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In JoVE (1)
Other Publications (13)
- Neuroreport
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Biochemical and Biophysical Research Communications
- BMC Cell Biology
- Development, Growth & Differentiation
- Development, Growth & Differentiation
- Cellular & Molecular Biology Letters
- Cellular & Molecular Biology Letters
- Metabolic Brain Disease
- The International Journal of Developmental Biology
- Brain Research
- Journal of Molecular Histology
Articles by Jiankai Luo in JoVE
JoVE General
Gene Transfer into Older Chicken Embryos by ex ovo Electroporation
1Albrecht-Kossel-Institute for Neuroregeneration, School of Medicine University of Rostock, 2Institute of Anatomy I, School of Medicine University of Jena
A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.
Other articles by Jiankai Luo on PubMed
Cadherin-20 Expression by Motor Neurons is Regulated by Sonic Hedgehog During Spinal Cord Development
During chicken spinal cord development, cadherin-20 is expressed by motor neurons of the lateral motor column and other cells in the basal and alar plates. To investigate the regulation of cadherin-20 expression, Sonic hedgehog (Shh) signaling was altered in chicken spinal cord and hindbrain by in-vivo electroporation. Our results show that, at an early embryonic stage 12, Shh induces cadherin-20 expression by motor neurons. Later, at stage 24 when the sorting of motor neuron pools begins, cadherin-20 expression is induced in the neural progenitors of the ventricular zone but not in motor neurons. Blockage of Shh signaling inhibits cadherin-20 expression in the motor column. Therefore, cadherin-20 expression in motor neurons is regulated by Shh in a time-dependent manner.
Expression of Seven Members of the ADAM Family in Developing Chicken Spinal Cord
The expression patterns of seven members of the ADAM (a disintegrin and metalloprotease) family, including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23, were analyzed in the developing chicken lumbar spinal cord by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the lumbar cord and its surrounding tissues. ADAM9, ADAM10, ADAM22, and ADAM23 are expressed predominantly by motoneurons in the motor column and by sensory neurons in the dorsal root ganglia, each with a different expression pattern. ADAM12 and ADAM13 are mainly expressed in the meninges around the lumbar cord and in the condensed sheets of chondroblasts around the vertebrae. ADAM17 expression is strong in the ventricular layer and limited to early stages. The differential expression of the ADAMs in the lumbar cord suggests that the ADAMs play a regulatory role in development of the spinal cord.
Regional Expression of the ADAMs in Developing Chicken Cochlea
The expression patterns of five members of the ADAM (a disintegrin and metalloprotease) family including ADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 were analyzed in different anatomical structures of the developing chicken cochlea by in situ hybridization and immunohistochemistry. Results show that ADAM9, ADAM10, and ADAM17 are widely expressed in the sensory epithelium of the basilar papilla, by homogene cells, spindle-shaped cells, and acoustic ganglion cells, and in the tegmentum vasculosum, each with a different pattern. ADAM22 expression is restricted to spindle-shaped cells and acoustic ganglion cells, while ADAM23 is prominently expressed by hair cells and acoustic ganglion cells. Furthermore, ADAM10 protein is coexpressed with several members of the classic cadherins, including cadherin-7, N-cadherin, and R-cadherin in distinct anatomical regions of the cochlea except for acoustic ganglion cells. The expression of the ADAMs in the developing cochlea suggests a contribution of the ADAMs to the development of distinct cochlear structures.
Differentiation of Human Neural Progenitor Cells Regulated by Wnt-3a
Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and β-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized β-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/β-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized β-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/β-catenin pathway in ReNcell VM cells.
Erythropoietin and the Effect of Oxygen During Proliferation and Differentiation of Human Neural Progenitor Cells
Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process.
Regional Expression of ADAM19 During Chicken Embryonic Development
ADAM19 (also named meltrin β) is a member of the ADAM (a disintegrin and metalloprotease) family of metalloproteases and is involved in morphogenesis and tissue formation during embryonic development. In the present study, chicken ADAM19 is cloned by reverse transcription-polymerase chain reaction and identified by sequencing. Its expression patterns in different parts of the developing chicken embryo are investigated by Western blot analysis and immunohistochemistry. Results show that ADAM19 protein is widely expressed in chicken embryos. It is detectable in the central nervous system, including the brain, spinal cord, cochlea, and retina. Furthermore, ADAM19 protein is also found in other tissues and organs such as digestive organs, the thymus, the lung bud, the dorsal aorta, the kidney, the gonad, muscles, and in the feather buds. All these data suggest that ADAM19 plays an important role in the embryonic development of chicken.
Differential Expression of the ADAMs in Developing Chicken Retina
The expression patterns of the seven members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 were analyzed in the developing chicken retina by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the developing retinal layers. ADAM9, ADAM10 and ADAM17 are widely expressed in the differential layers of the retina throughout the whole embryonic period, while ADAM12 and ADAM13 are mainly expressed in the ganglion cell layer at a later stage. ADAM22 and ADAM23 are restricted to the inner nuclear layer and the ganglion cell layer at a later stage. Furthermore, ADAM10 protein is co-expressed with the four members of the classic cadherins, N-cadherin, R-cadherin, cadherin-6B and cadherin-7 in distinct retinal layers. Therefore, the differential expression of the investigated ADAMs in the developing retina suggests the contribution of them to the retina development.
Quantitative and Dynamic Expression Profile of Premature and Active Forms of the Regional ADAM Proteins During Chicken Brain Development
The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins - ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 - in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.
Quantitative and Kinetic Profile of Wnt/β-catenin Signaling Components During Human Neural Progenitor Cell Differentiation
ReNcell VM is an immortalized human neural progenitor cell line with the ability to differentiate in vitro into astrocytes and neurons, in which the Wnt/β-catenin pathway is known to be involved. However, little is known about kinetic changes of this pathway in human neural progenitor cell differentiation. In the present study, we provide a quantitative profile of Wnt/β-catenin pathway dynamics showing its spatio-temporal regulation during ReNcell VM cell differentiation. We show first that T-cell factor dependent transcription can be activated by stabilized β-catenin. Furthermore, endogenous Wnt ligands, pathway receptors and signaling molecules are temporally controlled, demonstrating changes related to differentiation stages. During the first three hours of differentiation the signaling molecules LRP6, Dvl2 and β-catenin are spatio-temporally regulated between distinct cellular compartments. From 24 h onward, components of the Wnt/β-catenin pathway are strongly activated and regulated as shown by mRNA up-regulation of Wnt ligands (Wnt5a and Wnt7a), receptors including Frizzled-2, -3, -6, -7, and -9, and co-receptors, and target genes including Axin2. This detailed temporal profile of the Wnt/β-catenin pathway is a first step to understand, control and to orientate, in vitro, human neural progenitor cell differentiation.
Decreased Expression of Myelin Gene Regulatory Factor in Niemann-Pick Type C 1 Mouse
Niemann-Pick type C 1 (NPC1) disease is an autosomal recessive cholesterol transport defect resulting in a neurodegenerative process in patients mainly at an early age, although some patients may start with manifestation in adult. Since loss of myelin is considered as a main pathogenetic factor, the precise mechanism inducing dysmylination in NPC1 disease is still unclear. In the present study, a quantitative evaluation on the myelin protein and its regulatory factors of oligodendrocytes, such as SRY-related HMG-box 10 (Sox10), Yin Yang 1 factor (YY1) and myelin gene regulatory factor (MRF), in different parts of the brain and spinal cord was performed in NPC1-mutant mice. The results showed that NPC1 protein was expressed in oligodendrocytes and the amount of myelin protein was generally decreased in all parts of the brain and spinal cord in NPC1-mutant mice. Compared to wild type, the amount of Sox10 and YY1 was not different in NPC1-mutant mice, but MRF was significantly decreased, suggesting a possible mechanism perturbing differentiation of oligodendrocytes and the myelination process in the NPC1-mutant mouse.
ADAM17 Overexpression Promotes Angiogenesis by Increasing Blood Vessel Sprouting and Pericyte Number During Brain Microvessel Development
The angiogenic process is precisely regulated by different molecular mechanisms, with a balance between stimulatory and inhibitory factors in embryonic development. Transmembrane proteins of the ADAM (a disintegrin and metalloprotease) family play a critical role in embryogenesis and are involved in protein ectodomain shedding, as well as cell-cell and cell-matrix interactions. In the present study, we found that ADAM17 is expressed spatiotemporally in the tectal layers during chicken embryonic development. To investigate the effect of ADAM17 overexpression on angiogenesis, chicken ADAM17 plasmids were transfected into the developing tectum in vivo by electroporation. Results showed that overexpression of ADAM17 induces morphological changes of brain microvessels, such as an increase in diameter, of capillary sprouting from radial microvessels and an increase in the number of pericytes, but not of endothelial cells. Our data suggest that overexpression of ADAM17 in the developing tectum promotes angiogenesis by increasing the number of pericytes and capillary sprouting in the radial vessels.
Increased Excitability and Compromised Long-term Potentiation in the Neocortex of NPC1(-/-) Mice
Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the NPC1 gene which encodes a transmembrane protein of the acidic compartment. Albeit the NPC1(-/-) mouse is available serving as an appropriate animal model of the human disease, the precise function of this protein remains obscure. Here, we investigated the synaptic consequences of this disease and explored long-term potentiation (LTP) in slices taken from the hippocampal CA1 region, the dorsomedial striatum as well as the somatosensory neocortex in NPC1(-/-) mice using extracellular field potential recordings. We did not observe significant changes in synaptic excitability as well as LTP in the hippocampal CA1 region and the dorsomedial striatum of NPC1(-/-) mice when compared to wildtype littermates. However, neocortical excitability was significantly enhanced while LTP was abolished. These results suggest that at least in the somatosensory neocortex NPC1 protein is instrumental in synaptic function.
Expression Patterns of ADAMs in the Developing Chicken Lens
In the present study the expression patterns of ADAM (a disintegrin and metalloprotease) genes in the chicken developing lens were analyzed. Using in situ hybridization, we found that seven members of the ADAM family including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 are expressed in the developing embryonic lens. From embryonic incubation day (E) 2 to E3, most of the ADAMs investigated here are expressed in the lens placode and lens vesicle. From E5 to E7, all seven ADAMs, but predominantly ADAM9 and ADAM10, are throughly expressed in the central epithelium, as well as in the proliferating lens epithelium and the equatorial lens epithelium. From E9 to E14, expression of ADAM9, ADAM10, and ADAM17 decreases moderately in these regions. ADAM12 and ADAM13 are weakly expressed in the central epithelium and the lens epithelium, and are not detectable from E14 onward. ADAM22 and ADAM23 are expressed in the central epithelium, the lens epithelium and the equatorial lens epithelium at E5 and decrease gradually afterwards in the same regions. At E16, only weak ADAM9, ADAM10 and ADAM17 signals are found in the anterior lens epithelium. The changing spatiotemporal expression of the seven ADAMs suggests a regulatory role for these molecules during chicken lens development.
