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

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 All-trans Retinoic acid 9-cis Retinoic acid

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 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 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 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 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 RAR-ATRA All trans RA RARE mRNA Transcriptional Activation Levels of ATRA influence gene expression during development, making ATRA a morphogen but also a potential teratogen.

Signficance 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 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 % Failed Hatching Dose of Retinoid (mM)

Treatment of Embryos 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 Control ATRA 9-cis RA 10nM 100nM 1mM

Cardiac Morphology Is Dramatically Altered by 100nM ATRA Treatment Control 100nM ATRA

Effects of ATRA on Cardiogenesis and Vasculogenesis Control ATRA 10X 10X Day 6 10X 10X

Range of Cardiac Malformations

Your Experiment – Lasting 6 days 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)