the secret life of vitamin a holly boettger-tong, wesleyan college department of biology
TRANSCRIPT
The Secret Life of The Secret Life of Vitamin AVitamin A
Holly Boettger-Tong, Wesleyan College Holly Boettger-Tong, Wesleyan College Department of BiologyDepartment of Biology
What is Vitamin A ?What is Vitamin A ?Required for normal visionRequired for normal visionreproduction,differentiation, reproduction,differentiation, homeostasishomeostasisDeficiency leads to blindness, Deficiency leads to blindness, sterilitysterilityRetinol, Retinal, Retinoic acid Retinol, Retinal, Retinoic acid all have biological activity. all have biological activity. Nature of function depends Nature of function depends upon form, converting upon form, converting enzymes, expression of enzymes, expression of receptors for each retinoidreceptors for each retinoid
Retinoic AcidRetinoic Acid
Metabolite of Vitamin AMetabolite of Vitamin A
Organic compound that is produced endogenously and whose Organic compound that is produced endogenously and whose production is dependent upon tissue-specific expression of production is dependent upon tissue-specific expression of enzymes (retinaldehyde dehydrogenases, to be specific) enzymes (retinaldehyde dehydrogenases, to be specific)
Affects vertebrate physiological processes Affects vertebrate physiological processes DevelopmentDevelopment Cell growth and differentiationCell growth and differentiation MorphogenesisMorphogenesis Adult tissue functionAdult tissue function
Retinoid Isomer StructuresRetinoid Isomer Structures
All-trans Retinoic acid
9-cis Retinoic acid
How Retinoic Acid Effects How Retinoic Acid Effects Gene ExpressionGene Expression
Gene product
Retinoid binding protein
Retinoic AcidReceptor – bindsligand and DNA, Influencing gene expression
What is the Effect of Retinoic Acid What is the Effect of Retinoic Acid During Early Development? During Early Development?
What are the dose-response characteristics of What are the dose-response characteristics of retinoic acid’s effects on retinoic acid’s effects on Oryzias latipesOryzias latipes development?development?What insight into the role of retinoic acid in What insight into the role of retinoic acid in cardiogenesis might be gleaned from these cardiogenesis might be gleaned from these experiments? experiments? What are the gene expression changes associated What are the gene expression changes associated with retinoid-altered developmentwith retinoid-altered developmentHow does the stage of development effect How does the stage of development effect sensitivity to retinoid effects?sensitivity to retinoid effects?
Retinoids and DevelopmentRetinoids and DevelopmentSpecification of primary antero-Specification of primary antero-posterior axis, forming nervous system, posterior axis, forming nervous system, establishing limbsestablishing limbsControl of gene expression – produced Control of gene expression – produced at specific developmental times to yield at specific developmental times to yield a gradient of retinoic acida gradient of retinoic acidDefects in retinoic acid production or in Defects in retinoic acid production or in receptor expression result in defective receptor expression result in defective development. Excess retinoic acid is development. Excess retinoic acid is teratogenic, causing malformations teratogenic, causing malformations which are informative as to retinoid which are informative as to retinoid action in specific tissues. action in specific tissues.
The Japanese KillifishThe Japanese Killifish
Oryzias latipesOryzias latipes are native to Japan, Taiwan, and are native to Japan, Taiwan, and southeastern Asiasoutheastern Asia
Freshwater and common to rice paddiesFreshwater and common to rice paddies
Rapid development (ranges from 1-3 weeks) in Rapid development (ranges from 1-3 weeks) in a translucent egg makes them ideal for a translucent egg makes them ideal for determining potential teratologic effects of determining potential teratologic effects of chemicals of interestchemicals of interest
Good for students due to ease of cultureGood for students due to ease of culture
All-Trans Retinoic AcidAll-Trans Retinoic Acid
All trans retinoic acid is a derivative of vitamin AAll trans retinoic acid is a derivative of vitamin A
Essential for normal growth and developmentEssential for normal growth and development
Affect gene expression by binding to specific receptors; liganded Affect gene expression by binding to specific receptors; liganded receptors act as transcription factorsreceptors act as transcription factors
All Trans Retinoic Acid
Gene Expression and All-Trans Retinoic AcidGene Expression and All-Trans Retinoic Acid
RAR-ATRA
All trans RA
Transcriptional Activation
Levels of ATRA influence gene expression during development,
making ATRA a morphogen but also a potential teratogen.
RARE
mRNA
SignficanceSignficance
In humans ATRA is present in the plasma at In humans ATRA is present in the plasma at concentrations of 2-4ng/mlconcentrations of 2-4ng/mlAcne MedicationAcne Medication AccutaneAccutane DifferinDifferinAnti-cancer DrugsAnti-cancer Drugs IsotretinoinIsotretinoinOrganochlorine PesticidesOrganochlorine Pesticides ChlordaneChlordane Dieldrin and AldrinDieldrin and Aldrin EndosulfanEndosulfan MethopreneMethoprene
Experimental DesignExperimental Design
Modified FETAX – treat embryos for three Modified FETAX – treat embryos for three days with different concentrations of days with different concentrations of retinoid (or vehicle control), then retinoid (or vehicle control), then discontinue use and observe effects of discontinue use and observe effects of treatmenttreatment
Monitor embryos daily, observing Monitor embryos daily, observing morphologymorphology
Determine effects on hatchingDetermine effects on hatching
1 6
3 3 3 3
8 3
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
0 0 . 0 0 1 0 . 0 1 0 . 1 1
Dose Dependent Inhibition of Dose Dependent Inhibition of Medaka Hatching by ATRA and 9-Medaka Hatching by ATRA and 9-
cis RAcis RA
Dose of Retinoid (M)
% F
aile
d H
atch
ing
Treatment of EmbryosTreatment of Embryos
Treat embryo at 100nM level for 3 daysTreat embryo at 100nM level for 3 days
Incubate for an additional 3 daysIncubate for an additional 3 days
Take pictures of the embryos during the Take pictures of the embryos during the incubation periodincubation period
Embryonic Morphology on Embryonic Morphology on Experimental Day 6Experimental Day 6
Control
ATRA
9-cis RA
10nM 100nM 1M
Cardiac MorphologyCardiac MorphologyIs Dramatically Altered by 100nM ATRA Is Dramatically Altered by 100nM ATRA
TreatmentTreatment
100nM ATRA100nM ATRAControlControl
Day 6
Control ATRA
Effects of ATRA on Cardiogenesis and Vasculogenesis
10X
10X10X
10X
Range of Cardiac MalformationsRange of Cardiac Malformations
Normally Chambered
Hearts
Heart Looping
Heart Strings/ Cardiac Tissue
Masses
Control
10nM ATRA/9-cis
50nM ATRA/9-cis
100nM ATRA/9-cis
1µM ATRA/9-cis
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Your Experiment – Lasting 6 daysYour Experiment – Lasting 6 daysMake 25mL (final volume) of 100nM solutions of ATRA (from Make 25mL (final volume) of 100nM solutions of ATRA (from stock of 6.65mM in DMSO); light protect in 50mL vialsstock of 6.65mM in DMSO); light protect in 50mL vialsDilute DMSO accordingly for vehicle controlDilute DMSO accordingly for vehicle controlStage embryos/ take picturesStage embryos/ take picturesDistribute embryos into 100nM or Control vialsDistribute embryos into 100nM or Control vialsTreat animals for three days (fresh dilutions of ATRA every Treat animals for three days (fresh dilutions of ATRA every day)day)Take pix on day four, five, six of treated and control embryosTake pix on day four, five, six of treated and control embryosQuantitate differences between control and treated embryos Quantitate differences between control and treated embryos on days 4, 5, 6 (eg., # with heartbeat on day 4 in Control vs on days 4, 5, 6 (eg., # with heartbeat on day 4 in Control vs ATRA treated; # of somites on day 4 in Control vs ATRA)ATRA treated; # of somites on day 4 in Control vs ATRA)Write up results (carefully, as this is writeup is worth 24 Write up results (carefully, as this is writeup is worth 24 points)points)