Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices.

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 ß) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ventral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments.

Wild-Type and Mutant FUS Expression Reduce Proliferation and Neuronal Differentiation Properties of Neural Stem Progenitor Cells.

Nervous system development involves proliferation and cell specification of progenitor cells into neurons and glial cells. Unveiling how this complex process is orchestrated under physiological conditions and deciphering the molecular and cellular changes leading to neurological diseases is mandatory. To date, great efforts have been aimed at identifying gene mutations associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the RNA/DNA binding protein Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) have been associated with motor neuron degeneration in rodents and humans. Furthermore, increased levels of the wild-type protein can promote neuronal cell death. Despite the well-established causal link between FUS mutations and ALS, its role in neural cells remains elusive. In order to shed new light on FUS functions we studied its role in the control of neural stem progenitor cell (NSPC) properties. Here, we report that human wild-type Fused in Sarcoma (WT FUS), exogenously expressed in mouse embryonic spinal cord-derived NSPCs, was localized in the nucleus, caused cell cycle arrest in G1 phase by affecting cell cycle regulator expression, and strongly reduced neuronal differentiation. Furthermore, the expression of the human mutant form of FUS (P525L-FUS), associated with early-onset ALS, drives the cells preferentially towards a glial lineage, strongly reducing the number of developing neurons. These results provide insight into the involvement of FUS in NSPC proliferation and differentiation into neurons and glia.

Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

Prenatal ultrasound diagnosis of neural tube defects in the era of intrauterine repair - Eleven years' experiences.

OBJECTIVE: To modify the current neural tube defect (NTD) classification for fetal medicine specialists, and to investigate the impact of prenatal ultrasound conus medullaris position screening on the detection rate of closed spinal dysraphism and pregnancy outcomes. MATERIALS AND METHODS: The clinical data of 112 patients prenatally diagnosed with neural tube defects in Taiji clinic from 2008 to 2018 were retrospectively analyzed. All cases were classified following the modified classification. We compared the detection rate before and after introducing the conus medullaris screening and pregnancy outcomes for NTD types. RESULTS: Closed spinal dysraphism type prevailed in our sample (43.8%). The median gestational age at the time of detection for cranial dysraphism was 13.3 weeks, open spinal dysraphism was 22.0 weeks, and closed spinal dysraphism was 22.6 weeks. All cranial dysraphism (n = 43) and open spinal dysraphism cases (n = 20) had pregnancies terminated. For closed spinal dysraphism Class 1, the live-birth rate was 100.0% in the cases without other anomalies and 33.3% in the cases with other anomalies, respectively (X2 = 17.25, p < 0.001). Similarly, for Class 2, pregnancy continuation rate was 50.0% in cases without other anomalies and 20.0% in cases with other anomalies, yet it failed to reach statistical significance (X2 = 0.9, p = 0.524). CONCLUSION: Our case series may help to improve early screening and prenatal diagnosis of NTDs. Modified classification is adjusted for use in ultrasound fetal care facilities, which could be used for predicting pregnancy outcome. We suggest promoting first-trimester anatomical screening in order to make an earlier diagnosis and therefore provide better prenatal care for open spinal dysraphism cases in the era of intrauterine repair. Our findings imply that the use of fetal conus medullaris position as a marker for closed spinal dysraphism improves the detection rate and would unlikely lead to a higher termination rate.

Zygotic Vsx1 Plays a Key Role in Defining V2a Interneuron Sub-Lineage by Directly Repressing tal1 Transcription in Zebrafish.

In the spinal cord, excitatory V2a and inhibitory V2b interneurons are produced together by the final division of common P2 progenitors. During V2a and V2b diversification, Tal1 is necessary and sufficient to promote V2b differentiation and Vsx2 suppresses the expression of motor neuron genes to consolidate V2a interneuron identity. The expression program of Tal1 is triggered by a Foxn4-driven regulatory network in the common P2 progenitors. Why the expression of Tal1 is inhibited in V2a interneurons at the onset of V2a and V2b sub-lineage diversification remains unclear. Since transcription repressor Vsx1 is expressed in the P2 progenitors and newborn V2a cells in zebrafish, we investigated the role of Vsx1 in V2a fate specification during V2a and V2b interneuron diversification in this species by loss and gain-of-function experiments. In vsx1 knockdown embryos or knockout Go chimeric embryos, tal1 was ectopically expressed in the presumptive V2a cells, while the generation of V2a interneurons was significantly suppressed. By contrast, in vsx1 overexpression embryos, normal expression of tal1 in the presumptive V2b cells was suppressed, while the generation of V2a interneuron was expanded. Chromatin immunoprecipitation and electrophoretic mobility shift assays in combination with core consensus sequence mutation analysis further revealed that Vsx1 can directly bind to tal1 promoter and repress tal1 transcription. These results indicate that Vsx1 can directly repress tal1 transcription and plays an essential role in defining V2a interneuron sub-lineage during V2a and V2b sub-lineage diversification in zebrafish.

Differentiation and localization of interneurons in the developing spinal cord depends on DOT1L expression.

Genetic and epigenetic factors contribute to the development of the spinal cord. Failure in correct exertion of the developmental programs, including neurulation, neural tube closure and neurogenesis of the diverse spinal cord neuronal subtypes results in defects of variable severity. We here report on the histone methyltransferase Disruptor of Telomeric 1 Like (DOT1L), which mediates histone H3 lysine 79 (H3K79) methylation. Conditional inactivation of DOT1L using Wnt1-cre as driver (Dot1l-cKO) showed that DOT1L expression is essential for spinal cord neurogenesis and localization of diverse neuronal subtypes, similar to its function in the development of the cerebral cortex and cerebellum. Transcriptome analysis revealed that DOT1L deficiency favored differentiation over progenitor proliferation. Dot1l-cKO mainly decreased the numbers of dI1 interneurons expressing Lhx2. In contrast, Lhx9 expressing dI1 interneurons did not change in numbers but localized differently upon Dot1l-cKO. Similarly, loss of DOT1L affected localization but not generation of dI2, dI3, dI5, V0 and V1 interneurons. The resulting derailed interneuron patterns might be responsible for increased cell death, occurrence of which was restricted to the late developmental stage E18.5. Together our data indicate that DOT1L is essential for subtype-specific neurogenesis, migration and localization of dorsal and ventral interneurons in the developing spinal cord, in part by regulating transcriptional activation of Lhx2.

Expression of Pax6 and Pax7 proteins during the central nervous system development in human embryos / Expresión de proteínas Pax6 y Pax7 durante el desarrollo del sistema central nervioso en embriones humanos

The family of paired box (Pax) genes encodes the transcription factors that have been emphasized for the particular importance to embryonic development of the CNS, with the evidence obtained from various animal models. Human embryos have rarely been available for the detection of the expression of Pax family members. In this study 32 human embryos of Carnegie (CS) stages 10-20 were investigated to find the differences in the expression of Pax6 and Pax7 proteins in different regions of the neural tube and the caudal spinal cord. The expression of Pax6 and Pax7, as determined by immunohistochemistry, showed a tendency to increase in the later stages of the development both in the spinal cord and the brain. Significantly weaker expression of Pax6 and Pax7 was observed at CS 10 as compared to the later stages. At CS 10-12 weak expression of Pax6 was noticed in both dorsal and ventral parts of the developing spinal cord, while the expression of Pax7 was restricted to the cells in the roof plate and the dorsal part of the spinal cord. At CS 14-20 in the developing spinal cord Pax6 and Pax7 were detected mostly in the neuroepithelial cells of the ventricular layer, while only weak expression characterized the mantle and the marginal layers. At the same stages in the developing brain Pax6 and Pax7 were expressed in the different regions of the forebrain, the midbrain and the hindbrain suggesting for their involvement in the differentiation of neurons in specific parts of the developing brain.

La familia de genes Pax del inglés (Paired box) codifica los factores de transcripción debido a la particular importancia en el desarrollo embrionario del SNC, con la evidencia obtenida de varios modelos animales. Rara vez han estado disponibles embriones humanos para la detección de la expresión de genes de la familia Pax. En este estudio, se investigaron 32 embriones humanos de Carnegie (CS) etapas 10-20 para encontrar las diferencias en la expresión de las proteínas Pax6 y Pax7 en diferentes regiones del tubo neural y la médula espinal caudal. La expresión de Pax6 y Pax7, según la inmunohistoquímica, se observó una tendencia a aumentar en las etapas posteriores del desarrollo, tanto en la médula espinal como en el cerebro. Se observó una expresión significativamente más débil de Pax6 y Pax7 en CS 10 en comparación con las etapas posteriores. En CS 10-12 se notó una expresión débil de Pax6 en las partes dorsal y ventral de la médula espinal en desarrollo, mientras que la expresión de Pax7 se limitó a células en la placa del techo y dorsal de la médula espinal. En CS 14-20 en la médula espinal en desarrollo, Pax6 y Pax7 se observó principalmente en las células neuroepiteliales de la capa ventricular, mientras que expresión débil se caracterizó en las capas marginales. En las mismas etapas en el cerebro en desarrollo, Pax6 y Pax7 se expresaron en las diferentes áreas del prosencéfalo, el mesencéfalo y el mesencéfalo, lo que sugiere su participación en la diferenciación de las neuronas en partes específicas del cerebro en desarrollo.

BMP7 is Downregulated in Lumbosacral Spinal Cord of Rat Embryos With Anorectal Malformation.

BACKGROUND: Bone morphogenetic proteins (BMPs) comprise a highly conserved signaling protein family, which are involved in spinal cord formation, development and differentiation. Malformations of the lumbosacral spinal cord are associated with postoperation complications of anorectal malformation (ARM). However, the mechanism underlying the development of these malformations remains elusive. MATERIALS AND METHODS: Embryonic rat ARM model induced by ethylenethiourea (ETU) was introduced to investigate BMP7 expression in lumbosacral spinal cord. BMP7 expression was analyzed by immunohistochemical staining, qRT-PCR, and Western blot analysis on embryonic (E) days 16, 17, 19, and 21. The expression of the neuronal marker neurofilament (NF) and pSmad1/5 was determined by immunofluorescence double staining and Western blot analysis during peak BMP7 expression. RESULTS: BMP7 mRNA (E16, 1.041 ± 0.169 versus 0.758 ± 0.0423, P < 0.05; E17, 1.889 ± 0.444 versus 1.601 ± 0.263, P < 0.05; E19, 2.898 ± 0.434 versus 1.981 ± 0.068, P < 0.01; and E21, 2.652 ± 0.637 versus 1.957 ± 0.09, P < 0.05;) and protein (E16, 1.068 ± 0.065 versus 0.828 ± 0.066, P < 0.01; E17, 1.728 ± 0.153 versus1.4 ± 0.148, P < 0.05; E19, 2.313 ± 0.141 versus 1.696 ± 0.21, P < 0.01; and E21, 2.021 ± 0.13 versus 1.43 ± 0.128, P < 0.01) were downregulated, and their expressions were specifically low in interneurons (IN) located in the dorsal horn of the lumbosacral spinal cord in embryos with ARM. On E19, Western blot analysis revealed reduced P-Smad1/5(1.13 ± 0.08 versus 0.525 ± 0.06, P < 0.01). CONCLUSIONS: An implication of this study is the possibility that BMP7 downregulation contributes to maldevelopment of the lumbosacral spinal cord during embryogenesis in fetal rats with ARM, indicating that BMP7 may play an important role in ARM pathogenesis and the complications thereof.

Measurement of fetal conus distance with 3D ultrasonography as a reliable prenatal diagnosis method for tethered cord syndrome.

AIM: The aim of this study is to investigate if three-dimensional (3D) ultrasonography is a reliable diagnosis method for prenatal tethered cord syndrome (TCS) by measuring fetal conus distance (CD) in comparison to two-dimensional (2D) ultrasonography and magnetic resonance imaging (MRI). METHODS: This retrospective study included 468 normal fetuses as control group and 14 TCS fetuses as tethered group. CD were measured by 2D, 3D ultrasonography and MRI, and the reliability and repeatability of CD measurement was compared between two experienced ultrasound specialists or among the multiple measurements for each specialist. RESULTS: The results showed that 3D ultrasonography was superior to 2D ultrasonography in the repeatability of measuring CD. The CD were positively correlated to gestational ages in control group (P < 0.05). The CD of TCS group (-1.342 ± 0.124) was significantly shorter than that of normal control group (0.013 ± 0.965) (P < 0.01). There were no significant differences in the effectiveness of locating conus medullaris and measuring CD between 3D ultrasonography and MRI.Conclusion Measurement of fetal CD with 3D ultrasonography can serve as a reliable and cost-effective prenatal diagnosis method for TCS.

Cell necrosis, intrinsic apoptosis and senescence contribute to the progression of exencephaly to anencephaly in a mice model of congenital chranioschisis.

Exencephaly/anencephaly is one of the leading causes of neonatal mortality and the most extreme open neural tube defect with no current treatments and limited mechanistic understanding. We hypothesized that exencephaly leads to a local neurodegenerative process in the brain exposed to the amniotic fluid as well as diffuse degeneration in other encephalic areas and the spinal cord. To evaluate the consequences of in utero neural tissue exposure, brain and spinal cord samples from E17 exencephalic murine fetuses (maternal intraperitoneal administration of valproic acid at E8) were analyzed and compared to controls and saline-injected shams (n = 11/group). Expression of apoptosis and senescence genes (p53, p21, p16, Rbl2, Casp3, Casp9) was determined by qRT-PCR and protein expression analyzed by western blot. Apoptosis was measured by TUNEL assay and PI/AV flow cytometry. Valproic acid at E8 induced exencephaly in 22% of fetuses. At E17 the fetuses exhibited the characteristic absence of cranial bones. The brain structures from exencephalic fetuses demonstrated a loss of layers in cortical regions and a complete loss of structural organization in the olfactory bulb, hippocampus, dental gyrus and septal cortex. E17 fetuses had reduced expression of NeuN, GFAP and Oligodendrocytes in the brain with primed microglia. Intrinsic apoptotic activation (p53, Caspase9 and 3) was upregulated and active Caspase3 localized to the layer of brain exposed to the amniotic fluid. Senescence via p21-Rbl2 was increased in the brain and in the spinal cord at the lamina I-II of the somatosensory dorsal horn. The current study characterizes CNS alterations in murine exencephaly and demonstrates that degeneration due to intrinsic apoptosis and senescence occurs in the directly exposed brain but also remotely in the spinal cord.

Notch signalling maintains Hedgehog responsiveness via a Gli-dependent mechanism during spinal cord patterning in zebrafish.

Spinal cord patterning is orchestrated by multiple cell signalling pathways. Neural progenitors are maintained by Notch signalling, whereas ventral neural fates are specified by Hedgehog (Hh) signalling. However, how dynamic interactions between Notch and Hh signalling drive the precise pattern formation is still unknown. We applied the PHRESH (PHotoconvertible REporter of Signalling History) technique to analyse cell signalling dynamics in vivo during zebrafish spinal cord development. This approach reveals that Notch and Hh signalling display similar spatiotemporal kinetics throughout spinal cord patterning. Notch signalling functions upstream to control Hh response of neural progenitor cells. Using gain- and loss-of-function tools, we demonstrate that this regulation occurs not at the level of upstream regulators or primary cilia, but rather at the level of Gli transcription factors. Our results indicate that Notch signalling maintains Hh responsiveness of neural progenitors via a Gli-dependent mechanism in the spinal cord.

Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification.

During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

Inferring Regulatory Programs Governing Region Specificity of Neuroepithelial Stem Cells during Early Hindbrain and Spinal Cord Development.

Neuroepithelial stem cells (NSC) from different anatomical regions of the embryonic neural tube's rostrocaudal axis can differentiate into diverse central nervous system tissues, but the transcriptional regulatory networks governing these processes are incompletely understood. Here, we measure region-specific NSC gene expression along the rostrocaudal axis in a human pluripotent stem cell model of early central nervous system development over a 72-h time course, spanning the hindbrain to cervical spinal cord. We introduce Escarole, a probabilistic clustering algorithm for non-stationary time series, and combine it with prior-based regulatory network inference to identify genes that are regulated dynamically and predict their upstream regulators. We identify known regulators of patterning and neural development, including the HOX genes, and predict a direct regulatory connection between the transcription factor POU3F2 and target gene STMN2. We demonstrate that POU3F2 is required for expression of STMN2, suggesting that this regulatory connection is important for region specificity of NSCs.

The people behind the papers - Shai Eyal and Elazar Zelzer.

Most bones in the vertebrate skeleton are made in the same way - endochondrial ossification - yet they display a variety of shapes and sizes. The question of how these unique bone morphologies, including the superstructures that protrude from their surfaces, arise during development is still unclear, and the subject of a new paper in Development We caught up with first author Shai Eyal and his supervisor Elazar Zelzer, Professor in the Department of Molecular Genetics at the Weizmann Institute of Science in Rehovot, Israel, to find out more about the story.

The Neuromodulator Adenosine Regulates Oligodendrocyte Migration at Motor Exit Point Transition Zones.

During development, oligodendrocyte progenitor cells (OPCs) migrate extensively throughout the spinal cord. However, their migration is restricted at transition zones (TZs). At these specialized locations, unique glial cells in both zebrafish and mice play a role in preventing peripheral OPC migration, but the mechanisms of this regulation are not understood. To elucidate the mechanisms that mediate OPC segregation at motor exit point (MEP) TZs, we performed an unbiased small-molecule screen. Using chemical screening and in vivo imaging, we discovered that inhibition of A2a adenosine receptors (ARs) causes ectopic OPC migration out of the spinal cord. We provide in vivo evidence that neuromodulation, partially mediated by adenosine, influences OPC migration specifically at the MEP TZ. This work opens exciting possibilities for understanding how OPCs reach their final destinations during development and identifies mechanisms that could promote their migration in disease.

Subtype Diversification and Synaptic Specificity of Stem Cell-Derived Spinal Interneurons.

Neuronal diversification is a fundamental step in the construction of functional neural circuits, but how neurons generated from single progenitor domains acquire diverse subtype identities remains poorly understood. Here we developed an embryonic stem cell (ESC)-based system to model subtype diversification of V1 interneurons, a class of spinal neurons comprising four clades collectively containing dozens of molecularly distinct neuronal subtypes. We demonstrate that V1 subtype diversity can be modified by extrinsic signals. Inhibition of Notch and activation of retinoid signaling results in a switch to MafA clade identity and enriches differentiation of Renshaw cells, a specialized MafA subtype that mediates recurrent inhibition of spinal motor neurons. We show that Renshaw cells are intrinsically programmed to migrate to species-specific laminae upon transplantation and to form subtype-specific synapses with motor neurons. Our results demonstrate that stem cell-derived neuronal subtypes can be used to investigate mechanisms underlying neuronal subtype specification and circuit assembly.

Secretory pathway calcium ATPase 1 (SPCA1) controls mouse neural tube closure by regulating cytoskeletal dynamics.

Neural tube closure relies on the apical constriction of neuroepithelial cells. Research in frog and fly embryos has found links between the levels of intracellular calcium, actomyosin dynamics and apical constriction. However, genetic evidence for a role of calcium in apical constriction during mammalian neurulation is still lacking. Secretory pathway calcium ATPase (SPCA1) regulates calcium homeostasis by pumping cytosolic calcium into the Golgi apparatus. Loss of function in Spca1 causes cranial exencephaly and spinal cord defects in mice, phenotypes previously ascribed to apoptosis. However, our characterization of a novel allele of Spca1 revealed that neurulation defects in Spca1 mutants are not due to cell death, but rather to a failure of neuroepithelial cells to apically constrict. We show that SPCA1 influences cell contractility by regulating myosin II localization. Furthermore, we found that loss of Spca1 disrupts actin dynamics and the localization of the actin remodeling protein cofilin 1. Taken together, our results provide evidence that SPCA1 promotes neurulation by regulating the cytoskeletal dynamics that promote apical constriction and identify cofilin 1 as a downstream effector of SPCA1 function.

Bone morphogenetic protein 4 expression in the developing lumbosacral spinal cord of rat embryos with anorectal malformations.

Although there are improvements in treatment of anorectal malformations (ARMs), patients can still develop fecal incontinence, constipation, and soiling with loss in quality of life. Recent evidence suggests that malformations in the lumbosacral spinal cord are one of the factors that affect postoperative anorectal function. However, the underlying mechanism that produces these malformations has yet to be elucidated. The bone morphogenetic proteins (BMPs) comprise a large group of highly conserved molecules that are involved in multiple processes and play important roles in the formation, development, and differentiation of the spinal cord. This study was designed to investigate the levels of BMP4 expression in the lumbosacral spinal cord in ARMs rat embryos induced by ethylenethiourea (ETU). Specifically, we assessed the association of BMP4 levels with the maldevelopment of the lumbosacral spinal cord and whether BMP4 acted through the canonical intracellular pathway in embryonic rats with ARMs. BMP4 expression was confirmed with immunohistochemical staining, RT-qPCR and western blot analyses of embryonic day (E) 16, E17, E19 and E21 embryos, moreover Smad1/5 and pSmad1/5 expression were confirmed with western blot analyses at peak time point of BMP4 expression. Our results reveal that BMP4 expression in the lumbosacral spinal cord of ARMs rat embryos is decreased at both the mRNA and protein levels and could decrease the phosphorylation of smad1/5, when compared with their expression levels in normal tissue. These results also suggest that reductions in BMP4 expression were possibly responsible for dysfunction of the lumbosacral spinal cord during late developmental stages in ARMs fetal rats. Taken together, we conclude a role for BMP4 in the pathogenesis of lumbosacral spinal cord maldevelopment in developing ARMs rats.

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse.

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms.

Vascularization of the Damaged Nerve under the Effect of Experimental Cell Therapy.

Quantitative analysis of blood vessels in the distal segment of rat sciatic nerve after its ligation for 40 sec and subperineurial administration of mesenchymal stem cells or dissociated cells of rat embryonic spinal cord was carried our by immunohistochemical tracing of von Willebrand factor, a marker of endothelial cells of blood vessels. It was found that the number of blood vessels per unit area of the nerve trunk in 21 days after injury and administration of mesenchymal stem cells increased by more than 1.5 times in comparison with the control (damaged nerve). After administration of dissociated cells of the embryonic spinal cord, this effect was not observed. It is assumed that mesenchymal stem cells stimulate the growth of vessels of the damaged nerve via production of angiogenic factors.