Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling.

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.

Nuclear transfer technology in mammalian cloning.

The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

Cellular interactions in the development of the olfactory system: an ablation and homotypic transplantation analysis.

In the current study, we addressed two questions: First, is the olfactory placode necessary for the development of the olfactory bulb and the entire telencephalon? Second, does the olfactory placode contribute cells to the olfactory bulb? We addressed these questions by unilaterally ablating the olfactory placode in chick embryos before an olfactory nerve was produced and, in a second series of experiments, by replacing the ablated chick olfactory placode with a quail olfactory placode. Our results indicate that the olfactory placode is critical for olfactory bulb development, but is not necessary for the development of the rest of the telencephalon. Further, our results support the hypothesis that LHRH neurons and olfactory nerve glia originate in the olfactory placode, but do not support an olfactory placodal origin for other cell types within the olfactory bulb.

TTF-1, a homeodomain gene required for diencephalic morphogenesis, is postnatally expressed in the neuroendocrine brain in a developmentally regulated and cell-specific fashion.

TTF-1 is a member of the Nkx family of homeodomain genes required for morphogenesis of the hypothalamus. Whether TTF-1, or other Nkx genes, contributes to regulating differentiated hypothalamic functions is not known. We now report that postnatal hypothalamic TTF-1 expression is developmentally regulated and associated with the neuroendocrine process of female sexual development. Lesions of the hypothalamus that cause sexual precocity transiently activate neuronal TTF-1 expression near the lesion site. In intact animals, hypothalamic TTF-1 mRNA content also increases transiently, preceding the initiation of puberty. Postnatal expression of the TTF-1 gene was limited to subsets of hypothalamic neurons, including LHRH neurons, which control sexual maturation, and preproenkephalinergic neurons of the lateroventromedial nucleus of the basal hypothalamus, which restrain sexual maturation and facilitate reproductive behavior. TTF-1 mRNA was also detected in astrocytes of the median eminence and ependymal/subependymal cells of the third ventricle, where it colocalized with erbB-2, a receptor involved in facilitating sexual development. TTF-1 binds to and transactivates the erbB-2 and LHRH promoters, but represses transcription of the preproenkephalin gene. The singular increase in hypothalamic TTF-1 gene expression that precedes the initiation of puberty, its highly specific pattern of cellular expression, and its transcriptional actions on genes directly involved in neuroendocrine reproductive regulation suggest that TTF-1 may represent one of the controlling factors that set in motion early events underlying the central activation of mammalian puberty.

Disruption of the olfactory placode and brain conditioned medium increase the number of LHRH immunostained neurons in explants.

The olfactory placode gives rise to both olfactory receptor neurons, which remain as a component of the peripheral nervous system, and to luteinizing hormone-releasing hormone (LHRH) neurons, which migrate to the central nervous system. In this study, we used chick olfactory placode explants to ask several questions regarding LHRH neuronal differentiation. We found that explants of ectoderm from the fronto-nasal region of embryos as early as Hamilton & Hamburger (HH) stage 12 gave rise to LHRH neurons, that explants from all regions of the olfactory placode were able to generate LHRH neurons, that both brain conditioned medium and disruption of the olfactory placode increase the number of LHRH neurons observed in explants, and that the combination of these two manipulations results in the production of more LHRH neurons than either treatment alone. We conclude that LHRH neurons originate in the olfactory epithelium and that some of the same factors which influence olfactory receptor neuron development also affect LHRH neuronal development.

LHRH neuronal migration: heterotypic transplantation analysis of guidance cues.

During embryonic development, the olfactory placode (OP) differentiates into the olfactory epithelium (OE). Luteinizing hormone-releasing hormone (LHRH) neurons migrate out of the OE in close association with the olfactory nerve (ON) to the telencephalon. LHRH neuronal migration and ON extension to the telencephalon may be independent events which are correlated but do not represent a causal relationship. However, we hypothesize that LHRH neurons are dependent on ON axons to migrate to the brain. To test this hypothesis, we ablated the right trigeminal placode and replaced it with an OP from another chick embryo. After several days' additional incubation, the embryos were fixed, sectioned, and immunostained with antibodies against LHRH or N-CAM. The ectopic OPs were well integrated into the host and developed into relatively normal appearing OEs. The ONs extended from the OE to several different sites: the lateral rectus of the eye, the ciliary ganglion, and the trigeminal ganglion. In all cases, LHRH neurons were found in the OE and ON, regardless of where the ON terminated. When the ON extended to the trigeminal ganglion, LHRH neurons could clearly be seen entering the metencephalon. Our results support the idea that LHRH neurons are dependent on the ON for guidance as they appear to follow the nerve even when it extends away from the brain. The cues which direct the ON and LHRH neurons to the telencephalon do not appear to be unique to this brain region.

Herpes simplex virus as a transneuronal tracer.

Determining the connections of neural systems is critical for determining how they function. In this review, we focus on the use of HSV-1 and HSV-2 as transneuronal tracers. Using HSV to examine neural circuits is technically simple. HSV is injected into the area of interest, and after several days, the animals are perfused and processed for immunohistochemistry with antibodies to HSV proteins. Variables which influence HSV infection include species of host, age of host, titre of virus, strain of virus and phenotype of infected cell. The choice of strain of HSV is critically important. Several strains of HSV-1 and HSV-2 have been utilized for purposes of transneuronal tract-tracing. HSV has been used successfully to study neuronal circuitry in a variety of different neuroanatomical systems including the somatosensory, olfactory, visual, motor, autonomic and limbic systems.

FUSE-binding protein is developmentally regulated and is highly expressed in mouse and chicken embryonic brain.

GAP-43 modulates axon guidance and neuronal plasticity. In vitro, FUSE-binding protein (FBP) binds to a segment of GAP-43 mRNA which regulates the stability of the transcript. FBP has also been shown to bind to a c-myc cis element and regulate transcription. In the current work, analysis of RNA and protein expression indicated that FBP is expressed in a distinct spatial temporal pattern during embryonic development. Expression was particularly high in the brain. In the adult, expression was not detected in most tissues but was still prominent in the brain and teste. This finding is consistent with a dual role of the protein as a single-strand polynucleotide-binding protein.

Survival and functional demonstration of interregional pathways in fore/midbrain slice explant cultures.

An important general question in neurobiology concerns the development and expression of the rich context of neuronal phenotypes, especially in relation to the diverse patterns of connectivity. Organotypic cultures of brain slices may offer distinct advantages for such studies if such a preparation survives, maintains a wide diversity of neuronal phenotypes and displays appropriate synaptic connections between regions. To address these requirements, we utilized long-term organotypic cultures of intact horizontal slices of rat forebrain and midbrain and assessed a variety of markers of phenotype in combination with functional tests of connectivity. This explant preparation displayed a distinct viability requirement such that the greatest explant survival was seen in slices taken from pups of less than postnatal day 7 and was independent of N-methyl-D-aspartate channel blockade. The anatomical features of the major brain regions (e.g., neocortex, striatum, septum, hippocampus, diencephalon and midbrain) were observed in their normal boundaries. The presence of cholinergic and catecholaminergic neurons was demonstrated with acetylcholinesterase histochemistry and tyrosine hydroxylase immunohistochemistry. Labelled neurons displayed multiple, regionally-appropriate cytoarchitectures and, in some cases, could be seen to project to brain regions in a manner quite similar to that seen in vivo. Finally, the direct demonstration of spontaneous and evoked interregional excitatory synaptic transmission was made using whole-cell patch-clamp recordings from striatal neurons which revealed an intact glutamate-using corticostriatal pathway. This simple explant preparation appears to contain a rich diversity of neuronal types and synaptic organization. Therefore, this preparation appears to have several distinct advantages for basic neurobiologic research since it combines long-term culture viability and many features of mature brain including complex interregional neuronal systems.

Subpopulations of migrating neurons express different levels of LHRH in quail and chick embryos.

LHRH neurons of the septal-preoptic area originate in the olfactory placode and migrate in the olfactory nerve into the brain during embryonic development. In adult birds, LHRH neurons have been found in the septal-preoptic area, mesencephalon and more recently in the lateral anterior nucleus of the thalamus (LA). LHRH neurons of the LA do not originate in the olfactory placode. Using immunocytochemistry, we examined the distribution of LHRH neurons in the embryonic and adult quail nervous system. The pattern of LHRH immunostaining in quail embryos was similar to that seen in chick embryos. However, there were many fewer neurons immunostained for LHRH from the olfactory placode to the septal-preoptic area in quail than in chick embryos. In contrast, there were more labeled neurons and more intense LHRH immunostaining in the thalamus of the quail than in the thalamus of chick embryos. In agreement with other studies, our data suggest that there are species differences in LHRH expression in migrating neurons. The current results should also be considered for quail-chick chimeras involving the olfactory placode.

Tangential migration of luteinizing hormone-releasing hormone (LHRH) neurons in the medial telencephalon in association with transient axons extending from the olfactory nerve.

During embryonic development, luteinizing hormone-releasing hormone (LHRH) neurons migrate to the brain from the medial olfactory epithelium through the olfactory nerve. LHRH neurons enter the brain and migrate tangentially along the medial edge of the telencephalon in close association with a neural cell adhesion molecule (N-CAM) enriched fiber bundle. In the current work we wished to determine whether this N-CAM enriched fiber bundle is an extension of the olfactory nerve. Ablation experiments, immunocytochemistry and diI implants all suggest that LHRH neurons migrate in association with a very small subset of transient N-CAM enriched neuronal processes which extend out of the olfactory nerve proper to the septal-preoptic area.

Cerebrospinal fluid of Parkinson's disease patients inhibits the growth and function of dopaminergic neurons in culture.

We report the possible existence of an inhibitory factor in the CSF of Parkinson's disease patients that inhibits the function and growth of dopaminergic neurons in rat mesencephalic culture. After 40 hours' exposure to the < 10 kd fraction of CSF from PD patients, the high-affinity dopamine uptake was 66% of that of cultures exposed to CSF from controls. However, the number of dopaminergic neurons remained unchanged at this time. After 90 hours' exposure to the < 10 kd fraction of CSF from PD patients, the number of dopaminergic neurons decreased to 10% of that in cultures exposed to CSF from controls, and the size of the remaining dopaminergic neurons in the culture became smaller. This inhibitory factor did not affect the growth of other types of neurons. The chemical nature of this inhibitory factor is under investigation.

LHRH neuronal subtypes have multiple origins in chickens.

Previous studies indicate that LHRH neurons of the septal-preoptic area originate in the olfactory epithelium. In the current study, we found that LHRH neurons entering the telencephalon from the olfactory nerve were not continuous with LHRH neurons in the thalamus. In addition, ablation of the olfactory placode eliminated LHRH neurons in the telencephalon but did not eliminate LHRH neurons in the thalamus. We propose that there are at least two sites of LHRH neuron production, the olfactory epithelium and the diencephalon.

Single- and double-label immunocytochemical study of the ovine suprachiasmatic nucleus (SCN): GABAergic and peptidergic relationships.

This study evaluated the neuropeptide and neurotransmitter content of the ovine suprachiasmatic nucleus (SCN) using both single- and double-label immunocytochemical methods. Single-label immunocytochemistry identified a few lightly labeled gamma aminobutyric acid (GABA) cells within the SCN as well as a dense plexus of fibers staining positive for the GABA biosynthetic enzyme, glutamic acid decarboxylase (GAD). Vasoactive intestinal polypeptide (VIP) fibers exhibited a similar distribution to GAD fibers; VIP cells were found throughout the SCN, as well as in the paraventricular (PVN) and supraoptic nuclei. Both GAD and VIP fibers exited dorsally from the SCN towards the PVN. Neurophysin (NP) and neuropeptide-Y (NPY) fibers were sparsely distributed throughout the SCN. Double-label immunocytochemistry revealed that GAD varicosities were often in close apposition to VIP cells. These results confirm the presence of GABAergic elements within the sheep SCN. Furthermore, they raise the possibility of a GABAergic modulation of VIP neuronal activity within the ovine SCN.

Cell adhesion molecules and the migration of LHRH neurons during development.

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.

Sensitization of c-fos expression in rat striatum following multiple challenges with D-amphetamine.

D-Amphetamine transiently stimulates the expression of the immediate-early response gene, c-fos, in rat striatal cell nuclei. D-Amphetamine (2.5 mg/kg i.p.) induced a significantly greater expression of Fos-like immunoreactivity in striatum of rats treated three days previously with D-amphetamine compared to rats treated three days previously with saline. This increase in the expression of Fos-like immunoreactivity in rat striatum was characterized by a significantly greater number of immunoreactive nuclei and a significant increase in the intensity of the immunoreactivity. This sensitization of c-fos expression following a repeated administration of D-amphetamine indicates an increased activation of post-synaptic elements in rat striatum.

Magnetic resonance imaging of neural transplants in rat brain using a superparamagnetic contrast agent.

Rat fetal brain tissue was incubated in vitro with superparamagnetic ferrite particles covalently coupled to the lectin wheat germ agglutinin (WGA) and transplanted into the adult rat striatum. At 6 days and at 3 weeks post-surgery the transplants were observed on T1 weighted magnetic resonance (MR) images of the rat head as an area of relatively low signal intensity which could be clearly differentiated from the higher signal intensity produced by the host brain. Histological analysis revealed that the ferrite particles were largely restricted to the transplant in a patchy distribution. The ferrite particles were associated with cells having an apparent normal morphology. Superparamagnetic ferrite particles act as potent MR contrast agents and can be used to label transplanted cells. The labeled cells are apparently not adversely affected by the WGA-ferrite particles and can be monitored for at least three weeks in vivo using noninvasive MR imaging.

Development of olfactory nerve glia defined by a monoclonal antibody specific for Schwann cells.

Although there is considerable interest in the possible role of olfactory glia in the pathfinding abilities of olfactory nerve axons, the complete development of these glia in vivo has not been described. Using a specific Schwann cell marker, the 1E8 antibody, we have found that olfactory nerve glia can be identified throughout development. These glia appear to originate in the olfactory placode and migrate initially into the periphery of the olfactory nerve, and later into the center of the nerve. Olfactory nerve glia enter the presumptive olfactory bulb with the olfactory receptor neuron axons and distribute themselves along the edge of the olfactory nerve layer. The fact that olfactory nerve glia are specifically immunostained by the 1E8 monoclonal antibody, which recognizes the Schwann cell-specific protein P0, suggests that these cells more closely resemble Schwann cells than astrocytes or enteric glia. These results support and extend previous findings suggesting that olfactory nerve glia have distinctive developmental and anatomical features which may be important to the regenerative capacity of the olfactory system.

Anterograde transport of HSV-1 and HSV-2 in the visual system.

The anterograde spread of herpesvirus in the visual system subsequent to retinitis has been observed clinically. We compared the ability of two well-studied Herpes simplex virus (HSV) strains to be transported in the anterograde direction in the hamster visual system: strain McIntyre, representing HSV-1, and strain 186, representing HSV-2. Intravitreal injection of HSV-2 labeled more retinorecipient neurons than did HSV-1, suggesting important type differences in the ability of HSV to infect retinorecipient neurons after intravitreal injection. The most likely explanation for our results is that HSV-2 is more efficiently adsorbed than HSV-1 in the retinal ganglion cells. Our results also suggest that HSV may be useful as an anterograde transneuronal tracer for neuroanatomical studies of the visual system.