Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue.

Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.

Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.

High-throughput quantitative histological analysis of Alzheimer's disease pathology using a confocal digital microscanner.

To develop a rapid method of quantifying immunohistochemical information in tissue sections, we tested a confocal laser fluorescence microscanner initially designed for DNA microarray analysis. This instrument collects digital images at multiple wavelengths, scans entire sections at a resolution of 5 or 10 microm in less than 10 min, and quantifies structures labeled with fluorescent or nonfluorescent probes. We assessed the microscanner by studying immunostained amyloid plaques in the Alzheimer's disease (AD) brain and in the brain of a transgenic mouse model of AD amyloidosis, as efforts to correlate measures of amyloid plaques in brain sections with behavioral impairments are impeded by limitations in current morphometric methods. Microscanner analysis was used to determine amyloid burden in the occipital and entorhinal cortices of the mouse (3.7%) and human AD brain (1.6%). We also quantified the colocalization of plaque beta-amyloid (Abeta) with glial fibrillary acidic protein, a marker of gliosis (mouse 0.9%, human AD 3.7%). The microscanner may be generally applicable to a wide variety of human histopathologies and their animal models, wherever rapid unbiased quantitative analysis is needed.

At the interface of the immune system and the nervous system: how neuroinflammation modulates the fate of neural progenitors in vivo.

Neural stem and progenitor cells express a variety of receptors that enable them to sense and react to signals emanating from physiological and pathophysiological conditions in the brain as well as elsewhere in the body. Many of these receptors and were first described in investigations of the immune system, particularly with respect to hematopoietic stem cells. This emerging view of neurobiology has two major implications. First, many phenomena known from the hematopoietic system may actually be generalizable to stem cells from many organ systems, reflecting the cells' progenitor-mediated regenerative potential. Second, regenerative interfaces may exist between diverse organ systems; populations of cells of neuroectodermal and hematopoietic origin may interact to play a crucial role in normal brain physiology, pathology, and repair. An understanding of the origins of signals and the neural progenitors' responses might lead to the development of effective therapeutic strategies to counterbalance acute and chronic neurodegenerative processes. Such strategies may include modifying and modulating cells with regenerative potential in subtle ways. For example, stem cells might be able to detect pathology-associated signals and be used as "interpreters" to mediate drug and other therapeutic interventions. This review has focused on the role of inflammation in brain repair. We propose that resident astroglia and blood-born cells both contribute to an inflammatory signature that is unique to each kind of neuronal degeneration or injury. These cells play a key role in coordinating the neural progenitor cell response to brain injury by exerting direct and indirect environmentally mediated influence on neural progenitor cells. We suggest that investigations of the neural progenitor-immunologic interface will provide valuable data related to the mechanisms by which endogenous and exogenous neural progenitor cells react to brain pathology, ultimately aiding in the design of more effective therapeutic applications of stem cell biology. Such improvements will include: (1) ascertaining the proper timing for implanting exogenous neural progenitor cells in relation to the administration of anti-inflammatory agents; (2) identifying what types of molecules might be administered during injury to enhance the mobilization and differentiation of endogenous and exogenous neural progenitor cells while also inhibiting the detrimental aspects of the inflammatory reaction; (3) divining clues as to which molecules may be required to change the lesioned environment in order to invite the homing of reparative neural progenitor cells.

Rational design of an animal model for Alzheimer's disease: introduction of multiple human genomic transgenes to reproduce AD pathology in a rodent.

A major obstacle to understanding the pathogenesis of Alzheimer's disease is the lack of easily studied animal models. Our approach is to apply transgenic methods to humanize mice and rats, employing methods that introduce large genomic transgenes, because this improves the level of transgene protein expression and the tissue specificity of expression. Our plan is to reproduce AD pathology in rodents by making them transgenic for several human proteins involved in AD. This report describes transgenic animal lines that we have produced, and summarizes our current approach and future plans. Two human genes known to be involved in AD pathology are the amyloid precursor protein (APP) and the E4 isoform of apolipoprotein E (apoE4). So far, we have produced and analyzed a transgenic line carrying the entire human APP gene cloned in a yeast artificial chromosome. We have also produced but not yet analyzed a mouse carrying the human apoE4 gene. Work is in progress to produce a transgenic line carrying a disease-causing mutation in the human APP gene. As we produce these animals, we are breeding them together, and also breeding them with a mouse line that lacks endogenous apoE, to produce an animal model carrying several human proteins whose interaction is believed to be instrumental in development of AD pathology. These transgenic animals will be useful for dissecting the biochemical and physiological steps leading to AD, and for development of therapies for disease intervention.

Twofold overexpression of human beta-amyloid precursor proteins in transgenic mice does not affect the neuromotor, cognitive, or neurodegenerative sequelae following experimental brain injury.

By using transgenic mice that overexpress human beta-amyloid precursor proteins (APPs) at levels twofold higher than endogenous APPs, following introduction of the human APP gene in a yeast artificial chromosome (YAC), we examined the effects of controlled cortical impact (CCI) brain injury on neuromotor/cognitive dysfunction and the development of Alzheimer's disease (AD)-like neuropathology. Neuropathological analyses included Nissl-staining and immunohistochemistry to detect APPs, beta-amyloid (Abeta), neurofilament proteins, and glial fibrillary acidic protein, whereas Abeta levels were measured in brain homogenates from mice subjected to CCI and control mice by using a sensitive sandwich enzyme-linked immunosorbent assay. Twenty APP-YAC transgenic mice and 17 wild type (WT) littermate controls were anesthetized and subjected to CCI (velocity, 5 m/second; deformation depth, 1 mm). Sham (anesthetized but uninjured) controls (n = 10 APP-YAC; n = 8 WT) also were studied. Motor function was evaluated by using rotarod, inclined-plane, and forelimb/hindlimb flexion tests. The Morris water maze was used to assess memory. Although CCI induced significant motor dysfunction and cognitive deficits, no differences were observed between brain-injured APP-YAC mice and WT mice at 24 hours and 1 week postinjury. By 1 week postinjury, both cortical and hippocampal CA3 neuron loss as well as extensive astrogliosis were observed in all injured animals, suggesting that overexpression of human APPs exhibited no neuroprotective effects. Although AD-like pathology (including amyloid plaques) was not observed in either sham or brain-inj ured animals, a significant decrease in brain concentrations of only Abeta terminating at amino acid 40 (Abeta x-40) was observed following brain injury in APP-YAC mice (P < 0.05 compared with sham control levels). Our data show that the APP-YAC mice do not develop AD-like neuropathology following traumatic brain injury. This may be because this injury does not induce elevated levels of the more amyloidogenic forms of human Abeta (i.e., Abeta x-42/43) in these mice.

A gene expression profile of embryonic stem cells and embryonic stem cell-derived neurons.

Embryonic stem (ES) cells have the ability to differentiate into a variety of cell lineages. We are examining ES cell differentiation in vitro by using cDNA microarrays to generate a molecular phenotype for each cell type. El4 ES cells induced by retinoic acid after forming embryoid bodies differentiate almost exclusively to neurons. We obtained expression patterns for about 8500 gene sequences by comparing mRNAs from undifferentiated ES cells and their differentiated derivatives in a competitive hybridization. Our results indicate that the genes expressed by ES cells change dramatically as they differentiate (58 gene sequences up-regulated, 34 down-regulated). Most notably, totipotent ES cells expressed high levels of a repressor of Hox expression (the polycomb homolog Mphl) and a co-repressor (CTBP2). Expression of these genes was undetectable in differentiated cells; the ES cell-derived neurons expressed a different set of transcriptional regulators, as weil as markers of neurogenesis. The gene expression profiles indicate that ES cells actively suppress differentiation by transcriptional repression; cell-cell contact in embryoid bodies and retinoic acid treatment may overcome this suppression, allowing expression of Hox genes and inducing a suite of neuronal genes. Gene expression profiles will be a useful outcome measure for comparing in vitro treatments of differentiating ES cells and other stem cells. Also, knowing the molecule phenotype of transplantable cells will allow correlation of phenotype with the success of the transplant.

A gene expression profile of Alzheimer's disease.

Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

Designing Animal Models of Alzheimer's Disease with Amyloid Precursor Protein (APP) Transgenes.

Amyloid precursor protein (APP) and Alzheimer's disease are irrevocably linked, as APP is cleaved to form the Aß peptides that are the major components of amyloid plaques. One of the most resilient hypotheses about the cause of AD centers on the Aß peptide; all genetic causes and risk factors can be fitted into a general "amyloid cascade hypothesis," which maintains that all pathology is initiated by an abnormal accumulation of Aß amyloid (1).

Banking placental tissue: an optimized collection procedure for genome-wide analysis of nucleic acids.

INTRODUCTION: Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection in a typical clinical setting are lacking. METHODS: To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial timepoints in a 2-h window after delivery, simultaneously comparing the traditional snap-freeze technique to commercial solutions designed to preserve RNA (RNAlater™), and DNA (DNAgard(®)). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide microarray profiling for gene expression and DNA methylation. RESULTS: We found that samples collected in RNAlater had higher and more consistent RINs compared to snap-frozen tissue. Similar RINs were obtained for tissue collected in RNAlater as large (1 cm(3)) and small (∼0.1 cm(3)) pieces. RNAlater appeared to better stabilize the time zero gene expression profile compared to snap-freezing for first trimester placenta. DNA methylation profiles remained quite stable over a 2 h time period after removal of the placenta from the uterus, with DNAgard being superior to other treatments. DISCUSSION AND CONCLUSION: The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is higher than samples collected using the snap-freeze method and easier to implement in busy clinical environments.

Neural crest cell migratory pathways in the trunk of the chick embryo.

Neural crest cells migrate during embryogenesis to give rise to segmented structures of the vertebrate peripheral nervous system: namely, the dorsal root ganglia and the sympathetic chain. However, neural crest cell arise from the dorsal neural tube where they are apparently unsegmented. It is generally agreed that the somites impose segmentation on migrating crest cells, but there is a disagreement about two basic questions: exactly pathways do neural crest cells use to move through or around somites, and do neural crest cells actively migrate or are they passively dispersed by the movement of somite cells? The answers to both questions are critically important to any further understanding of the mechanisms underlying the precise distribution of the neural crest cells that develop into ganglia. We have done an exhaustive study of the locations of neural crest cells in chick embryos during early stages of their movement, using antibodies to neural crest cells (HNK-1), to neural filament-associated protein in growing nerve processes (E/C8), and to the extracellular matrix molecule laminin. Our results show that Some neural crest cells invade the extracellular space between adjacent somites, but the apparent majority move into the somites themselves along the border between the dermatome/myotome (DM) and the sclerotome. Neural crest cells remain closely associated with the anterior half of the DM of developing somites as they travel, suggesting that the basal lamina of the DM may be used as a migratory substratum. Supporting this idea is our observation that the development of the DM basal lamina coincides in time and location with the onset of crest migration through the somite. The leading front of neural crest cells advance through the somite while the length of the DM pathway remains constant, suggesting active locomotion, at least in this early phase of development. Neural crest cells leave the DM at a later stage of development to associate with the dorsal aorta, where sympathetic ganglia form, and to associate with newly emerging fibers of the ventral root nerve, where they presumably give rise to neuronal supportive cells. Thus we propose that the establishment of the segmental pattern of the peripheral ganglia and nerves depends on the timely development of appropriate substrata to guide and distribute migrating neural crest cells during the early stages of embryogenesis.

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest.

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.

Migratory pathways of HNK-1-immunoreactive neural crest cells in the rat embryo.

In avian embryos, the precursors of the peripheral nervous system, the neural crest cells, migrate along precise pathways limited to the anterior half of each somite and the intersomitic space. This segmental migration foreshadows the development of segmented peripheral ganglia and thus may be critical to normal neuronal development. We report here that a remarkably similar pattern of migration of HNK-1-immunoreactive cells, which we believe to be neural crest cells, exists in the rat embryo, suggesting that the underlying mechanisms of neural crest guidance may be the same in avian and mammalian embryonic development.

Survival and function of aggregate cultures of rat fetal dopamine neurons grafted in a rat model of Parkinson's disease.

The ability to maintain tissue in culture prior to grafting would greatly facilitate the widespread application of graft therapy to neurological diseases such as Parkinson's disease. However, neurons cultured on planar substrata can be easily damaged when they are removed from the substrata and redissociated for use in grafting procedures. To overcome this limitation we utilized aggregate tissue culture methods, which allowed dopamine (DA)-rich neuronal tissue to be grafted directly following culture, without an additional redissociation. Fetal rat dopamine-neuron-containing ventral mesencephalon was cultured for 9 days in rotating flasks. The cells formed many small spheres (280 microns mean diameter), each estimated to contain about 10,000 cells. Forty such aggregate spheres were injected via a 22G needle into the DA-denervated striata of host Parkinsonian rats. A significant reduction of amphetamine-induced rotation was seen onward from 6 weeks post-transplantation, with a complete reversal of rotational asymmetry by 15 weeks post-transplantation. Well placed, surviving grafts were found in all behaviorally compensated rats (N = 6). Grafts contained an average of 517 tyrosine hydroxylase (TH)-positive neurons, as well as TH-positive fibers seen extending into the host striatum. These results suggest that aggregate culture methods are a promising means to maintain and deliver tissue for transplant therapy.

Expression of APP in brains of transgenic mice containing the entire human APP gene.

A major component of amyloid deposits found in Alzheimer disease and Down syndrome is the beta/A4 peptide, which is derived from the Alzheimer amyloid protein precursor (APP). Recent evidence indicates that increases in APP expression and/or beta/A4 peptide accumulation may underlie the amyloidosis characteristic of these diseases. In the present study, transgenic mice carrying the entire human APP gene were studied for expression of human APP. Significant expression of human APP protein was observed in these animals, and this expression paralleled the expression of endogenous APP. These results, which represent a first demonstration of significant human APP expression in transgenic animals, support the use of such animals to study human APP expression and processing in vivo and possibly as models for the amyloidosis associated with Alzheimer disease.

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy.

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus.

B lymphocyte differentiation is characterized by an ordered series of Ig gene assembly and expression events. In the majority of normal B cells, assembly and expression of Ig heavy (H) chain genes precedes that of light (L) chain genes. To determine the role of the Ig heavy chain protein in B cell development and L chain gene rearrangement, we have generated mice that cannot assemble Ig H chain genes as a result of targeted deletion of the JH gene segments in embryonic stem cells. Mice homozygous for this deletion are devoid of slg+ B cells in the bone marrow and periphery. B cell differentiation in these mice is blocked at the large, CD43+ precursor stage. However, these precursor B cells do assemble kappa L chain genes at a low level in the absence of mu H chain proteins. These data demonstrate that rearrangement and expression of the mu H chain gene is not absolutely required for kappa L chain gene rearrangement in vivo. Expression of mu chains may facilitate either efficient L chain gene rearrangement or the survival of cells that have rearranged light chain genes by promoting the differentiation of large, CD43+ to small, CD43- pre-B cells.

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.