Visualization of Gli Activity in Craniofacial Tissues of Hedgehog-Pathway Reporter Transgenic Zebrafish

Schwend, Tyler; Loucks, Evyn J.; Ahlgren, Sara

doi:10.1371/journal.pone.0014396

Cited by 40 publications

(46 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Nuclear mCherry fluorescence could be first detected by the onset of somitogenesis (11 hpf), several hours earlier than in previous Hh reporter lines [15], [16]. Mesodermal adaxial cells and overlying neural plate cells in the midline exhibited reporter expression, which progressively increased during somite formation and differentiation (Figure 3A–D and Movie S1).…”

Section: Resultsmentioning

confidence: 65%

“…For example, somitic tissues form slow-twitch muscle fibers and muscle pioneer cells in response to moderate and high levels of notochord-derived Hh signals, respectively [31]. In contrast to these endogenous processes, the previously described EGFP and mCherry reporter lines do not exhibit Gli-dependent fluorescence until mid-somitogenesis (approximately 17 hpf) [15], [16]. Nor can they clearly resolve the differences in Hh pathway activity that give rise to distinct muscle cell types during somite development.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

In Vivo Imaging of Hedgehog Pathway Activation with a Nuclear Fluorescent Reporter

et al. 2014

View full text Add to dashboard Cite

The Hedgehog (Hh) pathway is essential for embryonic development and tissue regeneration, and its dysregulation can lead to birth defects and tumorigenesis. Understanding how this signaling mechanism contributes to these processes would benefit from an ability to visualize Hedgehog pathway activity in live organisms, in real time, and with single-cell resolution. We report here the generation of transgenic zebrafish lines that express nuclear-localized mCherry fluorescent protein in a Gli transcription factor-dependent manner. As demonstrated by chemical and genetic perturbations, these lines faithfully report Hedgehog pathway state in individual cells and with high detection sensitivity. They will be valuable tools for studying dynamic Gli-dependent processes in vertebrates and for identifying new chemical and genetic regulators of the Hh pathway.

show abstract

Section: Resultsmentioning

confidence: 65%

Section: Introductionmentioning

confidence: 99%

In Vivo Imaging of Hedgehog Pathway Activation with a Nuclear Fluorescent Reporter

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Because of the accessibility of the embryo, high number of eggs laid for each clutch, and high reproducibility of the results, these vertebrates are ideal for teratogenic studies. Fish can also be manipulated to contain fluorescent molecules to use as a visual reporter for a particular gene or pathway of interest 19 . In the ethanol studies, the doses used appear quite high compared to mammalian blood alcohol levels.…”

Section: Discussionmentioning

confidence: 99%

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Loucks

Ahlgren

2012

JoVE

View full text Add to dashboard Cite

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue 2 . In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study [3][4][5][6][7][8] . Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model. Video LinkThe . Embryos are maintained at 28 °C for the desired length of time. After the desired length of ethanol exposure, the ethanol-water solution is removed and replaced with three washes of regular zebrafish water and maintained for the desired length of time. The specificity of the defect depends on the time of onset, the dose, and the length of the pulse of ethanol. Collection of Embryos for RNA, in situ Hybridization, Antibody, and Cartilage Staining1. To collect mRNA for qPCR, age matched embryos are collected in eppendorf tubes. After removal of the zebrafish water, lysis buffer with TCEP is added. A motorized pulverizer is used to physically dissociate the embryos in the lysis solution. This is then passed through a preclear column, which removes any undissociated large pieces. The resulting solution contains all the macromolecules released by the lysis process. RNA is then extracted by running this solution on a column, followed by washes and DNAse treatment. Elution is then performed using either water or an elution solution provided by the manufacturer (5'-Prime). The RNA can be converted to cDNA using standard techniques for qPCR or used in microarray analysis. 2. For in situ hybridization and antibody staining, embryos from 6 to 24 hpf were fixed in 4% paraformaldehyde overnight at 4 °C. This is followed by 3 washes in PBS + 0.1% Tween (PBT), to keep embryos from sticking together. Embryos are then transferred through a series of 3 methanol:PBT washes into 100% methanol, at which point they can be stored at -20 °C. These embryos can be used for in situ hybridization or antibody staining. Some antibodies do not work after methanol tre...

show abstract

“…Similar to studies of the FEZ in chick, bidirectional Shh signaling between neural and oral epithelium and NC-derived palate progenitors are critical for primary palate formation in zebrafish [97,98]. A glid:mCherry reporter for Hh signaling is first detected in this region around 48 h postfertilization, after cranial NCC have completed migration and just prior to the onset of cartilage differentiation [101]. miR-140 overexpression or pharmacological inhibition of Shh signaling results in clefting and a failure of NCC to form the ethmoid [99].…”

Section: Midfacial Integrationmentioning

confidence: 52%