Differential propagation of stroma and cancer stem cells dictates tumorigenesis and multipotency.

Glioblastoma Multiforme (GBM) is characterized by high cancer cell heterogeneity and the presence of a complex tumor microenvironment. Those factors are a key obstacle for the treatment of this tumor type. To model the disease in mice, the current strategy is to grow GBM cells in serum-free non-adherent condition, which maintains their tumor-initiating potential. However, the so-generated tumors are histologically different from the one of origin. In this work, we performed high-throughput marker expression analysis and investigated the tumorigenicity of GBM cells enriched under different culture conditions. We identified a marker panel that distinguished tumorigenic sphere cultures from non-tumorigenic serum cultures (high CD56, SOX2, SOX9, and low CD105, CD248, αSMA). Contrary to previous work, we found that 'mixed cell cultures' grown in serum conditions are tumorigenic and express cancer stem cell (CSC) markers. As well, 1% serum plus bFGF and TGF-α preserved the tumorigenicity of sphere cultures and induced epithelial-to-mesenchymal transition gene expression. Furthermore, we identified 12 genes that could replace the 840 genes of The Cancer Genome Atlas (TCGA) used for GBM-subtyping. Our data suggest that the tumorigenicity of GBM cultures depend on cell culture strategies that retain CSCs in culture rather than the presence of serum in the cell culture medium.

Modeling psychiatric disorders: from genomic findings to cellular phenotypes.

Major programs in psychiatric genetics have identified >150 risk loci for psychiatric disorders. These loci converge on a small number of functional pathways, which span conventional diagnostic criteria, suggesting a partly common biology underlying schizophrenia, autism and other psychiatric disorders. Nevertheless, the cellular phenotypes that capture the fundamental features of psychiatric disorders have not yet been determined. Recent advances in genetics and stem cell biology offer new prospects for cell-based modeling of psychiatric disorders. The advent of cell reprogramming and induced pluripotent stem cells (iPSC) provides an opportunity to translate genetic findings into patient-specific in vitro models. iPSC technology is less than a decade old but holds great promise for bridging the gaps between patients, genetics and biology. Despite many obvious advantages, iPSC studies still present multiple challenges. In this expert review, we critically review the challenges for modeling of psychiatric disorders, potential solutions and how iPSC technology can be used to develop an analytical framework for the evaluation and therapeutic manipulation of fundamental disease processes.

"The developmental and functional logic of neuronal circuits": commentary on the Kavli Prize in Neuroscience.

The first Kavli Prize in Neuroscience recognizes a confluence of career achievements that together provide a fundamental understanding of how brain and spinal cord circuits are assembled during development and function in the adult. The members of the Kavli Neuroscience Prize Committee have decided to reward three scientists (Sten Grillner, Thomas Jessell, and Pasko Rakic) jointly "for discoveries on the developmental and functional logic of neuronal circuits". Pasko Rakic performed groundbreaking studies of the developing cerebral cortex, including the discovery of how radial glia guide the neuronal migration that establishes cortical layers and for the radial unit hypothesis and its implications for cortical connectivity and evolution. Thomas Jessell discovered molecular principles governing the specification and patterning of different neuron types and the development of their synaptic interconnection into sensorimotor circuits. Sten Grillner elucidated principles of network organization in the vertebrate locomotor central pattern generator, along with its command systems and sensory and higher order control. The discoveries of Rakic, Jessell and Grillner provide a framework for how neurons obtain their identities and ultimate locations, establish appropriate connections with each other, and how the resultant neuronal networks operate. Their work has significantly advanced our understanding of brain development and function and created new opportunities for the treatment of neurological disorders. Each has pioneered an important area of neuroscience research and left a legacy of exceptional scientific achievement, insight, communication, mentoring and leadership.

C fragment of tetanus toxin hybrid proteins evaluated for muscle-specific transsynaptic mapping of spinal motor circuitry in the newborn mouse.

We investigated whether the non-toxic C fragment of tetanus toxin (TTC) fused to either beta-galactosidase or green fluorescent protein could be utilized to transsynaptically trace muscle-specific spinal circuitry in the neonatal mouse after i.m. injection into a single hindlimb muscle. We found that even with careful low volume injection (0.2-1.0 microl) into a single muscle (medial gastrocnemius), the TTC hybrid proteins spread rapidly to many other hindlimb muscles and to trunk musculature such that retrograde labeling of motoneurons could not be constrained to a single motoneuron pool. Retrogradely labeled motoneurons in the lower lumbar segments harboring the medial gastrocnemius motoneuron pool were first observed two hours after the medial gastrocnemius injection. Within the next 10 h, additional lumbar and lower thoracic motoneurons became labeled, and punctate labeling in the neuropil surrounding the motoneurons appeared. Many of the TTC hybrid protein-labeled puncta in the neuropil co-localized synaptotagmin, indicating that they represent presynaptic axon terminals onto motoneurons. Although this is consistent with retrograde transsynaptic passage, we found no evidence that the TTC hybrid proteins were transported further along premotor axons to label interneuron somata. The i.m. TTC injection procedure described here therefore provides an important tool for the study of presynaptic terminals onto motoneurons. However, additional technical modifications will be required to utilize TTC tracers for transsynaptic mapping of muscle-specific spinal motor circuitry in the neonatal mouse. We provide here a set of criteria for assessing the i.m. delivery of TTC tracers as a basis for future improvements in this technique.

Quantitative axial profiles of retinoic acid in the embryonic mouse spinal cord: 9-cis retinoic acid only detected after all-trans-retinoic acid levels are super-elevated experimentally.

Studies using bioassays in normal mice and gene activation in transgenic reporter mice have demonstrated peaks of retinoic acid receptor (RAR) signaling in the brachial and lumbar regions of the spinal cord. Recently, Solomin et al. (Solomin et al. [1998] Nature 395:398-402) detected a retinoid X receptor (RXR) signal in the same region of the developing spinal cord at a slightly later stage than the RAR signal. This finding raises the question of which retinoid ligands underlie RAR and RXR signaling in this part of the embryo. Quantitative measurements of regional differences in retinoid profiles have not been reported previously due to limitation in the sensitivity and specificity of available retinoid detection methods. Here, by using a recently developed ultrasensitive HPLC technique (Sakhi et al. [1998] J. Chromatogr. A 828:451-460), we address this question in an attempt to identify definitively the endogenous retinoids present in different regions of the spinal cord at the stages when regional differences in RAR and RXR signaling have been reported. We find a bimodal distribution of all-trans retinoic acid (at-RA), the ligand for RARs, and relate this to the expression of several retinoid-synthesizing enzymes. However, we do not detect 9-cis-retinoic acid (9-cis-RA), the putative RXR ligand, in any region of the spinal cord unless retinoid levels are massively increased experimentally by gavage feeding pregnant mice with teratogenic doses of at-RA. This study provides for the first time quantitative profiles of endogenous retinoids along the axis of the developing spinal cord, thereby establishing a foundation for more definitive studies of retinoid function in the future. It sets definite limits on how much 9-cis-RA potentially is present and demonstrates that at-RA predominates over 9-cis-RA by at least 30- to 180-fold in different spinal cord regions.

Correlated patterns of neuron differentiation and Hox gene expression in the hindbrain: a comparative analysis.

Hindbrain neurons are organized into coherent subpopulations with characteristic projection patterns and functions. Many of these serve vital functions that have been conserved throughout the vertebrate radiation, but diversification to modified or highly specialized functions has also occurred. The differentiation of identifiable neuron groups in specific spatial domains must involve the regional expression of determinants within the hindbrain neuroepithelium. The Hox genes are involved in longitudinal regionalization of the neural tube, and their expression patterns in the hindbrain are closely related to the rhombomeres which partition the hindbrain into morphogenetic units. Hox gene expression also exhibits conserved patterning as well as phylogenetic variation. One plausible mechanism that may have contributed to evolutionary diversification in hindbrain neuron populations is therefore the emergence of species-specific differences in Hox gene expression. This article presents a comparative overview of the regional patterning of selected Hox genes and hindbrain neuron populations in several embryologically important species. Although tantalizing correlations exist, the relationship between Hox genes and neuronal patterning is complex, and complicated by dynamic features in each. Much more comparative and developmental data must be obtained before the link between Hox gene expression and hindbrain neuron patterning can be elucidated satisfactorily in an evolutionary context.

Quantitative assessment of retinoid signaling pathways in the developing eye and retina of the chicken embryo.

Retinoid signaling has been implicated as an important regulator of retinal development and differentiation. We have used state of the art high-pressure liquid chromatography to identify and quantitate biologically active retinoids, immunohistochemistry to localize the retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 (RALDH2), and nucleic acid assays to quantitate and localize retinoid receptor gene transcripts in the developing eye and retina of the chicken. Our results demonstrate spatial distinctions in retinoid synthesis and signaling that may be related to laminar differentiation in the developing retina. Retinoic acids (RAs) and their precursor retinols (ROHs) are the predominant retinoids in the developing eye. All-trans-RA and all-trans-3,4-didehydro-RA are present in the neuroepithelium in approximately equal amounts from early stages of neurogenesis until shortly before hatching. The retinoid X receptor (RXR) ligand 9-cis-RA is undetectable at all stages; if present, it cannot exceed a small percentage of the total RA content. RAs are not detected in the pigment epithelium. All-trans-ROH is present in the neuroepithelium and pigment epithelium, whereas all-trans-3,4-didehydro-ROH is detected only in the pigment epithelium and/or the choroid and sclera. RALDH2 immunoreactivity is intense in the choroid, low or absent in the pigment epithelium, and moderate in the neuroepithelium, where it is highest in the outer layers. Transcripts of all five chicken retinoid receptor genes are present in the neural retina and eye throughout development. During the period of neurogenesis, at least three of the receptors (RAR gamma, RXR gamma, RXRalpha), exhibit dynamic patterns of differential localization within the depths of the neural retina.

[New knowledge of brain repair]. / Ny kunnskap om reparasjon av hjernen.

BACKGROUND: Injury to the brain and spinal cord has long been considered particularly refractory to recovery because of the distinct lack of regenerative capacity in the central nervous system. In recent years, however, new knowledge has been gained about what limits this capacity, and increasing evidence has indicated that the potential for regrowth and functional reorganization is much greater than previously deduced. MATERIAL AND METHODS: This review presents historical background and a selection of recent studies that illustrate some of the emerging principles and the outlook for future clinical developments. RESULTS AND INTERPRETATION: Two potential restitutive mechanisms have been identified in the adult brain that open new horizons for the clinical promotion of brain repair. These art the presence of proliferative neural stem cells and the capacity for cortical reorganization within and across functional modalities.

Antecedents, descriptions and consequences of wandering in cognitively-impaired adults and the Safe Return (SR) program.

Unattended wandering is a major problem in cognitively impaired (CI) individuals and can result in those individuals becoming lost in the community. The purpose of this study was to identify important characteristics of unattended wandering and important prevention strategies. Data were compiledfrom registration files and missing and discovery reports collected through the Safe Return (SR) program. These data were analyzed to determine where individuals were found, who found them, from what setting they left, what mode of transportation they used, and what circumstances surrounded the unattended wandering. The study highlights the unpredictable and varied nature of unattended wandering. Recommendations are provided for communities to develop strategies to minimize unattended wandering and to determine effective methods of locating CI individuals when they become lost.

Neuroepithelial 'compartments' and the specification of vestibular projections.

The implication that there exist coherent vestibulo-ocular neuron pools with specific functions may provide new insight into how conjugate eye movements are synthesized within the vestibulo-ocular reflex. The systematic relationship between pool position and synergistic principle terminations, the 'hodological mosaic' suggests, moreover, a determinate groundplan established by developmental mechanisms operative at early stages in the hindbrain neuroepithelium. From such a groundplan, evolutionary and use-dependent modifications could mold connectivity patterns functionally appropriate for each species and individual. How the expression of developmentally regulatory genes contributes to establishing the mosaic organization of the vestibular system is the current focus of our research.

Development of specific connectivity between premotor neurons and motoneurons in the brain stem and spinal cord.

Astounding progress has been made during the past decade in understanding the general principles governing the development of the nervous system. An area of prime physiological interest that is being elucidated is how the neural circuitry that governs movement is established. The concerted application of molecular biological, anatomical, and electrophysiological techniques to this problem is yielding gratifying insight into how motoneuron, interneuron, and sensory neuron identities are determined, how these different neuron types establish specific axonal projections, and how they recognize and synapse upon each other in patterns that enable the nervous system to exercise precise control over skeletal musculature. This review is an attempt to convey to the physiologist some of the exciting discoveries that have been made, within a context that is intended to link molecular mechanism to behavioral realization. The focus is restricted to the development of monosynaptic connections onto skeletal motoneurons. Principal topics include the inductive mechanisms that pattern the placement and differentiation of motoneurons, Ia sensory afferents, and premotor interneurons; the molecular guidance mechanisms that pattern the projection of premotor axons in the brain stem and spinal cord; and the precision with which initial synaptic connections onto motoneurons are established, with emphasis on the relative roles played by cellular recognition versus electrical activity. It is hoped that this review will provide a guide to understanding both the existing literature and the advances that await this rapidly developing topic.

RXR gamma gene is expressed by discrete cell columns within the alar plate of the brainstem of the chicken embryo.

With in situ hybridization assays, we mapped the distribution of retinoid X receptor gamma (RXR gamma) gene transcripts in the central nervous system of the chicken embryo. Previous studies have demonstrated the presence of RXR gamma transcripts in migrating neural crest and in neural crest derivatives throughout the peripheral nervous system, implicating RXR gamma as an early pan-neural crest marker (Rowe et al. 1991. Development 111:771-778), and in the retina (Hoover et al. 1998. J Comp Neurol 391:204-213). Here we report the presence of RXR gamma transcripts in discrete regions of the developing neural tube, within the hindbrain, the cerebellar plate, the optic tectum, and the diencephalon. At stage 10, when migrating neural crest expresses RXR gamma transcripts, we detect no transcripts in the neural tube. By stage 13, RXR gamma transcripts accumulate to detectable levels along the midline of the posterior optic tectum, where the neural crest-derived sensory neurons of the mesencephalic trigeminal nucleus are located. By stage 15, RXR gamma transcripts also appear in an intermittent longitudinal cell column within the mantle zone of the alar plate of the hindbrain, eventually extending into the cerebellar plate rostrally and into the cervical spinal cord caudally, with a gap at about rhombomere 3. By stage 19, transcripts appear in a discrete population of cells within the diencephalon. Expression in these cell populations continues until at least stage 22.5, when many neuron populations have been generated in the hindbrain. The localization of the RXR gamma-positive cells to the mantle zone suggests that they are postmitotic and are probably neurons. Their specific alar locations indicate that they reside within sensory columns and potential downstream targets, evidently corresponding to some of the central components of the trigeminal system.

Regional patterning of reticulospinal and vestibulospinal neurons in the hindbrain of mouse and rat embryos.

The dispositions and axonal trajectories of bulbospinal neurons in the pons and medulla of mouse and rat embryos is described from the earliest times these projections can be labelled retrogradely from the cervical spinal cord. Reticulospinal and vestibulospinal neurons are clustered into identifiable groups, each with a characteristic combination of spatial domain and axon trajectory. The various groups can be labelled retrogradely in a specific developmental sequence. The position of some groups shifts from medial to lateral with development, apparently through cell migration. These observations show that the basic regional organization of the reticulospinal and vestibulospinal projections is similar in mouse and rat and is already established during early stages of axon outgrowth.

The relationship between rhombomeres and vestibular neuron populations as assessed in quail-chicken chimeras.

The aim of this study was to evaluate the role segmentation plays in the determination of neuronal identity in the hindbrain. We focused on two specific sets of hindbrain neurons, namely, the vestibulospinal and vestibulo-ocular neurons, which comprise distinct groups that can be identified and distinguished by virtue of their axonal projection pathways. The relationship between rhombomeres and the vestibular neuron groups was assessed by a combination of quail-chicken chimeric grafting and selective retrograde axonal tracing. Individual quail hemirhombomeres were transplanted homotopically and isochronically into a chicken embryo host. Subsequently, vestibulospinal and vestibulo-ocular neurons with specific axon trajectories were labeled retrogradely with biotin-conjugated dextran-amines. The relationship between the spatial domains of the vestibular neuron groups and rhombomere-derived domains had the following features: (1) some groups were derived from single rhombomeres; (2) some groups were derived from multiple contiguous rhombomeres; (3) two groups occupied domains that could not be defined in terms of whole rhombomere lengths; (4) some groups spanning multiple rhombomeres exhibited an internal cytoarchitectonic organization that related to individual rhombomeres; and (5) some groups exhibited limited boundary violation. These results support the notion that positional information within defined domains of the neural tube provides a groundplan for the regional determination of neuronal identity and axon pathfinding, and that hindbrain segmentation contributes to this process. But they also indicate that segmentation is not the only mechanism that defines the rostrocaudal domains of neuron types. Moreover, they emphasize that the relationship between rhombomeres and neuronal determination cannot be couched simply in terms of segmental iteration or of bimeric (paired rule) specification.

Segment-specific pattern of sympathetic preganglionic projections in the chicken embryo spinal cord is altered by retinoids.

Sympathetic preganglionic neurons exhibit segment-specific projections. Preganglionic neurons located in rostral spinal segments project rostrally within the sympathetic chain, those located in caudal spinal segments project caudally, and those in midthoracic segments project either rostrally or caudally in segmentally graded proportions. Moreover, rostrally and caudally projecting preganglionic neurons are skewed toward the rostral and caudal regions, respectively, of each midthoracic segment. The mechanisms that establish these segment-specific projections are unknown. Here we show that experimental manipulation of retinoid signaling in the chicken embryo alters the segment-specific pattern of sympathetic preganglionic projections and that this effect is mediated by the somitic mesoderm. Application of exogenous retinoic acid to a single rostral thoracic somite decreases the number of rostrally projecting preganglionic neurons at that level. Conversely, disrupting endogenous synthesis of retinoic acid in a single caudal thoracic somite increases the number of rostrally projecting preganglionic neurons at that level. The number of caudally projecting neurons does not change in either case, indicating that the effect is specific for rostrally projecting preganglionic neurons. These results indicate that the sizes of the rostrally and caudally projecting populations may be independently regulated by different factors. Opposing gradients of such factors along the longitudinal axis of the thoracic region of the embryo could be sufficient, in combination, to determine the segment-specific identity of preganglionic projections.

Regional pattern of retinoid X receptor-alpha gene expression in the central nervous system of the chicken embryo and its up-regulation by exposure to 9-cis retinoic acid.

We have investigated the expression of the retinoid X receptor-alpha (RXRalpha) gene in the developing chicken embryo by using nonradioactive wholemount in situ hybridization. At the earliest stage of development examined (stage 9; Hamburger and Hamilton [1951] J. Morphol. 88:49-92), we detect RXRalpha transcripts in a stretch of neuroepithelium corresponding roughly to the presumptive caudal hindbrain. Upon formation of the rhombomeres at stage 12, a strongly RXRalpha-positive region extends from a sharp rostral limit at the boundary between rhombomeres 6 and 7 caudad to at least the level of somite 9. This pattern of highest expression continues at least until stage 22 but with some variability in the caudal extent. A lower level of expression extends throughout the spinal cord. Transverse sections show that RXRalpha transcripts are expressed in a gradient, with the highest levels near the roof plate and decreasing toward the floor plate. At later stages, the level of expression is highest in the proliferative ventricular zone. However, at reduced levels, RXRalpha transcripts are also detectable in the mantle zone as well as outside the developing central nervous system, for example, in the neural crest and the limb buds. Nine-cis-retinoic acid up-regulates RXRalpha transcripts at stages 19.5-22.0 within a few hours, augmenting but not expanding the expression pattern. Northern blots demonstrate the potential expression of multiple RXRalpha isoforms in the central nervous system at posthatch stages. These results implicate the RXRalpha receptor in both rostrocaudal and transverse patterning of the neural tube.

Retinoid X receptor gamma gene transcripts are expressed by a subset of early generated retinal cells and eventually restricted to photoreceptors.

We have examined the distribution of the retinoid X receptor gamma (RXRgamma) in the developing chicken retina by using in situ hybridization and RNase protection assays. We detected RXRgamma transcripts as early as 4 days of embryonic development (d4) in central regions of the retina, spreading to more peripheral regions by d8. The first few RXRgamma-positive cells were scattered within the depth of the retinal neuroepithelium, but as they increased in number they became localized predominantly to the apical (outer, ventricular) layer. The identity of the RXRgamma-positive cells at these stages is unknown, due to the lack of cell type-specific markers. By d10, when photoreceptors and ganglion cells have been generated and begun to establish their definitive layers, RXRgamma-positive cells were virtually restricted to the photoreceptor layer, and maintained this distribution to posthatch stages. RNase protection assays were performed on whole retinae to verify the temporal pattern of in situ hybridization results and showed that between d5 and d16 there was a significant increase in the mRNA levels of the RXRgamma2 isoform. Between d16 and early posthatch stages the level of RXRgamma2 mRNA did not change significantly. Consistent with previous studies, mRNA levels of the RXRgamma1 isoform were substantially lower than mRNA levels of the RXRgamma2 isoform at all time points examined. These results demonstrate that RXRgamma mRNA is expressed in photoreceptors in the developing chicken retina and implicate RXRgamma as the earliest marker of photoreceptor differentiation documented to date.