Post-gastrulation transition from whole-body to tissue-specific intercellular calcium signaling in the appendicularian tunicate Oikopleuradioica.

We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres. Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na+ â€‹channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.

Transcriptome analysis reveals disparate expression of inflammation-related miRNAs and their gene targets in iPSC-astrocytes from people with schizophrenia.

Despite the high heritability of schizophrenia (SCZ), details of its pathophysiology and etiology are still unknown. Recent findings suggest that aberrant inflammatory regulation and microRNAs (miRNAs) are involved. Here we performed a comparative analysis of the global miRNome of human induced pluripotent stem cell (iPSC)-astrocytes, derived from SCZ patients and healthy controls (CTRLs), at baseline and following inflammatory modulation using IL-1ß. We identified four differentially expressed miRNAs (miR-337-3p, miR-127-5p, miR-206, miR-1185-1-3p) in SCZ astrocytes that exhibited significantly lower baseline expression relative to CTRLs. Group-specific differential expression (DE) analyses exploring possible distinctions in the modulatory capacity of IL-1ß on miRNA expression in SCZ versus CTRL astroglia revealed trends toward altered miRNA expressions. In addition, we analyzed peripheral blood samples from a large cohort of SCZ patients (n = 484) and CTRLs (n = 496) screening for the expression of specific gene targets of the four DE miRNAs that were identified in our baseline astrocyte setup. Three of these genes, LAMTOR4, IL23R, and ERBB3, had a significantly lower expression in the blood of SCZ patients compared to CTRLs after multiple testing correction. We also found nominally significant differences for ERBB2 and IRAK1, which similarly displayed lower expressions in SCZ versus CTRL. Furthermore, we found matching patterns between the expressions of identified miRNAs and their target genes when comparing our in vitro and in vivo results. The current results further our understanding of the pathobiological basis of SCZ.

Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes.

Acid-sensing ion channels (ASICs) are proton-gated ion channels broadly expressed in the vertebrate nervous system, converting decreased extracellular pH into excitatory sodium current. ASICs were previously thought to be a vertebrate-specific branch of the DEG/ENaC family, a broadly conserved but functionally diverse family of channels. Here, we provide phylogenetic and experimental evidence that ASICs are conserved throughout deuterostome animals, showing that ASICs evolved over 600 million years ago. We also provide evidence of ASIC expression in the central nervous system of the tunicate, Oikopleura dioica Furthermore, by comparing broadly related ASICs, we identify key molecular determinants of proton sensitivity and establish that proton sensitivity of the ASIC4 isoform was lost in the mammalian lineage. Taken together, these results suggest that contributions of ASICs to neuronal function may also be conserved broadly in numerous animal phyla.

Gap junction-dependent coordination of intercellular calcium signalling in the developing appendicularian tunicate Oikopleura dioica.

We characterized spontaneous Ca2+ signals in Oikopleura dioica embryos from pre-fertilization to gastrula stages following injection of GCaMP6 mRNA into unfertilized eggs. The unfertilized egg exhibited regular, transient elevations in intracellular Ca2+ concentration with an average duration of 4-6 s and an average frequency of about 1 every 2.5 min. Fertilization was accompanied by a longer Ca2+ transient that lasted several minutes. Thereafter, regular Ca2+ transients were reinstated that spread within seconds among blastomeres and gradually increased in duration (by about 50%) and decreased in frequency (by about 20%) by gastrulation. Peak amplitudes also exhibited a dynamic, with a transitory drop occurring at about the 4-cell stage and a subsequent rise. Each peak was preceded by about 15 s by a smaller and shorter Ca2+ increase (about 5% of the main peak amplitude, average duration 3 s), which we term the "minipeak". By gastrulation, Ca2+ transients exhibited a stereotyped initiation site on either side of the 32-64-cell embryo, likely in the nascent muscle precursor cells, and spread thereafter symmetrically in a stereotyped spatial pattern that engaged blastomeres giving rise to all the major tissue lineages. The rapid spread of the transients relative to the intertransient interval created a coordinated wave that, on a coarse time scale, could be considered an approximate synchronization. Treatment with the divalent cations Ni2+ or Cd2+ gradually diminished peak amplitudes, had only moderate effects on wave frequency, but markedly disrupted wave synchronization and normal development. The T-type Ca2+ channel blocker mibefradil similarly disrupted normal development, and eliminated the minipeaks, but did not affect wave synchronization. To assess the role of gap junctions in calcium wave spread and coordination, we first characterized the expression of two Oikopleura connexins, Od-CxA and Od-CxB, both of which are expressed during pre-gastrulation and gastrula stages, and then co-injected double-stranded inhibitory RNAs together with CGaMP6 to suppress connexin expression. Connexin mRNA knockdown led to a gradual increase in Ca2+ transient peak width, a decrease of interpeak interval and a marked disruption of wave synchronization. As seen with divalent cations and mibefradil, this desynchronization was accompanied by a disruption of normal development.

Wilhelm His' lasting insights into hindbrain and cranial ganglia development and evolution.

Wilhelm His (1831-1904) provided lasting insights into the development of the central and peripheral nervous system using innovative technologies such as the microtome, which he invented. 150 years after his resurrection of the classical germ layer theory of Wolff, von Baer and Remak, his description of the developmental origin of cranial and spinal ganglia from a distinct cell population, now known as the neural crest, has stood the test of time and more recently sparked tremendous advances regarding the molecular development of these important cells. In addition to his 1868 treatise on 'Zwischenstrang' (now neural crest), his work on the development of the human hindbrain published in 1890 provided novel ideas that more than 100 years later form the basis for penetrating molecular investigations of the regionalization of the hindbrain neural tube and of the migration and differentiation of its constituent neuron populations. In the first part of this review we briefly summarize the major discoveries of Wilhelm His and his impact on the field of embryology. In the second part we relate His' observations to current knowledge about the molecular underpinnings of hindbrain development and evolution. We conclude with the proposition, present already in rudimentary form in the writings of His, that a primordial spinal cord-like organization has been molecularly supplemented to generate hindbrain 'neomorphs' such as the cerebellum and the auditory and vestibular nuclei and their associated afferents and sensory organs.

Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury.

KEY POINTS: Spinal compression injury targeted to the neonatal upper lumbar spinal cord, the region of highest hindlimb locomotor rhythmogenicity, leads to an initial paralysis of the hindlimbs. Behavioural recovery is evident within a few days and approaches normal function within about 3 weeks. Fictive locomotion in the isolated injured spinal cord cannot be elicited by a neurochemical cocktail containing NMDA, dopamine and serotonin 1 day post-injury, but can 3 days post-injury as readily as in the uninjured spinal cord. Low frequency coordinated rhythmic activity can be elicited in the isolated uninjured spinal cord by NMDA + dopamine (without serotonin), but not in the isolated injured spinal cord. In both the injured and uninjured spinal cord, eliciting bona fide fictive locomotion requires the additional presence of serotonin. ABSTRACT: Following incomplete compression injury in the thoracic spinal cord of neonatal mice 1 day after birth (P1), we previously reported that virtually normal hindlimb locomotor function is recovered within about 3 weeks despite substantial permanent thoracic tissue loss. Here, we asked whether similar recovery occurs following lumbar injury that impacts more directly on the locomotor central pattern generator (CPG). As in thoracic injuries, lumbar injuries caused about 90% neuronal loss at the injury site and increased serotonergic innervation below the injury. Motor recovery was slower after lumbar than thoracic injury, but virtually normal function was attained by P25 in both cases. Locomotor CPG status was tested by eliciting fictive locomotion in isolated spinal cords using a widely used neurochemical cocktail (NMDA, dopamine, serotonin). No fictive locomotion could be elicited 1 day post-injury, but could within 3 days post-injury as readily as in age-matched uninjured control spinal cords. Burst patterning and coordination were largely similar in injured and control spinal cords but there were differences. Notably, in both groups there were two main locomotor frequencies, but injured spinal cords exhibited a shift towards the higher frequency. Injury also altered the neurochemical dependence of locomotor CPG output, such that injured spinal cords, unlike control spinal cords, were incapable of generating low frequency rhythmic coordinated activity in the presence of NMDA and dopamine alone. Thus, the neonatal spinal cord also exhibits remarkable functional recovery after lumbar injuries, but the neurochemical sensitivity of locomotor circuitry is modified in the process.

Gaskell revisited: new insights into spinal autonomics necessitate a revised motor neuron nomenclature.

Several concepts developed in the nineteenth century have formed the basis of much of our neuroanatomical teaching today. Not all of these were based on solid evidence nor have withstood the test of time. Recent evidence on the evolution and development of the autonomic nervous system, combined with molecular insights into the development and diversification of motor neurons, challenges some of the ideas held for over 100 years about the organization of autonomic motor outflow. This review provides an overview of the original ideas and quality of supporting data and contrasts this with a more accurate and in depth insight provided by studies using modern techniques. Several lines of data demonstrate that branchial motor neurons are a distinct motor neuron population within the vertebrate brainstem, from which parasympathetic visceral motor neurons of the brainstem evolved. The lack of an autonomic nervous system in jawless vertebrates implies that spinal visceral motor neurons evolved out of spinal somatic motor neurons. Consistent with the evolutionary origin of brainstem parasympathetic motor neurons out of branchial motor neurons and spinal sympathetic motor neurons out of spinal motor neurons is the recent revision of the organization of the autonomic nervous system into a cranial parasympathetic and a spinal sympathetic division (e.g., there is no sacral parasympathetic division). We propose a new nomenclature that takes all of these new insights into account and avoids the conceptual misunderstandings and incorrect interpretation of limited and technically inferior data inherent in the old nomenclature.

Sensing External and Self-Motion with Hair Cells: A Comparison of the Lateral Line and Vestibular Systems from a Developmental and Evolutionary Perspective.

Detection of motion is a feature essential to any living animal. In vertebrates, mechanosensory hair cells organized into the lateral line and vestibular systems are used to detect external water or head/body motion, respectively. While the neuronal components to detect these physical attributes are similar between the two sensory systems, the organizational pattern of the receptors in the periphery and the distribution of hindbrain afferent and efferent projections are adapted to the specific functions of the respective system. Here we provide a concise review comparing the functional organization of the vestibular and lateral line systems from the development of the organs to the wiring from the periphery and the first processing stages. The goal of this review is to highlight the similarities and differences to demonstrate how evolution caused a common neuronal substrate to adapt to different functions, one for the detection of external water stimuli and the generation of sensory maps and the other for the detection of self-motion and the generation of motor commands for immediate behavioral reactions.

Segmental organization of vestibulospinal inputs to spinal interneurons mediating crossed activation of thoracolumbar motoneurons in the neonatal mouse.

Vestibulospinal pathways activate contralateral motoneurons (MNs) in the thoracolumbar spinal cord of the neonatal mouse exclusively via axons descending ipsilaterally from the vestibular nuclei via the lateral vestibulospinal tract (LVST; Kasumacic et al., 2010). Here we investigate how transmission from the LVST to contralateral MNs is mediated by descending commissural interneurons (dCINs) in different spinal segments. We test the polysynaptic nature of this crossed projection by assessing LVST-mediated ventral root (VR) response latencies, manipulating synaptic responses pharmacologically, and tracing the pathway transynaptically from hindlimb extensor muscles using rabies virus (RV). Longer response latencies in contralateral than ipsilateral VRs, near-complete abolition of LVST-mediated calcium responses in contralateral MNs by mephenesin, and the absence of transsynaptic RV labeling of contralateral LVST neurons within a monosynaptic time window all indicate an overwhelmingly polysynaptic pathway from the LVST to contralateral MNs. Optical recording of synaptically mediated calcium responses identifies LVST-responsive ipsilateral dCINs that exhibit segmental differences in proportion and dorsoventral distribution. In contrast to thoracic and lower lumbar segments, in which most dCINs are LVST responsive, upper lumbar segments stand out because they contain a much smaller and more ventrally restricted subpopulation of LVST-responsive dCINs. A large proportion of these upper lumbar LVST-responsive dCINs project to contralateral L5, which contains many of the hindlimb extensor MNs activated by the LVST. A selective channeling of LVST inputs through segmentally and dorsoventrally restricted subsets of dCINs provides a mechanism for targeting vestibulospinal signals differentially to contralateral trunk and hindlimb MNs in the mammalian spinal cord.

Targeting PBK/TOPK decreases growth and survival of glioma initiating cells in vitro and attenuates tumor growth in vivo.

BACKGROUND: Glioblastomas are invasive therapy resistant brain tumors with extremely poor prognosis. The Glioma initiating cell (GIC) population contributes to therapeutic resistance and tumor recurrence. Targeting GIC-associated gene candidates could significantly impact GBM tumorigenicity. Here, we investigate a protein kinase, PBK/TOPK as a candidate for regulating growth, survival and in vivo tumorigenicity of GICs. METHODS: PBK is highly upregulated in GICs and GBM tissues as shown by RNA and protein analyses. We knocked down PBK using shRNA vectors and inhibited the function of PBK protein with a pharmacological PBK inhibitor, HITOPK-032. We assessed viability, tumorsphere formation and apoptosis in three patient derived GIC cultures. RESULTS: Gene knockdown of PBK led to decreased viability and sphere formation and in one culture an increase in apoptosis. Treatment of cells with inhibitor HITOPK-032 (5 µM and 10 µM) almost completely abolished growth and elicited a large increase in apoptosis in all three cultures. HI-TOPK-032 treatment (5 mg/kg and 10 mg/kg bodyweight) in vivo resulted in diminished growth of experimentally induced subcutaneous GBM tumors in mice. We also carried out multi-culture assays of cell survival to investigate the relative effects on GICs compared with the normal neural stem cells (NSCs) and their differentiated counterparts. Normal NSCs seemed to withstand treatment slightly better than the GICs. CONCLUSION: Our study of identification and functional validation of PBK suggests that this candidate can be a promising molecular target for GBM treatment.

Modification of the larval swimming behavior in Oikopleura dioica, a chordate with a miniaturized central nervous system by dsRNA injection into fertilized eggs.

Using RNA interference, we have selectively perturbed neurotransmitter-related features of the larval swimming behavior of Oikopleura dioica, a tunicate with a central nervous system comprising about 130 neurons. We injected dsRNA into fertilized eggs to knockdown the expression of the genes, respectively, encoding ChAT (choline acetyltransferase) and GAD (glutamic acid decarboxylase), enzymes critical for the biosynthesis of acetylcholine and GABA. These two neurotransmitters have conserved roles during evolution, particularly within chordate motor systems, where they mediate respectively neuromuscular and central inhibitory signals. In Oikopleura, interference with ChAT expression prevented the normal bidirectional, propagating tail movement characteristic of swimming, permitting only repeated unilateral tail bends. Proper swimming was never observed, and the resting period between episodes of activity was lengthened. This phenotype is most likely caused by the reduction of transcription observed for both the targeted ChAT gene and the VAChT gene (Vesicular Acetylcholine Transporter), both genes being transcribed from the same operon. Interference with GAD expression led to an uncoordinated version of swimming with a spiral movement trajectory, but with episodes similar in duration and cycle frequency to those of normal swimming. Our results suggest locomotor functions for ChAT and GABA that are more subtle than previously proposed for tunicates and opens the way for a genetic dissection of Oikopleura neuronal circuits, which are likely to be among the most simplified in the chordate phylum.

Developmental Characterization of Tail Movements in the Appendicularian Urochordate Oikopleura dioica.

Using high-speed video cinematography, we characterized kinematically the spontaneous tail movements made by the appendicularian urochordate Oikopleura dioica. Videos of young adult (1-day-old) animals discriminated 4 cardinal movement types: bending, nodding, swimming and filtering, each of which had a characteristic signature including cyclicity, event or cycle duration, cycle frequency, cycle frequency variation, laterality, tail muscle segment coordination and episode duration. Bending exhibited a more common, unilateral form (single bending) and a rarer, bilateral form (alternating bending). Videos of developing animals showed that bending and swimming appeared in rudimentary form starting just after hatching and exhibited developmental changes in movement excursion, duration and frequency, whereas nodding and filtering appeared in the fully mature form in young adults at the time of first house production. More complex behaviors were associated with inflating, entering and exiting the house. We also assessed the influence of descending inputs by separating the tail (which contains all muscles and most likely the neural circuits that generate most motor outputs) from the head. Isolated tails spontaneously generated either bending or swimming movements in abnormally protracted episodes. This together with other observations of interactions between bending and swimming behaviors indicates the presence of several types of descending inputs that regulate the activity of the pattern generating circuitry in the tail nervous system.

Organization of pontine reticulospinal inputs to motoneurons controlling axial and limb muscles in the neonatal mouse.

Using optical recording of synaptically mediated calcium transients and selective spinal lesions, we investigated the pattern of activation of spinal motoneurons (MNs) by the pontine reticulospinal projection in isolated brain stem-spinal cord preparations from the neonatal mouse. Stimulation sites throughout the region where the pontine reticulospinal neurons reside reliably activated MNs at cervical, thoracic, and lumbar levels. Activation was similar in MNs ipsi- and contralateral to the stimulation site, similar in medial and lateral motor columns that contain trunk and limb MNs, respectively, and similar in the L2 and L5 segments that predominantly contain flexor and extensor MNs, respectively. In nonlesioned preparations, responses in both ipsi- and contralateral MNs followed individual stimuli in stimulus trains nearly one-to-one (with few failures). After unilateral hemisection at C1 on the same side as the stimulation, responses had substantially smaller magnitudes and longer latencies and no longer followed individual stimuli. After unilateral hemisection at C1 on the side opposite to the stimulation, the responses were also smaller, but their latencies were not affected. Thus we distinguish two pontine reticulospinal pathways to spinal MNs, one uncrossed and the other crossed, of which the uncrossed pathway transmits more faithfully and appears to be more direct.

Connecting ears to eye muscles: evolution of a 'simple' reflex arc.

Developmental and evolutionary data from vertebrates are beginning to elucidate the origin of the sensorimotor pathway that links gravity and motion detection to image-stabilizing eye movements--the vestibulo-ocular reflex (VOR). Conserved transcription factors coordinate the development of the vertebrate ear into three functional sensory compartments (graviception/translational linear acceleration, angular acceleration and sound perception). These sensory components connect to specific populations of vestibular and auditory projection neurons in the dorsal hindbrain through undetermined molecular mechanisms. In contrast, a molecular basis for the patterning of the vestibular projection neurons is beginning to emerge. These are organized through the actions of rostrocaudally and dorsoventrally restricted transcription factors into a 'hodological mosaic' within which coherent and largely segregated subgroups are specified to project to different targets in the spinal cord and brain stem. A specific set of these regionally diverse vestibular projection neurons functions as the central element that transforms vestibular sensory signals generated by active and passive head and body movements into motor output through the extraocular muscles. The large dynamic range of motion-related sensory signals requires an organization of VOR pathways as parallel, frequency-tuned, hierarchical connections from the sensory periphery to the motor output. We suggest that eyes, ears and functional connections subserving the VOR are vertebrate novelties that evolved into a functionally coherent motor control system in an almost stereotypic organization across vertebrate taxa.

Hox genes and region-specific sensorimotor circuit formation in the hindbrain and spinal cord.

Homeobox (Hox) genes were originally discovered in the fruit fly Drosophila, where they function through a conserved homeodomain as transcriptional regulators to control embryonic morphogenesis. In vertebrates, 39 Hox genes have been identified and like their Drosophila counterparts they are organized within chromosomal clusters. Hox genes interact with various cofactors, such as the TALE homeodomain proteins, in recognition of consensus sequences within regulatory elements of their target genes. In vertebrates, Hox genes display spatially restricted patterns of expression within the developing hindbrain and spinal cord, and are considered crucial determinants of segmental identity and cell specification along the anterioposterior and dorsoventral axes of the embryo. Here, we review their later roles in the assembly of neuronal circuitry, in stereotypic neuronal migration, axon pathfinding, and topographic connectivity. Importantly, we will put some emphasis on how their early-segmented expression patterns can influence the formation of complex vital hindbrain and spinal cord circuitries.

Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment.

BACKGROUND: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. RESULTS: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC-enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor-atypical expression profiles of neuron-, glia-, stem cell-, and tumor-related genes. CONCLUSIONS: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti-tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment.

Gene networks and the evolution of olfactory organs, eyes, hair cells and motoneurons: a view encompassing lancelets, tunicates and vertebrates.

Key developmental pathways and gene networks underlie the formation of sensory cell types and structures involved in chemosensation, vision and mechanosensation, and of the efferents these sensory inputs can activate. We describe similarities and differences in these pathways and gene networks in selected species of the three main chordate groups, lancelets, tunicates, and vertebrates, leading to divergent development of olfactory receptors, eyes, hair cells and motoneurons. The lack of appropriately posited expression of certain transcription factors in lancelets and tunicates prevents them from developing vertebrate-like olfactory receptors and eyes, although they generate alternative structures for chemosensation and vision. Lancelets and tunicates lack mechanosensory cells associated with the sensation of acoustic stimuli, but have gravisensitive organs and ciliated epidermal sensory cells that may (and in some cases clearly do) provide mechanosensation and thus the capacity to respond to movement relative to surrounding water. Although functionally analogous to the vertebrate vestibular apparatus and lateral line, homology is questionable due to differences in the expression of the key transcription factors Neurog and Atoh1/7, on which development of vertebrate hair cells depends. The vertebrate hair cell-bearing inner ear and lateral line thus likely represent major evolutionary advances specific to vertebrates. Motoneurons develop in vertebrates under the control of the ventral signaling molecule hedgehog/sonic hedgehog (Hh,Shh), against an opposing inhibitory effect mediated by dorsal signaling molecules. Many elements of Shh-signaling and downstream genes involved in specifying and differentiating motoneurons are also exhibited by lancelets and tunicates, but the repertoire of MNs in vertebrates is broader, indicating greater diversity in motoneuron differentiation programs.

Proliferation and recapitulation of developmental patterning associated with regulative regeneration of the spinal cord neural tube.

Developmental patterning during regulative regeneration of the chicken embryo spinal neural tube was characterized by assessing proliferation and the expression of transcription factors specific to neural progenitor and postmitotic neuron populations. One to several segments of the thoracolumbar neural tube were selectively excised unilaterally to initiate regeneration. The capacity for regeneration depended on the stage when ablation was performed and the extent of tissue removed. 20% of surviving embryos exhibited complete regulative regeneration, wherein the missing hemi-neural tube was reconstituted to normal size and morphology. Fate-mapping of proliferative adjacent tissue indicated contributions from the opposite side of the neural tube and potentially from the ipsilateral neural tube rostral and caudal to the lesion. Application of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) demonstrated a moderate increase in cell proliferation in lesioned relative to control embryos, and quantitative PCR demonstrated a parallel moderate increase in transcription of proliferation-related genes. Mathematical calculation showed that such modest increases are sufficient to account for the amount of regenerated tissue. Within the regenerated neural tube the expression pattern of progenitor-specific transcription factors was recapitulated in the separate advancing ventral and dorsal fronts of regeneration, with no evidence of abnormal mixing of progenitor subpopulations, indicating that graded patterning mechanisms do not require continuity of neural tube tissue along the dorsoventral axis and do not involve a sorting out of committed progenitors. Upon completion of the regeneration process, the pattern of neuron-specific transcription factor expression was essentially normal. Modest deficits in the numbers of transcription factor-defined neuron types were evident in the regenerated tissue, increasing particularly in dorsal neuron types with later lesions. These results confirm the regulative potential of the spinal neural tube and demonstrate a capacity for re-establishing appropriate cellular patterning despite a grossly abnormal morphogenetic situation.

EGF signalling and rapamycin-mediated mTOR inhibition in glioblastoma multiforme evaluated by phospho-specific flow cytometry.

Development of novel patient stratification tools for cancer is a challenge that require advanced molecular screening and a detailed understanding of tumour signalling networks. Here, we apply phospho-specific flow cytometry for signal profiling of primary glioblastoma tumours after preservation of single-cell phosphorylation status as a strategy for evaluation of tumour signalling potential and assessment of rapamycin-mediated mTOR inhibition. The method has already enhanced insight into cancers and disorders of the immune system, and our study demonstrate a great potential to improve the understanding of aberrant signalling in glioblastoma and other solid tumours.

Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.