Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning.
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Cranial neural crest cells generate the distinctive bone and connective tissues in the vertebrate head. Classical models of craniofacial development argue that the neural crest is prepatterned or preprogrammed to make specific head structures before its migration from the neural tube. In contrast, recent studies in several vertebrates have provided evidence for plasticity in patterning neural crest populations. Using tissue transposition and molecular analyses in avian embryos, we reconcile these findings by demonstrating that classical manipulation experiments, which form the basis of the prepatterning model, involved transplantation of a local signaling center, the isthmic organizer. FGF8 signaling from the isthmus alters Hoxa2 expression and consequently branchial arch patterning, demonstrating that neural crest cells are patterned by environmental signals. (+info)
Biotinylated dextran amine as a marker for fetal hypothalamic homografts and their efferents.
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We have explored the use of biotinylated dextran amine (BDA) as a marker for labeling fetal brain grafts and their connections with the host. As a model system we used transplantation of the hamster suprachiasmatic nucleus, the site of an endogenous biological clock governing circadian rhythms. Similar transplants into arrhythmic hosts have been shown to restore behavioral function with a period specific to the donor. For locomotor rhythms, efferent connections are not necessary. For other responses, including endocrine rhythms, efferent connections may be necessary. In order to visualize homografts and their efferents, injections of BDA, an anterograde tracer, were made into the anterior hypothalamic (AH) region containing the SCN or into the dorsal cortex (CTX) of fetal hamster brains. The fetal AH or CTX was microdissected out and stereotaxically implanted into the third ventricle of intact, adult hamsters. After 2, 4, 8, or 12 weeks, hosts were sacrificed and their brains were processed for detection of BDA by either histochemistry or immunofluorescence. BDA intensely labeled graft neurons, their dendrites, and axons with minimal or no spread to the adjacent host brain. Labeled graft axons could be followed for long distances (>1 mm) into the host brain and graft-derived varicosities formed close contacts with host neurons. BDA-labeled graft neurons, located at the perimeter of the graft, also extended dendrite-like processes into the host parenchyma. We conclude that BDA is a useful marker for fetal homografts and their efferents for survival times of less than 2 months. (+info)
Reelin function in neural stem cell biology.
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In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients. (+info)
Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats.
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The potential use of in vitro-expanded precursor cells or cell lines in repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide "self-repair." Recently, we have reported that cells from the brain-derived cell line RN33B (derived from the embryonic rat medullary raphe and immortalized through retroviral transduction of the temperature-sensitive mutant of the simian virus 40 ([SV40] large T-antigen) survive for at least 4 weeks, integrate, and differentiate after subretinal grafting to normal adult rats. Here, we demonstrate that grafts of these cells survive for at least 4 months after subretinal transplantation to adult, normal immunosuppressed rats. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve. In addition, the RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A large fraction of the grafted cells differentiate into glial cells, as shown by double labeling of the reporter genes LacZ or green fluorescent protein, and several glial markers, including oligodendrocytes. However, the cells did not differentiate into retinal neurons, judging from their lack of expression of retinal neuronal phenotypic markers. A significant number of the implanted cells in the host retina were in a proliferative stage, judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. To conclude, the cells survived, integrated, and migrated over long distances within the host. Therefore, our results may be advantageous for future design of therapeutic strategies, since such cells may have the potential of being a source of, for example, growth factor delivery in experimental models of retinal degeneration. (+info)
Late-stage immature neocortical neurons reconstruct interhemispheric connections and form synaptic contacts with increased efficiency in adult mouse cortex undergoing targeted neurodegeneration.
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In the neocortex, the effectiveness of potential cellular repopulation therapies for diseases involving neuronal loss may depend critically on whether newly incorporated cells can differentiate appropriately into precisely the right kind of neuron, re-establish precise long-distance connections, and reconstruct complex functional circuitry. Here, we test the hypothesis that increased efficiency of connectivity could be achieved if precursors could be more fully differentiated toward desired phenotypes. We compared embryonic neuroblasts and immature murine neurons subregionally dissected from either embryonic day 17 (E17) (Shin et al., 2000) or E19 primary somatosensory (S1) cortex and postnatal day 3 (P3) purified callosal projection neurons (CPNs) with regard to neurotransmitter and receptor phenotype and afferent synapse formation after transplantation into adult mouse S1 cortex undergoing targeted apoptotic degeneration of layer II/III and V CPNs. Two weeks after transplantation, neurons from all developmental stages were found dispersed within layers II/III and V, many with morphological features typical of large pyramidal neurons. Retrograde labeling with FluoroGold revealed that 42 +/- 2% of transplanted E19 immature S1 neurons formed connections with the contralateral S1 cortex by 12 weeks after transplantation, compared with 23 +/- 7% of E17 neurons. A greater percentage of E19-derived neurons received synapses (77 +/- 1%) compared with E17-derived neurons (67 +/- 2%). Similar percentages of both E17 and E19 donor-derived neurons expressed neurotransmitters and receptors [glutamate, aspartate, GABA, GABA receptor (GABA-R), NMDA-R, AMPA-R, and kainate-R] appropriate for endogenous adult CPNs progressively over a period of 2-12 weeks after transplantation. Although P3 fluorescence-activated cell sorting-purified neurons also expressed these mature phenotypic markers after transplantation, their survival in vivo was poor. We conclude that later-stage and region-specific immature neurons develop a mature CPN phenotype and make appropriate connections with recipient circuitry with increased efficiency. However, at postnatal stages of development, limitations in survival outweigh this increased efficiency. These results suggest that efforts to direct the differentiation of earlier precursors precisely along specific desired neuronal lineages could potentially make possible the highly efficient reconstruction of complex neocortical and other CNS circuitry. (+info)
FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain.
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The segmentation of the vertebrate hindbrain into rhombomeres is highly conserved, but how early hindbrain patterning is established is not well understood. We show that rhombomere 4 (r4) functions as an early-differentiating signaling center in the zebrafish hindbrain. Time-lapse analyses of zebrafish hindbrain development show that r4 forms first and hindbrain neuronal differentiation occurs first in r4. Two signaling molecules, FGF3 and FGF8, which are both expressed early in r4, are together required for the development of rhombomeres adjacent to r4, particularly r5 and r6. Transplantation of r4 cells can induce expression of r5/r6 markers, as can misexpression of either FGF3 or FGF8. Genetic mosaic analyses also support a role for FGF signaling acting from r4. Taken together, our findings demonstrate a crucial role for FGF-mediated inter-rhombomere signaling in promoting early hindbrain patterning and underscore the significance of organizing centers in patterning the vertebrate neural plate. (+info)
Ensheathing cells and methylprednisolone promote axonal regeneration and functional recovery in the lesioned adult rat spinal cord.
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Axons fail to regenerate after spinal cord injury (SCI) in adult mammals, leading to permanent loss of function. After SCI, ensheathing cells (ECs) promote recovery in animal models, whereas methylprednisolone (MP) promotes neurological recovery in humans. In this study, the effectiveness of combining ECs and MP after SCI was investigated for the first time. After lesioning the corticospinal tract in adult rats, ECs were transplanted into the lesion, and MP was administered for 24 hr. At 6 weeks after injury, functional recovery was assessed by measuring successful performance of directed forepaw reaching (DFR), expressed as percentages. Axonal regeneration was analyzed by counting the number of corticospinal axons, anterogradely labeled with biotin dextran tetramethylrhodamine, caudal to the lesion. Lesioned control rats, receiving either no treatment or vehicle, had abortive axonal regrowth (1 mm) and poor DFR success (38 and 42%, respectively). Compared with controls, MP-treated rats had significantly more axons 7 mm caudal to the lesion, and DFR performance was significantly improved (57%). Rats that received ECs in combination with MP had significantly more axons than all other lesioned rats up to 13 mm. Successful DFR performance was significantly higher in rats with EC transplants, both without (72%) and with (78%) MP, compared with other lesioned rats. These data confirm previous reports that ECs promote axonal regeneration and functional recovery after spinal cord lesions. In addition, this research provides evidence that, when used in combination, MP and ECs improve axonal regrowth up to 13 mm caudal to the lesion at 6 weeks after injury. (+info)
Suppression of the melanogenic potential of migrating neural crest-derived cells by the branchial arches.
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The development of melanocytes from neural crest-derived precursors that migrate along the dorsolateral pathway has been attributed to the selection of this route by cells that are fate-restricted to the melanocyte lineage. Alternatively, melanocytes could arise from nonspecified cells that develop in response to signals encountered while these cells migrate, or at their final destinations. In most animals, the bowel, which is colonized by crest-derived cells that migrate through the caudal branchial arches, contains no melanocytes; however, the enteric microenvironment does not prevent melanocytes from developing from crest-derived precursors placed experimentally into the bowel wall. To test the hypothesis that the branchial arches remove the melanogenic potential from the crest-derived population that colonizes the gut, the Silky fowl (in which the viscera are pigmented) was studied. Sources of crest included Silky fowl and quail vagal and truncal neural folds/tubes, which were cultured or explanted to chorioallantoic membranes alone or together with branchial arches or limb buds from Silky fowl, White Leghorn, or quail embryos. Crest and mesenchyme-derived cells were distinguished by using the quail nuclear marker. Melanocytes developed from Silky fowl and quail crest-derived cells. Melanocyte development from both sources was inhibited by quail and White Leghorn branchial arches (and limb buds), but melanocyte development was unaffected by branchial arch (and limb buds) from Silky fowl. These observations suggest that a factor(s) that is normally expressed in the branchial arches, and is lacking in animals with the Silky mutation, prevents cells with a melanogenic potential from colonizing the bowel. (+info)