Optical instruments for space applications with improved performances (smaller pixels and spectral range extension) are becoming more and more sensitive to chemical contamination and particle sedimentation. Outgassing under vacuum conditions causes dramatic flux losses, especially in the UV bandwidth. Furthermore, it is difficult to perform physicochemical analyses of contaminated surfaces on flight models, in a clean room. Conventional analytical techniques such as FTIR (Fourier Transform Infrared interferometer) need the tool to be in contact with the studied area, which is forbidden when working on satellites. In addition, it does not give any information about the distribution of the contaminants in the field of view. The probed area is large, mono-pixel, and the sensitivity of the instrument is too low for hundred nanometer thin film deposits. A first study has shown that we could benefit from using the UV/visible fluorescence spectra to partially identify contaminants and polymer materials. The shape of the fluorescence spectra of adhesives, paints and varnishes have specific signatures that could be recorded into a designated reference database. The location of the presence of these contaminants on such sensitive optics is also relevant. To acquire both these parameters, we designed a specific compact hyperspectral instrument to remotely acquire cube images (500x500 pixels) in a 5 degree field of view, and on a wide range of continuous wavelengths from UV at 320 nm up to the near infrared at 1000 nm. This paper will present the chosen trade-off between different critical optics for a new portable version of this instrument. It is dedicated to space and cultural heritage applications and the first results on an engineering prototype will be shown.
In vertebrates, the sensory neurons of the epibranchial (EB) ganglia transmit somatosensory signals from the periphery to the CNS. These ganglia are formed during embryogenesis by the convergence and condensation of two distinct populations of precursors: placode-derived neuroblasts and neural crest- (NC) derived glial precursors. In addition to the gliogenic crest, chondrogenic NC migrates into the pharyngeal arches, which lie in close proximity to the EB placodes and ganglia. Here, we examine the respective roles of these two distinct NC-derived populations during development of the EB ganglia using zebrafish morphant and mutants that lack one or both of these NC populations. Our analyses of mutant and morphant zebrafish that exhibit deficiencies in chondrogenic NC at early stages reveal a distinct requirement for this NC subpopulation during early EB ganglion assembly and segmentation. Furthermore, restoration of wildtype chondrogenic NC in one of these mutants, prdm1a, is sufficient to restore ganglion formation, indicating a specific requirement of the chondrogenic NC for EB ganglia assembly. By contrast, analysis of the sox10 mutant, which lacks gliogenic NC, reveals that the initial assembly of ganglia is not affected. However, during later stages of development, EB ganglia are dispersed in the sox10 mutant, suggesting that glia are required to maintain normal EB ganglion morphology. These results highlight novel roles for two subpopulations of NC cells in the formation and maintenance of EB ganglia: chondrogenic NC promotes the early-stage formation of the developing EB ganglia while glial NC is required for the late-stage maintenance of ganglion morphology.
Background: Neuronal and glial differentiation in the murine hypothalamus is not complete at birth, but continues over the first two weeks postnatally. Nutritional status and Leptin deficiency can influence the maturation of neuronal projections and glial patterns, and hypothalamic gliosis occurs in mouse models of obesity. Gnasxl constitutes an alternative transcript of the genomically imprinted Gnas locus and encodes a variant of the signalling protein Ga-s, termed XLas, which is expressed in defined areas of the hypothalamus. Gnasxl-deficient mice show postnatal growth retardation and undernutrition, while surviving adults remain lean and hypermetabolic with increased sympathetic nervous system (SNS) activity. Effects of this knock-out on the hypothalamic neural network have not yet been investigated. Results: RNAseq analysis for gene expression changes in hypothalami of Gnasxl-deficient mice indicated Glial fibrillary acid protein (Gfap) expression to be significantly down-regulated in adult samples. Histological analysis confirmed a reduction in Gfap-positive glial cell numbers specifically in the hypothalamus. This reduction was observed in adult tissue samples, whereas no difference was found in hypothalami of postnatal stages, indicating an adaptation in adult Gnasxl-deficient mice to their earlier growth phenotype and hypermetabolism. Especially noticeable was a loss of many Gfap-positive alpha-tanycytes and their processes, which form part of the ependymal layer that lines the medial and dorsal regions of the 3rd ventricle, while beta-tanycytes along the median eminence (ME) and infundibular recesses appeared unaffected. This was accompanied by local reductions in Vimentin and Nestin expression. Hypothalamic RNA levels of glial solute transporters were unchanged, indicating a potential compensatory up-regulation in the remaining astrocytes and tanycytes. Conclusion: Gnasxl deficiency does not directly affect glial development in the hypothalamus, since it is expressed in neurons, and Gfap-positive astrocytes and tanycytes appear normal during early postnatal stages. The loss of Gfap-expressing cells in adult hypothalami appears to be a consequence of the postnatal undernutrition, hypoglycaemia and continued hypermetabolism and leanness of Gnasxl-deficient mice, which contrasts with gliosis observed in obese mouse models. Since alpha-tanycytes also function as adult neural progenitor cells, these findings might indicate further developmental abnormalities in hypothalamic formations of Gnasxl-deficient mice, potentially including neuronal composition and projections.
Transgenic multicolor fluorescence reporters enable the visualization of alternative splicing patterns at a single-cell resolution in living organisms and facilitate further genetic analyses to identify cis-elements and trans-acting factors involved in splicing regulation. In this paper, we describe a method of generating fluorescence alternative splicing reporters for the nematode Caenorhabditis elegans. We describe strategies for designing minigene reporters and methods for constructing them; DNA fragments ('modules', such as promoter/3' cassettes, a genomic fragment of interest and a fluorescent protein cassette) that exist in separate vectors are assembled using site-directed recombination. We also describe strategies and methods for mutant screening and single-nucleotide polymorphism mapping using fluorescence reporters. This is the first detailed description of the design and construction of fluorescence alternative splicing reporters for C. elegans and their use in subsequent genetic analyses. It takes 2-4 months to construct minigenes and generate extrachromosomal lines for visualizing spatiotemporal distribution of alternative splicing events in vivo. Identification of regulators by integration of transgenes, mutant screening and mapping of the responsible genes takes a further 6-12 months. The fluorescence-reporter construction described here can also be applied to the vertebrate cell culture system.
We have developed a four-part protocol to differentiate human embryonic stem cells (hESCESCESCs) to oligodendrocyte progenitor cells (OPCOPCOPCs) according to developmental principles. In the first 2 weeks, hESCESCESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLOLIG2-expressing progenitors in the presence of retinoic acid (RARA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLOLIG2 and NKX2.2-expressing pre-OPCOPCOPCs. Finally, the pre-OPCOPCOPCs take 8-9 weeks to differentiate into OPCOPCOPCs, which express additional markers of oligodendrocytes, such as SOSOX10, platelet-derived growth factor receptor alpha (PDGFR alpha) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCOPCOPCs in the third part and the removal of FGF2 during the transition of pre-OPCOPCOPCs to OPCOPCOPCs. This 3-month differentiation protocol consistently yields OPCOPCOPCs of high purity capable of producing myelin sheaths in vivo.
We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic, HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods, our protocol does not use feeder cells, has a minimum dependence on proteins (purmorphamine replacing SHH), has controllable adherent selection and is adaptable for scalable suspension culture.
As a step toward using two closely related members of the nuclear receptor family, RORα and RORβ, as markers and tools for genetic manipulations in mouse forebrain, we have used in situ hybridization to analyze their expression from E10.5 to P7. At later embryonic and early postnatal ages, RORα expression in dorsal thalamus is mainly limited to robust expression throughout the principal sensory nuclei. RORβ is expressed in a similar set of dorsal thalamic nuclei as RORα, but exhibits a more limited expression within the principal sensory nuclei. RORα is expressed as early as E12.5 in dorsal thalamus by presumptive ventroposterior neurons, whereas RORβ expression is not detected until later embryonic ages. RORβ is highly expressed in embryonic neocortex, and exhibits strongly graded rostrocaudal and lateromedial patterns of expression. Over the first postnatal week, the graded expression of RORβ gradually acquires a disjunctive pattern largely restricted to layers 4 and 5 of the primary sensory areas. In contrast, very weak RORα expression is first detected in the neocortex just around birth, and is limited to the middle layer of the cortical plate of the putative somatosensory area. Later, a limited and very weak RORα expression is evident mainly in layer 4 of more caudal areas. To determine whether patterned retinal input is required for the proper postnatal expression and patterning of RORα and RORβ, we performed neonatal bilateral enucleations. We did not detect any significant differences between normal and enucleated mice in expression in visual areas. Although TCA input may be required for proper regulation of the postnatal expression of RORα and RORβ, these findings suggest that aspects of the dynamic postnatal expression and patterning of these genes are regulated independently of patterned visual activity relayed by geniculocortical afferents. The patterned expression of RORα in dorsal thalamus suggests that this gene locus may be useful to genetically modify the development of dorsal thalamus and thalamocortical projections.
Generation of neuronal cultures from induced pluripotent stem cells (hiPSCs) serve the studies of human brain disorders. However we lack neuronal networks with balanced excitatory- inhibitory activities, which are suitable for single cell analysis. We generated low-density networks of hPSC-derived GABAergic and glutamatergic cortical neurons. We used two different co-culture models with astrocytes. We show that these cultures have balanced excitatory-inhibitory synaptic identities using confocal microscopy, electrophysiological recordings, calcium imaging and mRNA analysis. These simple and robust protocols offer the opportunity for single-cell to multi-level analysis of patient hiPSC-derived cortical excitatory- inhibitory networks; thereby creating advanced tools to study disease mechanisms underlying neurodevelopmental disorders.
We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.
Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein - MBP - and proteolipid protein - PLP - (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation.