Transgenic quails reveal dynamic TCF/β-catenin signaling during avian embryonic development
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Curated by eLife
Evaluation Summary:
The manuscript describes several optimizations of classic DNA reporter constructs to monitor closely the dynamics of Wnt/β-catenin signalling during development using transgenic avian lines. As Wnt signalling pathway is essential in the homeostasis of vertebrate and invertebrate organisms, a robust tool to analyse finely the dynamics of Wnt/β-catenin pathway is of broad interest for the biology/biomedicine scientific communities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway’s activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.
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Evaluation Summary:
The manuscript describes several optimizations of classic DNA reporter constructs to monitor closely the dynamics of Wnt/β-catenin signalling during development using transgenic avian lines. As Wnt signalling pathway is essential in the homeostasis of vertebrate and invertebrate organisms, a robust tool to analyse finely the dynamics of Wnt/β-catenin pathway is of broad interest for the biology/biomedicine scientific communities.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript describes several optimizations of classic DNA reporter constructs to monitor closely the dynamics of Wnt/β-catenin signalling during development using transgenic avian lines. The generation of these tool, and in particular the construct 16xTCF-VNP, will be of great value to the scientific community as it can be used in electroporations and also to generate transgenic in different organisms. Moreover, the stable quail transgenic line are useful to explore Wnt signalling function in labs interested in avian species as developmental biology models and also in labs interested in evo/devo comparative analysis.
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Reviewer #2 (Public Review):
This report describes the generation of two quail Tcf/beta catenin signalling reporter lines that differ in their exact makeup and are designed to enhance specifically sensitivity and dynamics of the response. Initial experiment are presented to try to asses these features. A few experiments are described to show that the activity of the reporter coincides with known areas of Wnt signalling activity in E2.5-3.5 embryos, such as the AER of limb buds, Pharyngeal arches, somites and the cephalic neural crest. Some activity is seen is area not previously described to be associated with Wnt signalling, which is interpreted as Wnt independent beta catenin signalling, while in other places known to be areas of canonical Wnt signalling such as the neural tube roof plate are devoid of reporter signal. Importantly it …
Reviewer #2 (Public Review):
This report describes the generation of two quail Tcf/beta catenin signalling reporter lines that differ in their exact makeup and are designed to enhance specifically sensitivity and dynamics of the response. Initial experiment are presented to try to asses these features. A few experiments are described to show that the activity of the reporter coincides with known areas of Wnt signalling activity in E2.5-3.5 embryos, such as the AER of limb buds, Pharyngeal arches, somites and the cephalic neural crest. Some activity is seen is area not previously described to be associated with Wnt signalling, which is interpreted as Wnt independent beta catenin signalling, while in other places known to be areas of canonical Wnt signalling such as the neural tube roof plate are devoid of reporter signal. Importantly it shown that the fluorescent protein expression is strong enough for live imaging in at least some cases. There is a nice demonstration of the aggregation of the precursors of the AER in the developing limb bud. In essence the paper describes the development of two transgenic Tcf/beta catenin reporter lines and provides evidence that they detect signals at least in some in places where activity is expected, providing the expectation that this will be a very useful tool for future studies.
More thorough and quantitative characterization of the reporter lines would have strengthened the paper.
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Reviewer #3 (Public Review):
The manuscript by Barzilai-Tutsch and colleagues describes a new transgenic reporter model in quail to monitor active beta-catenin transcriptional activity during embryonic development. The transgene presented here goes beyond previous mouse TCF/LEF transgenic reporter lines transgenic models to increase sensitivity by further concatemerising TCF/LEF binding sites, adding viral translational enhancers and using an unstable GFP to limit expression to cells with active b-cat signalling. The major weakness is that the analysis of cis regulatory elements is incomplete and only a single transgenic line produced for each construct.
The most successful aspects are the beautiful expression of the transgene reporter in the migratory neural crest, the dermomyotome, and the limb buds. The use of the syn21 and p10 viral …
Reviewer #3 (Public Review):
The manuscript by Barzilai-Tutsch and colleagues describes a new transgenic reporter model in quail to monitor active beta-catenin transcriptional activity during embryonic development. The transgene presented here goes beyond previous mouse TCF/LEF transgenic reporter lines transgenic models to increase sensitivity by further concatemerising TCF/LEF binding sites, adding viral translational enhancers and using an unstable GFP to limit expression to cells with active b-cat signalling. The major weakness is that the analysis of cis regulatory elements is incomplete and only a single transgenic line produced for each construct.
The most successful aspects are the beautiful expression of the transgene reporter in the migratory neural crest, the dermomyotome, and the limb buds. The use of the syn21 and p10 viral translational elements in the transgene is of interest. The quail produced containing this construct had increased expression of nuclear Venus FP (although not well described or shown to be nuclear) in the embryos. The observation of reduced expression of the reporter in the roofplate of the neural tube and increasing expression as the neural crest migrated from the neural tube is fascinating. The question to be answered is does this reflect a physiological difference or is it a characteristic of the transgene.
The major weakness of this analysis is that there is no substantial transgenic analysis of the elements in animals. Two different constructs were tested and a single transgenic quail produced for each line. Expression levels may not depend on transposon copy number; however, the expression level due to integration site is a potential problem. Drawing a conclusion is difficult when comparing a single transgenic line with a second transgenic line.
The neural tube electroporation analysis is not complete. A transcriptional analysis of the constructs in cells, or using their embryo transfection system in the neural tube to analyse the constructs with or without the addition of v10 and syn21. At that point, the authors can claim that the elements are actually useful for increasing reporter gene expression/stability/translation. -
