Temporal restriction of migratory and lineage potential in rhombomere 1 and 2 neural crest

Migratory cranial neural crest cells differentiate into a wide range of cell types, such as ectomesenchymal tissue (bone and connective tissues) ventrally in the branchial arches and neural tissue (neurons and glia) dorsally. We investigated spatial and temporal changes of migration and differentiation potential in neural crest populations derived from caudal midbrain and rhombomeres 1 and 2 by back-transplanting cells destined for the first branchial arch and trigeminal ganglion from HH8-HH19 quail into HH7-HH11 chicks. Branchial arch cells differentiated down ectomesenchymal lineages but largely lost both the ability to localize to the trigeminal position and neurogenic differentiation capacity by HH12-HH13, even before the arch is visible, and lost long distance migratory ability around HH17. In contrast, neural crest-derived cells from trigeminal ganglia lost ectomesechymal differentiation potential by HH17. Despite this, they retain the ability to migrate into the branchial arches until at least HH19. However, many of the neural crest-derived trigeminal ganglia cells in the branchial arch localized to the non-neural crest core of the arch from HH13 and older donors. These results suggest that long distance migration ability, finer scale localization, and lineage restriction may not be coordinately regulated in the cranial neural crest population.     

Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.     

Spatial and temporal segregation of auditory and vestibular neurons in the otic placode

The otic placode generates the auditory and vestibular sense organs and their afferent neurons; however, how auditory and vestibular fates are specified is unknown. We have generated a fate map of the otic placode and show that precursors for vestibular and auditory cells are regionally segregated in the otic epithelium. The anterior-lateral portion of the otic placode generates vestibular neurons, whereas the posterior-medial region gives rise to auditory neurons. Precursors for vestibular and auditory sense organs show the same distribution. Thus, different regions of the otic placode correspond to particular sense organs and their innervating neurons. Neurons from contiguous domains rarely intermingle suggesting that the regional organisation of the otic placode dictates positional cues to otic neurons. But, in addition, vestibular and cochlear neurogenesis also follows a stereotyped temporal pattern. Precursors from the anterior-lateral otic placode delaminate earlier than those from its medial-posterior portion. The expression of the proneural genes NeuroM and NeuroD reflects the sequence of neuroblast formation and differentiation. Both genes are transiently expressed in vestibular and then in cochlear neuroblasts, while differentiated neurons express Islet1, Tuj1 and TrkC, but not NeuroM or NeuroD. Together, our results indicate that the position of precursors within the otic placode confers identity to sensory organs and to the corresponding otic neurons. In addition, positional information is integrated with temporal cues that coordinate neurogenesis and sensory differentiation.     

A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells

Neural crest (NC) cells have been elegantly traced to follow stereotypical migratory pathways throughout the vertebrate embryo, yet we still lack complete information on individual cell migratory behaviors and how molecular mechanisms direct NC cell guidance. Here, we analyze the spatio-temporal migratory pattern of post-otic NC and the in vivo role of the small Rho GTPase, RhoA, using fluorescent cell labeling, molecular perturbation, and intravital 4D (3D+ time) confocal imaging in the intact chick embryo. We find that the post-otic NC cell migratory pattern is established in two phases with distinct cell migratory behaviors. An initial wide front of lateral-directed NC cells, led by NC from rhombomere 7 (r7), move as a distinct subpopulation. This is followed in time by fewer NC cells that migrate collectively from r7 to r8 in a follow-the-leader manner with extensive cellular extensions between cells. We show that post-otic migratory NC cells express RhoA, using RT-PCR on isolated, flow cytometry sorted NC cells and in neural tube culture explants. When RhoA function is altered by expression of a dominant negative or constitutively active form, or injection of C3, there are two major consequences. RhoA constitutively active expressing NC cells are less directional, slower and form fewer follow-the-leader chain assemblies. NC cells expressing RhoA-DN are less affective in retracting filopodia, migrate slower and also form fewer follow-the-leader chain assemblies. Together, these alterations to NC cell intrinsic signaling and cell-cell contact disrupt the precise spatio-temporal post-otic NC cell migratory pattern.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprung's disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.     

周围神经或其组织成分移植修复成年哺乳动物视网膜节细胞的损伤

本综述重点阐述了移植周围神经或其组织成分雪旺细胞、成纤维细胞和神经营养因子,改善成年哺乳动物中枢神经系统抑制神经再生的微环境、增强受损神经元的内在再生潜力,以促进细胞损伤后的存活和轴突再生。     

Effects of antibodies to nerve growth factor on intrauterine development of derivatives of cranial neural crest and placode in the guinea pig

Fetal guinea pigs transplacentally exposed to maternal nerve growth factor antibodies in the latter part of gestation show marked depletion of sensory neurons in the trigeminal ganglion. Sensory neurons of the nodose ganglion and spiral ganglion which are derived from placodes, and parasympathetic motor neurons of the ciliary, otic, and sphenopalatine ganglia which are derived, like the bulk of the trigeminal ganglion, from cranial neural crest, are unaffected by the antibodies. Previous studies showed that sensory and some sympathetic derivatives of spinal neural crest are effected but that more peripherally located structures of similar origin are not. The local microenvironment in the fetus appears to alter the NGF requirements of structures derived from the same primordia. The model described provides a useful means of studying the effect of trophic factor inhibition in the natural fetal setting and is free of many potential artifacts of tissue culture. Comparison of the animal results with the pathology of familial dysautonomia indicates that nerve growth factor dysfunction alone does not, in our current state of knowledge, adequately account for the etiology of the disease.     

Nodose placode provides ectomesenchyme to the developing chick heart in the absence of cardiac neural crest

Summary The cardiac neural crest provides both ectomesenchyme and parasympathetic postganglionic neurons to the developing heart. Ablation of cardiac neural crest results in persistent truncus arteriosus, a condition in which the conotruncal and aorticopulmonary septa do not form in the developing heart. Parasympathetic postganglionic neurons are abundantly present in hearts with persistent truncus arteriosus, which indicates a regeneration of the neural component of the cardiac neural crest without comparable restitution of the ectomesenchymal component. The neural component has been shown to be provided by cells from the nodose placode following ablation of the cardiac neural crest. This investigation has shown that ectomesenchymal cells are also supplied to a limited extent by the nodose placode which normally has no ectomesenchymal derivatives. Although placode-derived ectomesenchyme helps to strengthen the wall of the cardiac outflow vessel, it is not competent to induce conotruncal and aorticopulmonary septal closure.     

Molecular mechanisms of projection neuron production and maturation in the developing cerebral cortex

The cerebral cortex is a brain structure unique to mammals and highly adapted to process complex information. Through multiple developmental steps, the cerebral cortex is assembled as a huge diversity of neurons comprising a complex laminar structure, and with both local and long-distance connectivity within the nervous system. Key processes must take place during its construction, including: (i) regulation of the correct number of neurons produced by progenitor cells, (ii) temporal and spatial generation of neuronal diversity, and (iii) control of neuron migration and laminar positioning as well as terminal differentiation within the mature cortex. Here, we seek to highlight recent cellular and molecular findings underlying these sequential steps of neurogenesis, cell fate specification and migration during cortical development, with particular emphasis on cortical projection neurons.     

Enhanced nerve growth factor efficiency in neural cell culture by immobilization on the culture substrate

Nerve growth factor (NGF) immobilization on a culture substrate may dramatically reduce the amount of NGF required for pheochromocytoma (PC12) cell culture. Coverslips on which NGF had been immobilized, or with NGF added to the culture medium daily, were used to culture PC12 cells. We examined the effects of adding 5, 10, or 100 ng of NGF to cultures daily, and compared them to the effects of immobilizing 5, 10, or 100 ng of NGF on culture substrates in a single dose. Cultures with 10 or 5 ng NGF added daily showed dramatically decreased cell viability, mitochondrial metabolic activity, and neuronal differentiation compared to cultures with 100 ng NGF added daily, while also exhibiting increased apoptosis. In contrast, a single dose of 100 ng immobilized NGF yielded results similar to 100 ng NGF added daily (total: 300 ng over 3 days), and 10 or 5 ng immobilized NGF showed far better results than 10 or 5 ng NGF added daily. These results demonstrate that NGF immobilization can dramatically reduce the amount of NGF required in neuronal cell culture.     

Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats

To investigate the short-and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent market diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or in intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an inital abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A. persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons. © 1993 John Wiley & Sons, Inc.     

Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system

We have systematically examined the developmental potential of neural crest stem cells from the enteric nervous system (gut NCSCs) in vivo to evaluate their potential use in cellular therapy for Hirschsprung disease and to assess differences in the properties of postmigratory NCSCs from different regions of the developing peripheral nervous system (PNS). When transplanted into developing chicks, flow-cytometrically purified gut NCSCs and sciatic nerve NCSCs exhibited intrinsic differences in migratory potential and neurogenic capacity throughout the developing PNS. Most strikingly, gut NCSCs migrated into the developing gut and formed enteric neurons, while sciatic nerve NCSCs failed to migrate into the gut or to make enteric neurons, even when transplanted into the gut wall. Enteric potential is therefore not a general property of NCSCs. Gut NCSCs also formed cholinergic neurons in parasympathetic ganglia, but rarely formed noradrenergic sympathetic neurons or sensory neurons. Supporting the potential for autologous transplants in Hirschsprung disease, we observed that Endothelin receptor B (Ednrb)-deficient gut NCSCs engrafted and formed neurons as efficiently in the Ednrb-deficient hindgut as did wild-type NCSCs. These results demonstrate intrinsic differences in the migratory properties and developmental potentials of regionally distinct NCSCs, indicating that it is critical to match the physiological properties of neural stem cells to the goals of proposed cell therapies.     

A ciliary neuronotrophic factor from peripheral nerve and smooth muscle which is not retrogradely transported

We have found that a CNTF-like molecule which supports ciliary and sympathetic neurons is not retrogradely transported in either sympathetic or parasympathetic nerves. The factor has an apparent M_r of 21 kDa, a pI of 4.9, and is present in peripheral nerves and smooth muscle of the chick. Our experiments indicate that CNTF-like activity does not accumulate on the distal side of ligated chickexpansor nerves. In contrast, there is a clear accumulation of NGF. The activity further differs from NGF in that it is not removed from a smooth muscle of the chick wing by innervating sympathetic fibers. Transection of these fibers does not lead to an accumulation of ciliary activity in theexpansor secundariorum muscle, suggesting that neurons do not actively deplete the muscle of factor by retrograde transport. Finally, recombinant CNTF or semi-purified preparations of CNTF-like activity labelled with¹²⁵I were not transported to the ciliary ganglion of chicks following injection of biologically active material into the eye. Our results suggest either that endogenous CNTF does not act as a survival factorin vivo, or that retrograde transport is not a property inherent to all neuronotrophic molecules.Special issue dedicated to Dr. Lawrence Austin     

Alterations in the morphology of ganglion cell dendrites in the adult rat retina after optic nerve transection and grafting of peripheral nerve segments

Summary Transected ganglion cell axons from the adult retina are capable of reinnervating their central targets by growing into transplanted peripheral nerve (PN) segments. Injury of the optic nerve causes various metabolic and morphological changes in the retinal ganglion cell (RGC) perikarya and in the dendrites. The present work examined the dendritic trees of those ganglion cells surviving axotomy and of those whose severed axons re-elongated in PN grafts to reach either the superior colliculus (SC), transplanted SC, or transplanted autologous thigh muscle. The elaboration of the dendritic trees was visualized by means of the strongly fluorescent carbocyanine dye DiI, which is taken up by axons and transported to the cell bodies and from there to the dendritic branches. Alternatively, retinofugal axons regrowing through PN grafts were anterogradely filled from the eye cup with rhodamine B-isothiocyanate. The transection of the optic nerve resulted in characteristic changes in the ganglion cell dendrites, particularly in the degeneration of most of the terminal and preterminal dendritic branches. This occurred within the first 1 to 2 weeks following axotomy. The different types of ganglion cells appear to vary in their sensitivity to axotomy, as reflected by a rapid degeneration of certain cell dendrites after severance of the optic nerve. The most vulnerable cells were those with small perikarya and small dendritic fields (type II), whereas larger cells with larger dendritic fields (type I and III) were slower to respond and less dramatically affected. Regrowth of the lesioned axons in peripheral nerve grafts and reconnection of the retina with various tissues did not result in a significant immediate recovery of ganglion cell dendrites, although it did prevent some axotomized cells from further progression toward posttraumatic cell death.