Extra-Cutaneous Melanin

Extra-cutaneous melanocytes derive from either the neural crest, the outer wall of the optic cup, or the cranial neural tube. Those of neural crest origin reach most bodily regions, and may give rise to primary melanoma in various tissues. The Kupffer cell produces a form of melanin, but is hardly a melanocyte. Melanocytes of the internal ear may be concerned with secretion of endolymph, trans-epithelial ion transport, and with protection against ototoxic drugs and high-intensity noise damage. There is evidence from albino animals that retinal pigment epithelium determines co-ordinates of the neural retina, and its decussation pattern during development. Neuromelanin derives from Dopamine, and is found in dopaminergic neurons widely distributed throughout the brain-stem and hypothalamus, and which project to the striatum and limbic system. Parkinsonism is due to degeneration of melanin-containing dopaminergic neurons of locus coeruleus and substantia nigra, and MPTP provides an investigative probe for studying the causes of Parkinsonism. Neuromelanin should not be regarded as a waste-product, but as something which can affect the firing properties of neurons with specific functional effect.     

Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors

In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation.In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.     

Parkinson's disease: studies with an animal model

Parkinson' disease has been associated with degeneration of dopamine-containing neurons of the nigrostriatal bundle. Many neurological features of Parkinsonism can be produced in rats by selective destruction of central dopaminergic neurons using the neurotoxin 6-hydroxydopamine. In this review we discuss two aspects of Parkinson's disease that have been investigated in these animals. First, we consider why near-total degeneration of nigrostriatal bundle neurons is required before neurological symptoms emerge. It appears that the loss of dopaminergic neurons is accompanied by an exponential increase in the ratio of tyrosine hydroxylase activity to dopamine content. Thus, after the brain lesions there may be a compensatory increase in the capacity of residual dopaminergic neurons to synthesize and release transmitter. Second, we consider why stress produces severe neurological deficits in patients who are only mildly impaired otherwise. It appears that a variety of stressors produce an abrupt but transient increase in dopaminergic activity in the striatum of intact animals and that this increase is markedly attenuated by 6-hydroxydopamine treatment. Thus, stress-induced akinesia in animals with dopamine-depleting brain lesions and in Parkinsonian patients may result from the impaired ability of residual neurons to respond approximately to such stimuli.     

In vitro analysis of sympathetic neuron differentiation from chick neural crest cells

Sympathetic neuron differentiation was studied using a fluorescence histochemical assay to detect the appearance of cell-bound catecholamines. Results from in vitro organ cultures indicate that chick neural crest cells must interact with both ventral neural tube (defined throughout as the ventral neural tube plus the notochord) and somitic mesenchyme in order to differentiate into sympathoblasts. Somite, ventral neural tube, and crest were cultured transfilter in various combinations to define these tissue interactions more precisely. Results from these experiments indicate that neural crest cells must be contiguous to somite in order to differentiate into sympathoblasts, but ventral neural tube may act across a Millipore filter membrane (type TH, 25 μm thick) either on somite, crest, or both. To distinguish among these possibilities, somite was cultured transfilter to ventral tube for a short period, after which ventral tube was removed and fresh crest was added to the somite. The results from this and other experiments support the hypothesis that the ventral tube does not act directly on crest cells, but elicits a developmental change in somitic mesenchyme, which then promotes sympathoblast differentiation. To study the relationship of nerve growth factor (NGF) to the differentiation of sympathetic neurons, cultures of somite + crest were temporarily exposed transfilter to ventral tube, in the presence or the absence of exogenous NGF. The results of these and other experiments are consistent with the hypothesis that the continued presence of ventral tube is required to ensure the survival of the differentiating sympathetic neurons. With respect to this second function, ventral tube can be replaced by exogenous NGF.     

The control of avian cephalic neural crest cytodifferentiation. II. Neural tissues.

A series of neural crest transplantations has been performed to (1) analyze whether avian premigratory cranial neural crest cells are pluripotential or restricted to specific developmental pathways and (2) examine the ability of trunk neural crest cells to develop in an environment usually occupied by cranial crest cells. Quail embryos, the cells of which have a unique nuclear marker, were used as donors and chick embryos as hosts. Hindbrain crest cells grafted in the place of diencephalic crest cells failed to form neurons in all but one case, in which a small ectopic ganglion was found. In the reciprocal transplants, neural crest cells emigrating from a segment of forebrain crest tissue grafted in the place of metencephalic crest cells produced trigeminal and ciliary ganglia which were completely normal. Thus, crest cells which normally never form ganglionic neurons will do so if placed in a suitable neurogenic environment. These results prove that premigratory avian cranial crest cells are not restricted to specific developmental pathways, but are initially pluripotential. Trunk crest cells grafted in the place of metencephalic crest cells form neuronal ganglia along the proximal trigeminal motor roots but do not form normal trigeminal ganglia. These root ganglia do not display normal peripheral projections, and placode cells, a normal component of the trigeminal ganglion, form ganglia in ectopic locations. Thus, while trunk crest cells respond to the metencephalic environment and form neurons, their response is different from that of cranial crest cells in the same location. Whether this is due to differences in developmental potential or in initial population size is not known.     

Origin and evolution of the neural crest: a hypothetical reconstruction of its evolutionary history

The neural crest has long been regarded as one of the key novelties in vertebrate evolutionary history. Indeed, the vertebrate characteristic of a finely patterned craniofacial structure is intimately related to the neural crest. It has been thought that protochordates lacked neural crest counterparts. However, recent identification and characterization of protochordate genes such as Pax3/7, Dlx and BMP family members challenge this idea, because their expression patterns suggest remarkable similarity between the vertebrate neural crest and the ascidian dorsal midline epidermis, which gives rise to both epidermal cells and sensory neurons. The present paper proposes that the neural crest is not a novel vertebrate cell population, but may have originated from the protochordate dorsal midline epidermis. Therefore, the evolution of the vertebrate neural crest should be reconsidered in terms of new cell properties such as pluripotency, delamination-migration and the carriage of an anteroposterior positional value, key innovations leading to development of the complex craniofacial structure in vertebrates. Molecular evolutionary events involved in the acquisitions of these new cell properties are also discussed. Genome duplications during early vertebrate evolution may have played an important role in allowing delamination of the neural crest cells. The new regulatory mechanism of Hox genes in the neural crest is postulated to have developed through the acquisition of new roles by coactivators involved in retinoic acid signaling.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.     

Nigral degeneration correlates with persistent activation of cerebellar Purkinje cells in MPTP-treated monkeys

In the present work we analyze the cerebellum of chronic parkinsonian monkeys in order to clarify whether chronic mesencephalic depletion is associated with long term activation of the cerebellar neurons in chronic Parkinsonism. In our study, we observed a persistent activation of Purkinje cells in the cerebellum of chronic parkinsonian macaques, characterized by the expression of c-Fos, which correlated with dopaminergic degeneration. These results are compatible with the results observed in fMRI in Parkinson's disease patients, and may contribute to the understanding of additional alterations in the brain circuitry in Parkinsonism.     

Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons.

In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.     

Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis

Neural crest cells arising from different rostrocaudal axial levels form different sets of derivatives as diverse as ganglia, cartilage and cornea. These variations may be due to intrinsic properties of the cell populations, different environmental factors encountered during migration or some combination thereof. We test the relative roles of intrinsic versus extrinsic factors by challenging the developmental potential of cardiac and trunk neural crest cells via transplantation into an ectopic midbrain environment. We then assess long-term survival and differentiation into diverse derivatives, including cornea, trigeminal ganglion and branchial arch cartilage. Despite their ability to migrate to the periocular region, neither cardiac nor trunk neural crest contribute appropriately to the cornea, with cardiac crest cells often forming ectopic masses on the corneal surface. Similarly, the potential of trunk and cardiac neural crest to form somatosensory neurons in the trigeminal ganglion was significantly reduced compared with control midbrain grafts. Cardiac neural crest exhibited a reduced capacity to form cartilage, contributing only nominally to Meckle's cartilage, whereas trunk neural crest formed no cartilage after transplantation, even when grafted directly into the first branchial arch. These results suggest that neural crest cells along the rostrocaudal axis display a graded loss in developmental potential to form somatosensory neurons and cartilage even after transplantation to a permissive environment. Hox gene expression was transiently maintained in the cardiac neural tube and neural crest at 12 hours post-transplantation to the midbrain, but was subsequently downregulated. This suggests that long-term differences in Hox gene expression cannot account for rostrocaudal differences in developmental potential of neural crest populations in this case.     

Altered neuronal lineages in the facial ganglia of Hoxa2 mutant mice

Neurons of cranial sensory ganglia are derived from the neural crest and ectodermal placodes, but the mechanisms that control the relative contributions of each are not understood. Crest cells of the second branchial arch generate few facial ganglion neurons and no vestibuloacoustic ganglion neurons, but crest cells in other branchial arches generate many sensory neurons. Here we report that the facial ganglia of Hoxa2 mutant mice contain a large population of crest-derived neurons, suggesting that Hoxa2 normally represses the neurogenic potential of second arch crest cells. This may represent an anterior transformation of second arch neural crest cells toward a fate resembling that of first arch neural crest cells, which normally do not express Hoxa2 or any other Hox gene. We additionally found that overexpressing Hoxa2 in cultures of P19 embryonal carcinoma cells reduced the frequency of spontaneous neuronal differentiation, but only in the presence of cotransfected Pbx and Meis Hox cofactors. Finally, expression of Hoxa2 and the cofactors in chick neural crest cells populating the trigeminal ganglion also reduced the frequency of neurogenesis in the intact embryo. These data suggest an unanticipated role for Hox genes in controlling the neurogenic potential of at least some cranial neural crest cells.     

Early segregation of a neuronal precursor cell line in the neural crest as revealed by culture in a chemically defined medium

This article addresses the problem of the segregation of cell lines during the development of peripheral nervous system components from the neural crest. We show here that committed precursors of peripheral neurons are present in the crest before the migration of its cells has started. If cultured in a serum-deprived medium, a subpopulation of the crest cells readily differentiates into neurons without dividing. Neuronal markers such as neurofilament proteins and receptor sites for tetanus toxin are not expressed in the committed neuronal precursors, but appear after a few hours in culture. They are coexpressed in neurons with the mesenchymal intermediate filament protein, vimentin, which is common to all neural crest cells regardless of their prospective fate. A strong inhibitory effect of serum factor(s) on neurite outgrowth is demonstrated. We show also that conditions stimulating proliferation of crest cells are incompatible with promotion of neuronal differentiation and vice-versa.     

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.     

Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Recovery from Parkinsonism with N-acetylcysteine-differentiated neurons

The upregulation of dopaminergic neuronal differentiation is necessary for stem cell therapy in Parkinson’s disease (PD). In this study, neuronal differentiation efficiency increased by more than 2 times in P19 embryonic stem cells (ESCs) induced by N-acetylcysteine (NAC) and retinoic acid (RA) as compared to RA alone, with suppressed glial differentiation. The majority of NAC-treated stem cells grafted into brains of PD mice differentiated into dopaminergic neurons and persisted well for 6 weeks. Parkinsonism was also greatly improved after grafting NAC-treated cells in comparison to cells treated with only RA. Our results strongly suggest that NAC treatment may be an effective strategy for generating stem cells fated to become dopaminergic neurons for PD clinical therapy.