The Effect of Retinoids on Zebrafish Development

Author(s): Jasmine James

Mentor(s): Valerie Olmo, Biology

Abstract

Vitamin A is primarily composed of the organic compound retinol (R). R is essential at all stages of life, but it is actually the derivative retinoic acid (RA) which is used in biological processes. RA plays a major role in embryonic development by affecting somite formation, organogenesis, limb formation, and more. However, RA is a known teratogen so the synthetic, retinol palmitate (RP), was created and is used in its place in a number of cosmetic products despite there being little literature on the compound. In order to understand the effects R, RA, and RP have on embryonic development, zebrafish embryos were exposed in 4nM of a retinoid solution until 72hpf. Upon removal each embryo was imaged, and the angle of its tail was determined and analyzed. While R was found to be nonsignificant, the angles gained for the RA and RP exposure groups were found to be significant when compared to the negative control (4.4E-08 and 0.04 respectively). This data suggests RP can affect embryonic development, but at present its full effects or their degree are not yet understood.

Video Transcript

Slide 1: Title 

Hi, my name is Jasmine James and I worked with my mentor, Dr. Valerie Olmo and her zebrafish on my project titled: The Effect of Retinoids on Zebrafish Development.

Slide 2: Introduction

The images and data you will see were actually collected last semester. This semester, I spent my time reanalyzing my data and writing a manuscript with the goal of publication. So, with that said, let’s get into retinoids. 

Retinoids are compounds which are related to and act like Vitamin A. The first one I tested was the major component of Vitamin A, retinol. And upon being taken into the body retinol is converted into retinoic acid for biological processes. And retinoic acid plays a great many roles in embryonic development, including organ development, gene proliferation, and the development of the spine. But, at both too high and too low concentrations it acts as a teratogen- meaning it can not only cause harm to a developing embryo, but cause death as well. Lastly there is retinol palmitate,  a synthetic made to mimic the functions of retinoic acid, and has thus replaced it in sorts of cosmetic products from lotion to antiaging creams, to sunscreens, and more. 

Slide 3: The Question

But this begs the question, if retinoic acid is a known teratogen, what are retinol and retinol palmitate’s effect on embryonic development?

Slide 4: Methods

In order to test how these chemicals can affect development, and knowing retinoic acid can cause visible malformations in zebrafish embryos at concentrations as low as 4nM, I made chemical concentrations of the three at 4nM and then put in a 1:1 ratio with E3 media. This gave a final concentration of 2nM. Embryos were then exposed and left until they became 72 hours post fertilization and then they were imaged.

Slide 5: Results- Survival

The first thing that was analyzed was the survival data, which is expressed in the chart. For the E3, retinol, and retinol palmitate exposure groups 150 zebrafish embryos were tested, but for retinoic acid only 125 were. The chart shows the number of embryos which survived until the 72 hours post fertilization mark. And then these numbers then underwent a chi-square analysis giving the non-significant p-value of 0.7596. 

Slide 6: Results: Tails (1)

On this slide are some example of how the images were taken, and how the angles determined. After taking each image I used ImageJ to find the angles, and the images you can see on the screen represent the median of each exposure group. 

Slide 7:  Results: Tails (2)

On this slide the tail data is expressed in a box and whisker plot to show the entire distribution after the experiment. And the p-values after a Kruskal-Wallis analysis are shown on the right. Retinol and retinol palmitate were both found to be non-significant but the positive control retinoic acid was significant. 

Slide 8: Conclusions

So, what does all this data mean? The lack of significance in the survival data for one, allows all of the tail data to be equally compared, which is good. But when it comes to the tail data, only retinoic acid was found to be significant at the 2nM level, and that was with the very low p-value of 9.0E-10. The other two exposure groups were not found to be significant at this level, but at the same time there is still so much we don’t know, so much to learn. We don’t quite know where retinol palmitate acts upon within the embryo, if its location differs from retinoic acid, and that said we also don’t know the difference in which concentrations are deemed safe for it. So, all this really means, is that there’s more room for investigation in the future.

Slide 9: References

So, these are my references, but lastly, I’d like to acknowledge a few groups.

Slide 10: Acknowledgements

So, thank you to URSP and OSCAR, Mason’s College of Science, the Biology Department, and the members of Dr. Brewster’s laboratory at UMBC.

Slide 11: Thank You

And of course, a thank you to everybody watching.

4 replies on “The Effect of Retinoids on Zebrafish Development”

Well done, Jasmine. Nice presentation. I really enjoyed it. Do you know how clinically relevant the concentrations you used are for an embryo? If it’s known, what kind of concentrations would be a “normal” exposure? What is the next thing you would do with this system? Nice job.

Unfortunately I dont know what considered a normal exposure in zebrafish given its just based on vitamin intake, but what I would like to do next is see if there’s a difference between expression of genes affected by RA and RP

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