Modified Whole-Mount In situ Hybridization Protocol for the Detection of Transgene Expression in Electroporated Chick Embryos

Arede, Natacha; Tavares, Ana Teresa

doi:10.1371/journal.pone.0002638

Cited by 10 publications

(9 citation statements)

References 19 publications

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“…In situ hybridization for Sox10 (Betancur et al, 2010), Pax7 (Basch et al, 2006), FoxD3 (Kos et al, 2001), and Ets1 (Barembaum and Bronner, 2013) was performed using previously described protocol (Acloque et al, 2008). In situ hybridization for GFP was performed using a modified protocol which involved incubating the embryos in DNase I (Promega) at 37°C for 1 hour (Arede and Tavares, 2008). The DIG-labeled RNA probe against GFP was a gift from Marcos Simões-Costa.…”

Section: Methodsmentioning

confidence: 99%

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Gandhi

Piacentino

Vieceli

et al. 2017

Developmental Biology

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The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.

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Section: Methodsmentioning

confidence: 99%

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Gandhi

Piacentino

Vieceli

et al. 2017

Developmental Biology

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“…Whole mount in situ hybridization for eGFP was modified using the guidelines in [52]. Double fluorescence in situ hybridization was performed according to [53], and hybridization chain reaction (HCR) to detect endogenous FoxD3 was conducted according to [19].…”

Section: Methodsmentioning

confidence: 99%

Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

et al. 2012

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The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes.

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“…After washing in PBS, sections were blocked and incubated with Horseradish Peroxidase (HRP)-conjugated anti-Fluo antibody (1:1000, Roche), followed by fluorescein isothiocyanate (FITC)-Tyramide amplification, as recommended by the manufacturer (TSA™-Plus Fluorescein System Kit, PerkinElmer, Waltham, MA, USA). When in situ hybridizations were performed to detect VNP mRNA, the electroporated plasmids were removed with DNAseI (Roche) to prevent recognition of DNA by the RNA probe [ 53 ]. To generate VNP or Hes5-1 coding region probes, Venus-NLS-PEST [ 33 ] or Hes5-1 coding region [ 14 ] were subcloned in pBlueScript KS II and used to transcribe the RNA probes.…”

Section: Methodsmentioning

confidence: 99%

A novel reporter of notch signalling indicates regulated and random notch activation during vertebrate neurogenesis

et al. 2011

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BackgroundBuilding the complex vertebrate nervous system involves the regulated production of neurons and glia while maintaining a progenitor cell population. Neurogenesis starts asynchronously in different regions of the embryo and occurs over a long period of time, allowing progenitor cells to be exposed to multiple extrinsic signals that regulate the production of different cell types. Notch-mediated cell-cell signalling is one of the mechanisms that maintain the progenitor pool, however, little is known about how the timing of Notch activation is related to the cell cycle and the distinct modes of cell division that generate neurons. An essential tool with which to investigate the role of Notch signalling on cell by cell basis is the development a faithful reporter of Notch activity.ResultsHere we present a novel reporter for Notch activity based on the promoter of the well characterised Notch target chick Hes5-1, coupled with multiple elements that confer instability, including a destabilized nuclear Venus fluorescent protein and the 3' untranslated region (UTR) of Hes5-1. We demonstrate that this reporter faithfully recapitulates the endogenous expression of Hes5-1 and that it robustly responds to Notch activation in the chick neural tube. Analysis of the patterns of Notch activity revealed by this reporter indicates that although Notch is most frequently activated prior to mitosis it can be activated at any time within the cell cycle. Notch active progenitors undergoing mitosis generate two daughters that both continue to experience Notch signalling. However, cells lacking Notch activity before and during mitosis generate daughters with dissimilar Notch activity profiles.ConclusionsA novel Notch reporter with multiple destabilisation elements provides a faithful read-out of endogenous Notch activity on a cell-by-cell basis, as neural progenitors progress through the cell cycle in the chick neural tube. Notch activity patterns in this cell population provide evidence for distinct Notch signalling dynamics underlying different cell division modes and for the involvement of random initiation of Notch signalling within the neuroepithelium. These findings highlight the importance of single-cell analysis in the study of the complexity of Notch activity and provide new insights into the mechanisms underlying cell fate decisions in neural progenitors.

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Modified Whole-Mount In situ Hybridization Protocol for the Detection of Transgene Expression in Electroporated Chick Embryos

Cited by 10 publications

References 19 publications

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo

Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

A novel reporter of notch signalling indicates regulated and random notch activation during vertebrate neurogenesis

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