The Effect of Retinoic Acid on Axolotl Jaw and Tooth DevelopmentBy Rohit Singla

Axolotl Salamanders

Yes, they do have teeth.

known as the “walking fish”amphibiansfound in Mexico highly endangered

Axolotl Anatomy

Retinoic Acid (RA)

Required for growth and development in chordate animals (fish-humans)

Acts as a signaling molecule

Guides early development

RA

RA controls over 500 genes (532)

Some genes it directly controls, some genes it indirectly controls

Tooth Development It is said that tooth development is controlled by 4 gene signaling pathways but exactly which are under debate in the scientific community

Some genes that have been tossed around are BMP, FGF and WNT, which each have their own gene regulation pathway.

get more specific about how tooth development is controlled

Genes Affecting Tooth Development

Over-expression of BMP leads to less teeth

Inactivation of FGF leads to less teeth and slower tooth development

The Inhibitors Matter TooSometimes, it is not about if you increase the concentration of the target gene or activate its pathway, as much as it is about affecting its inhibitor.

Previous research states that if you inhibit the BMP inhibitor, you actually get more teeth as opposed to the expected less teeth.

This is why we are not only using RA, but also DEAB (Diethylaminobenzaldehyde) (RA’s inhibitor), to see all possible effects

live axolotls

Experimental Setup

3 different concentrations RA: 10E-9, 10E-10, and 10E-11. 3 different concentrations DEAB: 10E-5, 10E-6 and 10E-7) 1 Plain Media control1 DMSO control

Axolotl in 100% Ethanol Axolotl in 70% Ethanol

Experimental Setup (cont)8 tubes for each collection date

6 collection dates (from 1/29/15-2/22/15

48 tubes in total

about 5 axolotl in each treatment

almost 250 axolotl in total being tested

Axolotls in Alcian Blue Stain

What I Did So FarEssentially I set up a continuum of collections of axolotls at different ages, over a month so I could follow the early jaw and tooth development of axolotls

Started 1/29 and set up treatments every 2-4 days

I would raise and harvest live brine shrimp to feed the growing axolotl

collected embryosput them into solutions with different concentrationsraised at 14 degrees until they had jaws kept alive until jaw fully developed

What Else Will I Do?Stain all the treatments with Alcian blue (cartilage) and Alizarin red (bone) to better see their jaws and teeth.

Make slides for each Axolotl in each treatment (over 250 slides), and gather raw data

collect all the data and analyze it to try and come up with a trend/functional results

What Am I Looking At?

Tooth count

Tooth size (length in mm)

Jaw length/span/width

feedback?

What Do I Expect?Based on outside research, I expect RA and DEAB to have an effect on the tooth number, tooth size and jaw size of axolotl salamanders

I expect it will increase the number of teeth, decrease the tooth length and increase the jaw length

Why My Research is Importantcurrently, no consensus but we want to understand how this signaling molecule affects jaw development

RA is becoming more present in our environment through pimple creams, anti wrinkle creams, hand creams.

one thing it is known to do: cause birth defects in humans

I want to understand how exactly it affects jaw and tooth development (of both us and axolotl)

a lot of the basic early developmental pathways are the same

can be applied to us

Acknowledgements Molecular Genetics of Tooth DevelopmentNIHPA Author Manuscripts. 2009 Oct; 19(5)504

Gene expression regulation by retinoic acid.J Lipid Res. 2002 Nov ;43(11):1773-808.

The genetic control of early tooth development.Crit Rev Oral Biol Med. 1997 ;8(1):4-39.

Interactions between FGF and Wnt signals and Tbx3 gene expression in mammary gland initiation in mouse embryosJournal of Anatomy. 2004 Jul; 205(1)1

Dr. Andrea Ward

Questions?Feedback?