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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+++

++

- -

- -

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- - -

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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)