Wnt signal specifies the intrathalamic limit and its organizer properties by regulating Shh induction in the alar plate.

The structural complexity of the brain depends on precise molecular and cellular regulatory mechanisms orchestrated by regional morphogenetic organizers. The thalamic organizer is the zona limitans intrathalamica (ZLI), a transverse linear neuroepithelial domain in the alar plate of the diencephalon. Because of its production of Sonic hedgehog, ZLI acts as a morphogenetic signaling center. Shh is expressed early on in the prosencephalic basal plate and is then gradually activated dorsally within the ZLI. The anteroposterior positioning and the mechanism inducing Shh expression in ZLI cells are still partly unknown, being a subject of controversial interpretations. For instance, separate experimental results have suggested that juxtaposition of prechordal (rostral) and epichordal (caudal) neuroepithelium, anteroposterior encroachment of alar lunatic fringe (L-fng) expression, and/or basal Shh signaling is required for ZLI specification. Here we investigated a key role of Wnt signaling in the molecular regulation of ZLI positioning and Shh expression, using experimental embryology in ovo in the chick. Early Wnt expression in the ZLI regulates Gli3 and L-fng to generate a permissive territory in which Shh is progressively induced by planar signals of the basal plate.

Striatal allografts in patients with Huntington's disease: impact of diminished astrocytes and vascularization on graft viability.

Neuronal transplantation has been proposed as a potential therapy to replace lost neurons in Huntington's disease. Transplant vascularization and trophic support are important for graft survival. However, very few studies have specifically addressed graft vascularization in patients with neurological disorders. In the present study, we analysed the vasculature of the host putamen and solid grafts of foetal striatal tissue transplanted into patients with Huntington's disease 9 and 12 years previously. Grafts were characterized by a significantly reduced number of large calibre blood vessels in comparison with the host brain. There were also significantly fewer astrocytes and gap junctions, suggesting a lack of functional blood-brain barrier components within the grafted tissue. Additionally, grafts demonstrated a nearly complete absence of pericytes (compared with the striatum) that are considered important for vascular stabilization and angiogenesis. Finally, the host striatum had a marked increase in atrophic astrocytes in comparison with controls and grafts. The extent to which the lower number of large calibre vessels and astrocytes within the transplants contributed to suboptimal graft survival is unknown. The marked increase in atrophic astrocytes in the host brain surrounding the grafts suggests that reduced host trophic support may also contribute to poor graft survival in Huntington's disease. A better understanding of the way in which these components support allografted tissue is critical to the future development of cell-based therapies for the treatment of Huntington's disease.

Grafted cerebellar cells in a mouse model of hereditary ataxia express IGF-I system genes and partially restore behavioral function.

Fetal grafts of normal cerebellar tissue were implanted into the cerebellum of Purkinje cell degeneration mutant mice (pcd/pcd), a model of adult-onset recessively inherited cerebello-olivary atrophy, in an attempt at correcting their cellular and motor impairment. Donor cerebellar cells engrafted in the appropriate sites, as evidenced by the pattern of expression of insulin-like growth factor-I (IGF-I) system genes. Bilateral cerebellar grafts led to an improvement of motor behaviors in balance rod tests and in the open field, providing evidence for functional integration into the atrophic mouse cerebellum and underscoring the potential of neural transplantation for counteracting the human cerebellar ataxias.

Potential of visual cortex to develop an array of functional units unique to somatosensory cortex.

The identification of specialized areas in the mammalian neocortex, such as the primary visual or somatosensory cortex, is based on distinctions in architectural and functional features. The extent to which certain features that distinguish neocortical areas in rats are prespecified or emerge as a result of epigenetic interactions was investigated. Late embryonic visual cortex transplanted to neonatal somatosensory cortex was later assayed for "barrels," anatomically identified functional units unique to somatosensory cortex, and for boundaries of glycoconjugated molecules associated with barrels. Barrels and boundaries form in transplanted visual cortex and are organized in an array that resembles the pattern in the normal barrelfield. These findings show that different regions of the developing neocortex have similar potentials to differentiate features that distinguish neocortical areas and contribute to their unique functional organizations.

Brain control of embryonic circadian rhythms in the silkmoth Antheraea pernyi.

The clock protein PER is necessary for circadian control of egg-hatching behavior in the silkmoth Antheraea pernyi. Since the brain and midgut of the silkmoth embryo contain PER-positive cells, we examined the circadian clock potential of these embryonic tissues. Transplantation experiments indicate that the circadian clock controlling egg-hatching behavior resides in brain, and that a humoral factor mediates this circadian regulation. We also used ligation experiments on first instar larvae to show that the circadian control of PER movement into the nuclei of midgut epithelial cells is dependent on an intact (connected) brain. These results implicate a novel brain factor in the circadian regulation of egg-hatching behavior and provide further evidence for differing mechanisms of PER control among species.

Induction of a mesencephalic phenotype in the 2-day-old chick prosencephalon is preceded by the early expression of the homeobox gene en.

The homeobox gene en, homologous to the gene en-grailed of Drosophila, is expressed in the metencephalic-mesencephalic segment of the vertebrate neural tube. Using quail-chick chimeras, an antibody against en proteins, and cytoarchitectonic techniques, we demonstrate that metencephalon transplanted to prosencephalon, at E2, maintains a high level of en proteins and its presumptive cerebellar fate. The ectopic metencephalon induces in the contiguous host prosencephalon the expression of en and, subsequently, a mesencephalic phenotype. These related genetic and phenotypic expressions indicate that the transcriptional regulatory en gene is involved in cerebellar and mesencephalic cyto-differentiation. The expression of en can also be induced in chick prosencephalon by a mammalian metencephalic graft, indicating that the factors regulating the transcription of en are phylogenetically well conserved.

Specification of mouse telencephalic and mid-hindbrain progenitors following heterotopic ultrasound-guided embryonic transplantation.

We have demonstrated the utility of ultrasound backscatter microscopy for targeted intraparenchymal injections into embryonic day (E) 13.5 mouse embryos. This system has been used to test the degree of commitment present in neural progenitors from the embryonic ventral telencephalon and mid-hindbrain region. Many E13.5 ventral telencephalic progenitors were observed to integrate and adopt local phenotypes following heterotopic transplantation into telencephalic or mid-hindbrain targets, whereas mid-hindbrain cells of the same stage were unable to integrate and change fate in the telencephalon. In contrast, many mid-hindbrain cells from an earlier developmental stage (E10.5) were capable of integrating and adopting a forebrain phenotype after grafting into the telencephalon, suggesting that mouse mid-hindbrain progenitors become restricted in their developmental potential between E10.5 and E13.5.

Involvement of neuropeptide mechanisms in the process of integration of heterotopic dental fascia transplants with recipient brains.

Embryonic dentate fascia was transplanted into the somatosensory area of the neocortex of adult rats. Ultrastructural and morphometric analyses of giant synapses formed by the granule neurons of transplants with inappropriate neuronal targets in the recipient brains were performed after nine months. As compared with intact synaptic terminals in the control hippocampus, there were differences in the quantity and distribution of large synaptic vesicles with electron-dense centers storing neuropeptide cotransmitters. The proportion of peptidergic vesicles (of the total number of vesicles) in ectopic giant synapses was 5.8 +/- 0.6%, compared with 3.3 +/- 0.6% in controls. Accumulations of large, dense vesicles close to the active zones of aberrant connections were seen almost 7.9 times more often than in controls. These results show that neuropeptide transmitters are critical for maintaining synaptic connections between heterotopic dentate fascia transplants and recipient brains.

Transplantation of GABAergic neurons but not astrocytes induces recovery of sensorimotor function in the traumatically injured brain.

Embryonic stem (ES) cells have been investigated in many animal models of injury and disease. However, few studies have examined the ability of pre-differentiated ES cells to improve functional outcome following traumatic brain injury (TBI). The purpose of the present study was to compare the effect of murine ES cells that were pre-differentiated into GABAergic neurons or astrocytes on functional recovery following TBI. Neural and astrocyte induction was achieved by co-culturing ES cells on a bone marrow stromal fibroblast (M2-10B4) feeder layer and incubating them with various mitogenic factors. Rats were initially prepared with a unilateral controlled cortical contusion injury of the sensorimotor cortex or sham procedure. Rats were transplanted 7 days following injury with approximately 100K GABAergic neurons, astrocytes, fibroblasts, or media. Animals were assessed on a battery of sensorimotor tasks following transplantation. The stromal fibroblast cells (M2-10B4), as a control cell line, did not differ significantly from media infusions. Transplantation of GABAergic neurons facilitated complete and total recovery on the vibrissae-forelimb placing test as opposed to all other groups, which failed to show any recovery. It was also found that GABAergic neurons reduced the magnitude of the initial impairment on the limb use test. Histological analysis revealed infiltration of host brain with transplanted neurons and astrocytes. The results of the present study suggest that transplantation of pre-differentiated GABAergic neurons significantly induces recovery of sensorimotor function; whereas, astrocytes do not.

Amphetamine induced rotation in the assessment of lesions and grafts in the unilateral rat model of Parkinson's disease.

In the unilateral rat model of Parkinson's disease (PD), amphetamine induced rotation is widely used as an index of both lesion deficits and of graft-derived recovery. We have analysed the time course of the rotational response in lesioned rats, and in rats with lesions and dopamine grafts. In lesioned rats, the rotation exhibited a typical dose-dependent response, with low rates of rotation in the first 10 min after injection, rising gradually to a maximum after 20-30 min. Grafted rats exhibited a peak of rotation in the first 10 min after injection, which then fell to a minimum after 30 min. We demonstrate that the response seen in grafted rats is both drug and dose-dependent and show that the rotational profile results from interaction of the grafted and intact striata which exhibit differential temporal responses to the amphetamine.

Genetically modified cells: applications for intracerebral grafting.

Grafting cells to the CNS is a useful approach to address fundamental and clinical issues in neurobiology. Recently, a hybrid technique - the genetic modification of cells followed by intracerebral implantation - has emerged, which may potentially enhance the power of CNS grafting. However, several methodological considerations need to be addressed to test the reliability of this new approach. Progress in the gene transfer-grafting technique has implications for expanding the range of issues and problems that may be addressed in both the basic science and clinical arenas.

Transplantation of glial cells into the CNS.

Glial cell transplantation into the CNS offers an experimental approach to help us unravel the complex interactions that occur between CNS glia, Schwann cells and axons during repair and development. This article reviews recent advances that have been made in our understanding of the nature and potential of CNS repair using this approach, and introduces the idea of using transplantation to address broader issues in glial biology.

Identifying and manipulating neuronal stem cells.

Fetal brain tissue has been shown to have clear behavioral effects when transplanted into adult lesioned brains. These results have focused attention on the cell types of the embryonic brain. Transplantation experiments using primary cells are beginning to define the plasticity of these cells and the times when they become committed to specific neuronal fates. Growth factors have been defined that regulate the proliferation of these cells in culture. Cell lines have been established that express stem cell properties and that are capable of differentiation when implanted into the developing brain. In this article we review this work on mammalian neuroepithelial stem cells and discuss how these studies might contribute to the therapeutic use of brain transplants.

Transplants of embryonic motoneurones to adult spinal cord: survival and innervation abilities.

One goal of transplantation experiments involving damaged spinal cords is to reconstruct a functional innervation to muscles in the periphery. Embryonic spinal cord grafts have been shown to survive transplantation into adult spinal cord lacking motoneurones. Motoneurones from the graft appear to be able to innervate muscle tissue by being encouraged to grow across a bridge of peripheral nerve. Integration of grafted motoneurones appears to involve their migration from the graft into the host ventral horn, thus replacing depleted host neurones. These results suggest possible strategies of research that might lead to treatments of spinal cord injuries and disorders in which motoneurone loss occurs, such as amyotrophic lateral sclerosis, spinal muscular atrophies and poliomyelitis.

Transplantation: a new tool in the analysis of the mammalian hypothalamic circadian pacemaker.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of pacemaker cells that generate circadian rhythmicity in mammals. Transplantation of the nucleus into animals whose own nucleus has been ablated results in the restoration of overt rhythmicity to the arrhythmic host. By using donors and hosts with genetically different circadian characteristics, the unambiguous recognition of the donor rhythm expressed in a transplant recipient is possible. The reappearance of a rhythm indicates that not only has the grafted tissue survived the transplantation procedure, but that pacemaker cells that generate circadian rhythms were included in the graft; this is essential in interpreting results of such transplantation experiments. The restoration of circadian function by neural transplantation has become an important tool for studying the generation and expression of biological rhythms in mammals, and is being used in the investigation of basic questions in this field.

Prospects of transplantation in human neurodegenerative diseases.

Over the past decade experimental data obtained from animals have suggested that restoration or preservation of function through cell transplantation into the CNS might be developed into a useful therapeutic approach in human neurodegenerative disorders. Clinical trials in patients with Parkinson's disease have provided evidence that grafts of fetal dopaminergic neurons can survive and induce functional effects in the human brain, but no treatment based on transplantation is available yet. Initiation of studies of patients with striatal neural grafts in Huntington's disease is supported by findings in animal models, and is motivated by the lack of therapy and the severity of the symptoms in this disorder. Application of cell transplantation to other neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and hereditary ataxia is definitely premature. Further progress can be made only by systematic studies in animals of the scientific issues that can now be defined, but will also require clinical trials in a few well-monitored patients.

Neural transplantation studies reveal the brain's capacity for continuous reconstruction.

Basic research using cell transplantation indicates that structural developmental mechanisms seen in immature brains can also function in the adult brain. As the brain matures, cellular migration and axonal growth is impeded. However, fetal neural transplantation studies have shown that directional cues are available for fetal axons to find specific host neurons in the adult brain. By reaching specific and distant CNS target zones, donor tissue with extended axonal growth periods demonstrate both an abundance and specificity of CNS neurotropic signals. The presence of specific guidance cues, despite strong inhibition of regenerative long-distance axonal growth, suggests that these cues play other physiological roles in the adult CNS, and could be utilized therapeutically for reconnection of neuronal pathways.

The neuropathology of Alzheimer's disease investigated by transplantation of mouse trisomy 16 hippocampal tissues.

This article evaluates the novel application of neural transplantation as a model for studying the neuropathological events associated with Alzheimer's disease and those that have subsequently also been observed in Trisomy 21 (Down syndrome).

The reconstruction of cerebellar circuits.

Repair of adult 'point-to-point' systems by neural grafting is possible only when grafted neurons succeed in synaptically replacing the host's missing neurons, thus re-establishing the anatomical and functional integrity of the impaired circuits. Grafting experiments carried out on the cerebellum of the adult pcd (Purkinje-cell-degeneration) mutant mouse (an animal model of hereditary degenerative ataxia) reveal that embryonic Purkinje cells, by some unknown sorting mechanism, selectively invade the deprived cerebellar cortex. These neurons migrate to their proper domains and, inducing axonal sprouting of specific populations of host neurons, they become integrated synaptically within the pcd cerebellar cortex. However, the re-establishment of the corticonuclear projection is achieved only rarely, and this is the current experimental limit for the complete reconstruction of the cerebellar circuit.

Can fetal neural transplants restore function in monkeys with lesion-induced behavioural deficits?

Experiments are now being conducted in monkeys to see whether the transplantation of fetal neural tissue, rich in certain neurotransmitter-producing cells, can restore behaviour in animals with movement or learning impairments induced by lesions that have destroyed important neurotransmitter pathways. Transplantation of dopamine neurons in humans may prove to be a useful therapy in Parkinson's disease, in which a severe movement disorder is associated with degeneration of the dopamine system. Transplantation of cholinergic neurones in monkeys can overcome a severe learning impairment induced by lesion of the cholinergic system. Cholinergic transplantation may eventually be of use in a variety of neurodegenerative dementing illnesses.