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The Secret Life of Vitamin A Holly Boettger-Tong, Wesleyan College Department of Biology. What is Vitamin A ?. Required for normal vision reproduction,differentiation, homeostasis Deficiency leads to blindness, sterility Retinol, Retinal, Retinoic acid all have biological activity.

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The secret life of vitamin a holly boettger tong wesleyan college department of biology

The Secret Life of Vitamin AHolly Boettger-Tong, Wesleyan College Department of Biology


What is vitamin a
What is Vitamin A ?

  • Required for normal vision

    reproduction,differentiation,

    homeostasis

  • Deficiency leads to blindness, sterility

  • Retinol, Retinal, Retinoic acid all have biological activity.

  • Nature of function depends upon form, converting enzymes, expression of receptors for each retinoid


Retinoic acid
Retinoic Acid

  • Metabolite of Vitamin A

  • Organic compound that is produced endogenously and whose production is dependent upon tissue-specific expression of enzymes (retinaldehyde dehydrogenases, to be specific)

  • Affects vertebrate physiological processes

    • Development

    • Cell growth and differentiation

    • Morphogenesis

    • Adult tissue function


Retinoid isomer structures
Retinoid Isomer Structures

All-trans Retinoic acid

9-cis Retinoic acid


How retinoic acid effects gene expression
How Retinoic Acid Effects Gene Expression

Retinoid binding protein

Retinoic Acid

Receptor – binds

ligand and DNA,

Influencing gene

expression

Gene product


What is the effect of retinoic acid during early development
What is the Effect of Retinoic Acid During Early Development?

  • What are the dose-response characteristics of retinoic acid’s effects on Oryzias latipes development?

  • What insight into the role of retinoic acid in cardiogenesis might be gleaned from these experiments?

  • What are the gene expression changes associated with retinoid-altered development

  • How does the stage of development effect sensitivity to retinoid effects?


Retinoids and development
Retinoids and Development Development?

  • Specification of primary antero-posterior axis, forming nervous system, establishing limbs

  • Control of gene expression – produced at specific developmental times to yield a gradient of retinoic acid

  • Defects in retinoic acid production or in receptor expression result in defective development. Excess retinoic acid is teratogenic, causing malformations which are informative as to retinoid action in specific tissues.


The japanese killifish
The Japanese Killifish Development?

  • Oryzias latipes are native to Japan, Taiwan, and southeastern Asia

  • Freshwater and common to rice paddies

  • Rapid development (ranges from 1-3 weeks) in a translucent egg makes them ideal for determining potential teratologic effects of chemicals of interest

  • Good for students due to ease of culture


All trans retinoic acid
All-Trans Retinoic Acid Development?

  • All trans retinoic acid is a derivative of vitamin A

  • Essential for normal growth and development

  • Affect gene expression by binding to specific receptors; liganded receptors act as transcription factors

All Trans Retinoic Acid


Gene expression and all trans retinoic acid
Gene Expression and All-Trans Retinoic Acid Development?

RARE

RAR-ATRA

All trans RA

mRNA

Transcriptional Activation

Levels of ATRA influence gene expression during development,

making ATRA a morphogen but also a potential teratogen.


Signficance
Signficance Development?

  • In humans ATRA is present in the plasma at concentrations of 2-4ng/ml

  • Acne Medication

    • Accutane

    • Differin

  • Anti-cancer Drugs

    • Isotretinoin

  • Organochlorine Pesticides

    • Chlordane

    • Dieldrin and Aldrin

    • Endosulfan

    • Methoprene


Experimental design
Experimental Design Development?

  • Modified FETAX – treat embryos for three days with different concentrations of retinoid (or vehicle control), then discontinue use and observe effects of treatment

  • Monitor embryos daily, observing morphology

  • Determine effects on hatching


Dose dependent inhibition of medaka hatching by atra and 9 cis ra
Dose Dependent Inhibition of Development? Medaka Hatching by ATRA and 9-cis RA

% Failed Hatching

Dose of Retinoid (mM)


Treatment of embryos
Treatment of Embryos Development?

  • Treat embryo at 100nM level for 3 days

  • Incubate for an additional 3 days

  • Take pictures of the embryos during the incubation period


Embryonic morphology on experimental day 6
Embryonic Morphology on Experimental Day 6 Development?

Control

ATRA

9-cis RA

10nM 100nM 1mM


Cardiac morphology is dramatically altered by 100nm atra treatment
Cardiac Morphology Development? Is Dramatically Altered by 100nM ATRA Treatment

100nM ATRA

Control


The secret life of vitamin a holly boettger tong wesleyan college department of biology

Effects of ATRA on Cardiogenesis and Vasculogenesis Development?

ATRA

Control

10X

10X

Day 6

10X

10X



Your experiment lasting 6 days
Your Experiment – Lasting 6 days Development?

  • Make 25mL (final volume) of 100nM solutions of ATRA (from stock of 6.65mM in DMSO); light protect in 50mL vials

  • Dilute DMSO accordingly for vehicle control

  • Stage embryos/ take pictures

  • Distribute embryos into 100nM or Control vials

  • Treat animals for three days (fresh dilutions of ATRA every day)

  • Take pix on day four, five, six of treated and control embryos

  • Quantitate differences between control and treated embryos 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)

  • Write up results (carefully, as this is writeup is worth 24 points)