Dataset Information

Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

ABSTRACT: Somite myogenesis is one of the crucial early embryonic events that lead to the formation of muscular tissue. A complex of dynamic gene regulatory networks masters this event. To understand and analyze these networks, there remains a genuine need for the use of a reproducible and highly efficient gene transfer technique. In vivo electroporation has proven to be amongst the best approaches in achieving a high level of gene transfer. However, unoptimized electroporation conditions can directly cause varying degrees of cellular damage which may induce abnormal embryonic development as well as changes in the endogenous gene expression. Presegmented mesoderm and epithelial somites are not easy to electroporate. Chick neural tube has served in many functional studies as an ideal experimental model organ which is both robust and easily manipulated. In the current detailed protocol, the neural tube was used as a tool to optimize the electroporation conditions which were subsequently applied in the electroporation of the presegmented mesoderm and epithelial somites. The protocol highlights important notes and hints that enable reproducible results and could be applied in the in vivo electroporation of other chick embryo tissues.

SUBMITTER: Abu-Elmagd M

PROVIDER: S-EPMC6706099 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Abu-Elmagd Muhammad M

Journal of biological methods 20180607 2

Somite myogenesis is one of the crucial early embryonic events that lead to the formation of muscular tissue. A complex of dynamic gene regulatory networks masters this event. To understand and analyze these networks, there remains a genuine need for the use of a reproducible and highly efficient gene transfer technique. <i>In vivo</i> electroporation has proven to be amongst the best approaches in achieving a high level of gene transfer. However, unoptimized electroporation conditions can direc ...[more]

PMID: 31453243

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Project description:The proneural NEUROG2 is essential for neuronal commitment, cell cycle exit and neuronal differentiation. Characterizing genes networks regulated downstream of NEUROG2 is therefore of prime importance. To identify NEUROG2 early response genes, we combined gain of function in the neural tube with a global detection of modified transcripts using microarrays. We included in our study a mutant form of NEUROG2 (NEUROG2AQ) that cannot bind DNA and cannot trigger neurogenesis. Using this approach, we identified 942 genes modified at the onset of NEUROG2 activation. The global analysis of functions regulated by NEUROG2 allowed unmasking its rapid impact on cell cycle control. We found that NEUROG2 specifically represses a subset of cyclins acting at the G1 and S phases of the cell cycle, thereby impeding S phase re-entry. This repression occurs before modification of p27kip1, indicating that the decision to leave the cell cycle precedes the activation of this Cyclin-dependant Kinase Inhibitor. Moreover, NEUROG2 down-regulates only one of the D-type cyclins, cyclinD1, and maintaining cyclinD1 blocks the ability of the proneural to trigger cell cycle exit, without altering its capacity to trigger neuronal differentiation. The fact that NEUROG2 represses a subset but not all cell cycle regulators indicates that cell cycle exit is not an indirect consequence of neuronal differentiation but is precisely controlled by NEUROG2. Altogether our findings indicate that NEUROG2, by specifically repressing G1 and S cyclins, allows committed neuronal precursors to perform their last mitosis but blocks their re-entry in the cell cycle, thus favouring cell cycle exit. Stage HH10-11 embryos (11 to 15 somites) were electroporated with a control vector (pGIG-GFP), a NEUROG2-expressing vector (pCIGNEUROG2-GFP), or a NEUROG2AQ-expressing vector (pCIGNEUROG2AQ-GFP). For each biological replicate, neural tubes from 20 embryos were pooled for GFP+ cells collection. GFP+ cells were collected 6h later using FACS sorting (Epics Altra HSS cell sorter, Toulouse Rio platform) and processed for RNA probe preparation and hybridization on Affymetrix microarrays. For each experimental condition, four biological replicates were processed.

Dataset Information

Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Publications

Use of chick neural tube for optimizing the PSM and epithelial somites electroporation parameters: A detailed protocol.

Similar Datasets

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