loosening of gap junctions in the extracellular matrix surrounding the newly-formed neural tube and trunk of the embryo. This extracellular matrix has chemo-attractant molecules
The importance of Somitogenesis and Neural Crest come to a head when neural crest cells must migrate anteriorly past somites 18 to 24
Members of the SOX gene family have multiple key contributors to Neural Crest in the Adrenal Medulla.
Mouse models have demonstrated that SOX-10 knock-outs (mice whose DNA has been engineered to not express SOX-10) do not have an adrenal medulla. There has also been suggestion that SOX-8,9 and 10 are all strongly related to the migratory patterning of neural crest cells to the developing adrenal glands. 
The TH gene (for Tyrosine Hydroxylase) encodes the aforementioned enzyme in the adrenal medulla and central nervous system. Tyrosine Hydroxylase is crucial in biochemical pathways and cascades for the synthesis of catecholamines and other CNS neurotransmitters. The conversion from L-Tyrosine to L-DOPA is dependent upon the catalytic action of Tyrosine Hydroxylase. From there, L-DOPA can make Dopamine and eventually Epinephrine and Norepinephrine as well. 
Abnormalities of the Adrenal Medulla are much less common than its mesoderm-derived shell of the Adrenal Gland. Three of the most notorious abnormalities of the Adrenal Medulla include:
Apart from these abnormalities, there are other pathologies that may be connected to the adrenal medulla. One such example is a pathology of the Organ of Zuckerkandl. Also known as the para-aortic body, the Organ of Zuckerkandl is a primarily-gestational organ comprised of chromaffin cells.
Animal models are an integral part of research in embryology. In particular due to the moral concerns regarding the use of humans in scientific research. As a result different animals are used as substitutes to simulate the same environments and hope to achieve results that would also be able to represent humans as well. Prenatal adrenal gland development has been described in a number of scientific reports dating from the 1900s to studies in more recent years. These involved a variety of animal models such as ox, sheep, swine and most commonly mice.
There has been extensive research conducted into the adrenal chromaffin cells and their embryological developments. As explained above nueral crest cells are pluripotent meaning they are able to differentiate into multiple different cells. The neural crest cells give rise to chromaffin cells of the adrenal medulla.
Previous research has reported that nueral crest cells differentiate into chromaffin cells around 19 days of gestation before forming the adrenal medulla. Past research had also suggested that neural crest derived adrenal gland cells migrate using the sympathetic ganglia to the adrenal gland or preaortic ganglion, following different pathways, to reach their specific destination. However the results have shown that the pathways used to reach its destinations overlapped. This would suggest that there is a possibility of the cells using the same pathway to move from the beginning production to the destination that is the adrenal medulla (for chromaffin cells) and the preaortic ganglion (other cells involved in the sympathetic system).
One particular study done by M.Yamato in 2004, along with his fellow scientists attempted to study the migration of neural crest cells in Wistar rats. In order to observe the formation adrenal medulla from the neural crest cells during the embryonic period, Yamato used anti - tyrosine hydroxylase (TH) antiserum injected into the rat embryos between 13-17 days of gestation. The anti - TH antiserum stain allows the scientists to visualize the neural crest cells under a light microscope. Later three dimensional images were created. From the images observed between 13 to 15 days of gestation, the TH - positive cells can be seen to move from the dorsal to the adrenal primordium. After 16 days it was observed that a capsule had formed in some parts and TH - positive cells had penetrated the adrenal gland. At seventeen days, TH - positive cells can be seen collecting at the medial part of the adrenal gland. The reconstructed images also shows that at the later stages during the 16-17 days of gestation, the accumulation of TH - positive cells grow and in combination with cell division leads to the formation of the adrenal medulla.
Chicken and rat models are often the most commonly used models when dealing with neural crest cells. In this particular review, collates multiple researchers results and presents evidence to discuss previous information suggesting that sympathetic neurons and chromaffin cells do not share a common progenitor. The relevant study involved the use of cell cultures, Sympathoadrneal (SA) progenitor cells from embryonic(from chick embryos) and eary developmental stages were able to be procured. From these cultures it was easy to identify that glucocorticoids were crucial in the differentiation of SA cells into their derivatives, sympathetic neurons and chromaffin cells.
Another study conducted deals with the role of the BMP-4 protein and its importance in the development of the adrenal medullary cells, chromaffin cells. Previous research had shown that BMP - 4 a growth factor is plays an important role in the regulation of chromaffin cell production. The researchers incubated chick embryos for 3 - 9 embryonic days and dissected them to be used for tissue cultures. From these
Cell culture studies using isolated SA progenitor cells from embryonic and early postnatal sympathetic ganglia and adrenal gland have suggested that glucocorticoids play a crucial role in the diversification of SA cells into sympathetic neurons and chromaffin cells [22-24]; for reviews see [2,5,6,25], by suppressing a neuronal differentiation programme in SA cells destined to become chromaffin cells. However, analysis of mice deficient for the glucocorticoid receptor  or lacking an adrenal cortex  revealed that chromaffin cell differentiation was largely unimpaired suggesting that cues unrelated to glucocorticoid receptor signalling and the adrenal cortex triggered the chromaffin phenotype. This notion was corroborated and extended by studies showing that chick SA progenitors are already heterogeneous in terms of neurofilament-M (NF-M) expression prior to populating the adrenal gland and sympathetic ganglia  with presumptive neuronal cells co-expressing tyrosine hydroxylase (TH) and NF-M, and presumptive neuroendocrine cells being TH-positive and NF-M-negative. This indicated that specification of the respective phenotypes occurred either during migration or even prior to migration at the level of the NT.
A recent study by Krispin et al. [7,29] showed that in the trunk NT of avian embryos, precursors for sympathetic ganglia, Schwann cells, sensory dorsal root ganglionic neurons, and melanocytes leave the NT in successive, largely non-overlapping waves, some being already distinct while still residing in the NT. Although adrenal chromaffin cells were not addressed in this study, the results raised the possibility that sympathetic neurons and chromaffin cells might also already be distinct at the level of the NT. We therefore conducted single cell electroporations (EPs) of green fluorescent protein (GFP)-DNA into pre-migratory NC cells at the level of somites 18–24 (“adrenomedullary level”), where both chromaffin cells and sympathetic neurons arise . The timing and location of EPs was directed to label the earliest progenitors that undergo delamination, previously shown to generate the sympathetic lineage [7,29], and the progeny of labelled cells was then analysed at E6 following homing to their target areas. Our results indicate that in more than 80% of the cases the progeny of a single labelled cell ends up in both sympathetic ganglia and adrenal gland, being mostly NF-M-negative in the adrenal gland and NF-M-positive in sympathetic ganglia. This suggests that chromaffin cells and sympathetic neurons share a common progenitor in the NT and that diversification of the phenotypes occurs during migration or assembly in primary sympathetic ganglia at the dorsal aorta. The sympathoadrenal (SA) cell lineage is a major sub-lineage of the NC that gives rise to sympathetic neurons, intra- and extra-adrenal chromaffin cells, and the small intensely fluorescent (SIF) cells, which are intermediate between sympathetic neurons and chromaffin cells [2-5]. SA derivatives share several features, including the ability to synthesize, store, and release catecholamines, but also exhibit traits specific for each cell type, such as axons, dendrites, and neurofilaments, which are only found in sympathetic neurons but not chromaffin cells.
Prenatal adrenal gland development has been described in numerous published reports covering a wide variety of species, including the ox (Katznelson, 1966; Wrobel and Suss, 1999), sheep (Davies, 1950; Wintour et al., 1975; Upadhyay and Zamboni, 1982; Naaman‐Reperant and Durand, 1997; Grino, 2004), swine (Sokolov et al., 2006) and mouse (Waring, 1935; Ikeda et al., 1994; Sass, 1996; Nyska and Maronpot, 1999; Bland et al., 2003; Val et al., 2007). Adrenal gland development also has been extensively described for humans as well (Kolliker, 1879; Minot, 1892; Zuckerkandl, 1912; Wieman, 1920; Ikeda et al., 1981; Langlois et al., 2002; Hanley and Arit, 2006).
Current research (labs)
Sympathoadrenal neural crest cells
As explained above the adrenal medulla plays an important part in the autonomic system of an adult human. The chromaffin cells, in particular, are important neural crest derived cell that is closely related to the adrenal medulla. Previous findings have focused specifically on the mechanisms controlling the NCC migration and how these cells go on to form the different derivatives in their particular destinations in the embryo. For example, as previously explained studies have shown that chromaffin cells have been observed to move to the adrenal gland to form the medulla of the adrenal gland.
The findings presented until now have focused on the mechanisms controlling migration along the dorsolateral path. In comparison, our understanding of the mechanisms controlling NCC migration along the ventral path are complicated by the fact that this population comprises multiple precursors that form different derivatives in distinct regions of the embryo. For example, ventrally migrating NCCs either stop within the sclerotome in close association with the neural tube to form neurons and glia of the sensory nervous system (i.e. the dorsal root ganglia [DRG]), while other NCCs continue to migrate further ventrally to form the autonomic nervous system (i.e. sympathetic ganglia and chromaffin cells of the adrenal gland).
Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type.
The autonomic nervous system is very important in understanding the human body and in turn understanding health. This report goes through a recent study that involves the adrenal medullary cells, in particular chromaffin cells, and their potential to be used to model certain pathological diseases related to these cells. This is part of a study that also involves neural crest cells and their ability to differentiate into multiple different types of cells.
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