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.

Serotonin analog selectively ablates indentified neurons in the leech embryo.

Exposure of embryonic leeches to 5,7-dihydroxytryptamine a cytotoxic analog of the monoamine neurotransmitter serotonin, results in the selective ablation of serotonin-containing neurons in the ventral nerve cord. Other neurons appear to be unaffected by this treatment, including those that contain another monoamine neurotransmitter, dopamine. Embryos with ablations continue to develop into juvenile leeches, but as juveniles they are unable to make normal swimming movements. However, normal swimming movements can be instated in such leeches by injecting them with serotonin.

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.

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.

Serotonin storage and uptake by identified neurons in the leech Haementeria ghilianii.

Characterization of serotonin-containing neurons in the glossiphoniid leech Haementeria ghilianii was undertaken to provide a reference for developmental studies of their differentiation and for comparative studies of their distribution and function. Five types of serotonin-containing neurons were identified with an antiserum against serotonin and by radioenzymatic assay of individual isolated somata. They contain high concentrations of serotonin (in some cases exceeding saturation in aqueous solution) and their serotonin content increases with growth of the animal. Each type is capable of taking serotonin up from the extracellular fluid, as demonstrated autoradiographically. They exhibit segment-specific, and on comparison with hirudinid leeches, species-specific, differences in distribution, morphology, and the expression of serotonin metabolism.

Development of the longitudinal projection patterns of lumbar primary sensory afferents in the chicken embryo.

The literature on the anatomical organization of primary sensory afferents, though extensive, contains relatively little information about the longitudinal extent of the central collateral projections. Our understanding of intersegmental sensorimotor integration in the spinal cord and of the developmental mechanisms that establish its underlying circuitry could be significantly enhanced by a more complete description of these projections. To address this issue from a developmental perspective, we labeled the central projections of lumbar primary afferents in fixed preparations of the chicken embryo with the lipophilic tracer DiI. At late embryonic stages, the afferent projections had the following characteristics: Primary afferents originating from a single lumbar dorsal root ganglion bifurcated to project longitudinally in the dorsal funiculus or Lissauer's tract. Dorsal funiculus axons extended up to seven segments caudally and to at least ten segments rostrally, whereas axons in Lissauer's tract extended up to seven segments in each direction. Collaterals branched off the longitudinal axons over a range of about seven segments in each direction. Within this range, collaterals to specific terminal fields exhibited more restricted ranges. The development of these longitudinal patterns during earlier embryonic stages was followed from the time the afferents first reached the neural tube on day 4 of embryogenesis. The longitudinal axons lengthened as a single bundle up to day 10, with medial axons consistently longer than lateral axons. After day 10, the longitudinal axons were segregated into the dorsal funiculus and Lissauer's tract. Collaterals sprouted after about 2 days of longitudinal axon growth, by which time the axons had extended several segments in each direction. The segmental range over which collaterals were present reached a maximum of 20 segments at day 10. Collaterals to the different terminal areas differed in their segmental ranges already by this time. After day 10, the total segmental range of collaterals decreased to the stable level of about seven segments in each direction, which is characteristic of late-stage embryos.

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.

Pathway specificity of reticulospinal and vestibulospinal projections in the 11-day chicken embryo.

The organization of the axonal pathways of reticulospinal and vestibulospinal projections in the 11-day chicken embryo was ascertained through retrograde tracing experiments. An in vitro preparation of the brainstem and cervical spinal cord facilitated precisely localized tracer applications. Single- and double-labelling experiments involving high cervical injections of tracers in combination with selective lesions defined the specific pathways by which different brainstem neurons project to the spinal cord. Coherent, and in many cases distinct, groups of reticulospinal and vestibulospinal neurons could thus be identified on the basis of their position and projection pathway. The organization of these groups and their projections in the 11-day chicken embryo is similar to that in avian and other vertebrate adults and therefore serves as a reference point for studies of pathfinding by bulbospinal axons during early development.

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.

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.

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.

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.

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 second-order vestibular projections in the chicken embryo.

The results reviewed here demonstrate that vestibulospinal and vestibulo-ocular interneurons in the chicken embryo are organized into coherent clusters, each of which occupies a unique spatial domain and has a characteristic axon trajectory. Moreover, the vestibulo-ocular clusters have specific termination patterns in the extraocular motoneuron pools and may to some extent have specific functions. The axon trajectories and termination patterns are established accurately during early development. This suggests the presence of selective mechanisms that ensure the generation of an appropriately specific connectivity pattern. The correlation of cluster domains with unique fields of regulatory gene expression at early stages suggests a mechanistic link between the position of a vestibular interneuron and the differentiation of its axon trajectory, termination pattern, and neurotransmitter phenotype.

Fluorescent dextran-amines used as axonal tracers in the nervous system of the chicken embryo.

We have used recently developed fluorescein- and rhodamine-conjugated dextran-amines as axonal tracers in in vitro preparations of the nervous system of the chicken embryo. These substances are efficiently taken up by injured axons and transported rapidly to the cell bodies. They are also, albeit to a lesser degree, taken up by intact nerve terminals in skeletal muscle. They reveal the dendritic and axonal structure of labelled neurons, and are well suited for use in double-labelling experiments.

Evidence that intersegmental competition influences the segmental distribution of the central terminals of muscle sensory afferents in the chicken embryo.

The possibility that muscle sensory afferents that enter the spinal cord at different segmental levels compete for terminal space in the lateral motor column was tested by unilaterally ablating neural crest cells destined to generate dorsal root ganglia (DRGs). Newly migrating neural crest was surgically removed from several lumbosacral segments on one side of the spinal cord on day 3 of embryogenesis (d3). The resultant experimental preparations developed without DRGs on the operated side at the segmental levels where the ablation was performed. At d18 the central projection from the intact DRG immediately caudal to the operated segments was anatomically mapped with a lipophilic axonal tracer. The central projection from the same DRG on the opposite, unoperated side was mapped as a control. The density of terminals along the longitudinal axis of the lateral motor column was quantified in serial sections with an image analysis program. On both the operated and control sides, the majority of terminals were found within the segment of entry and the rostral and caudal neighboring spinal segments. However, the rostral neighboring segment received a larger proportion of terminals on the operated side compared to the control side. Thus, the absence of primary afferents entering the spinal cord at specific segments leads to a shift of the afferent projection to the lateral motor column from the neighboring segments towards the deafferented segments.

The development of brain stem projections to the spinal cord in the chicken embryo.

Recent studies of the development of brain stem projections to the spinal cord in the chicken embryo, with an emphasis on axon pathway selection, are reviewed. Neurons from medullary to mesencephalic levels project to the spinal cord along specific fiber tracts. Coherent, segregated neuron groups can be defined on the basis of which tract and which side of the brain stem they project on. The choice of axon trajectory is, therefore, correlated with neuron position. During development, these trajectory-defined brain stem groups project to the spinal cord in a stereotyped sequence. Early stages of this sequence reveal a potential homology between the reticulospinal systems of avians and lower vertebrates. The possibility that neuron position may be involved in determining axon pathway choice of brain stem projections is supported by complementary studies on vestibuloocular projections. The boundaries between vestibuloocular, vestibulospinal, and reticulospinal neuron groups coincide with rhombomere boundaries and boundaries between longitudinal cell columns. Axon trajectory-specific domains are, therefore, correlated with segmental and mediolateral patterns of differential gene expression.