Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

Loucks, Evyn J.; Ahlgren, Sara

doi:10.1002/bdra.20564

Cited by 70 publications

(100 citation statements)

References 96 publications

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“…We have documented abnormal development of axial tissues, including the notochord (data not shown 14 ). The developmental delay produced by ethanol apparently leads to disruption of proper notochord elongation, leading to a shortened and occasionally disrupted notochord (data not shown Another consequence of ethanol exposure that might be downstream of early developmental delay and later notochord defects is a shortened length of the embryo (Figure 3).…”

Section: Representative Resultsmentioning

confidence: 76%

“…To assess morphogenic development, embryos are collected at the desired time and examined under the dissecting microscope. For somite shape, live embryos are imaged 14 . For inter ocular distance and body length, embryos are fixed in PFA.…”

mentioning

confidence: 99%

“…For inter ocular distance and body length, embryos are fixed in PFA. In all cases, images of the desired region are captured on a dissecting microscope at a fixed magnification and measurements taken using Adobe Photoshop software 10,14 . 4.…”

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confidence: 99%

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Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Loucks

Ahlgren

2012

JoVE

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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...

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Section: Representative Resultsmentioning

confidence: 76%

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confidence: 99%

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Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Loucks

Ahlgren

2012

JoVE

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“…Loucks and Ahlgren 49 demonstrated that zebrafish exposed to 1% or 1.5% ethanol had small, but significant reductions to the overall size of the embryo. The effect of ethanol was more substantial as higher concentrations of ethanol were tested 50 and Ali et al 51 found that a 1-h pulse of 10% ethanol resulted in smaller larvae with numerous structural defects. However, given the kinetics and partitioning of ethanol, discussed earlier, this treatment is likely to be far outside the physiological norms in humans.…”

Section: Zebrafish Models Of Fasdmentioning

confidence: 99%

“…30,[79][80][81][82] These ethanol-induced phenotypes can be rescued by injection of shh mRNA. 50 In addition to altering retinoic acid signaling, one alternative (or additional) mechanism by which ethanol attenuates Shh is the disruption of cholesterol modification of Shh needed for proper signaling, as has been found in zebrafish. 82 Further support for an involvement of Shh signaling in ethanol teratogenesis comes from genetic analyses (as we discuss in the next section).…”

Section: Zebrafish Models Of Fasdmentioning

confidence: 99%

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis

2016

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Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology and cognitive impairments. It affects *1 in 100 children born in the United States each year. Due to the pleiotropic effects of ethanol, animal models have proven critical in characterizing the mechanisms of ethanol teratogenesis. In this review, we focus on the utility of zebrafish in characterizing ethanolinduced developmental defects. A growing number of laboratories have focused on using zebrafish to examine ethanol-induced defects in craniofacial, cardiac, ocular, and neural development, as well as cognitive and behavioral impairments. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene-ethanol interactions. With a high degree of genetic amenability, zebrafish is at the forefront of identifying and characterizing the gene-ethanol interactions that underlie FASD.

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Genesis of teratogen‐induced holoprosencephaly in mice

Lipinski

Godin

O’Leary‐Moore

et al. 2010

American J of Med Genetics Pt C

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Evidence from mechanical, teratological, and genetic experimentation demonstrates that holoprosencephaly (HPE) typically results from insult prior to the time that neural tube closure is completed and occurs as a consequence of direct or indirect insult to the rostral prechordal cells that induce the forebrain or insult to the median forebrain tissue, itself. Here, we provide an overview of normal embryonic morphogenesis during the critical window for HPE induction, focusing on the morphology and positional relationship of the developing brain and subjacent prechordal plate and prechordal mesoderm cell populations. Subsequent morphogenesis of the HPE spectrum is then examined in selected teratogenesis mouse models. The temporal profile of Sonic Hedgehog expression in rostral embryonic cell populations and evidence for direct or indirect perturbation of the Hedgehog pathway by teratogenic agents in the genesis of HPE is highlighted. Emerging opportunities based on recent insights and new techniques to further characterize the mechanisms and pathogenesis of HPE are discussed.

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Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

Cited by 70 publications

References 96 publications

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis

Genesis of teratogen‐induced holoprosencephaly in mice

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