Question

How would you determine which retinoid receptors were involved in mediating the effects of RA (retonic acid) signaling in fresh water planaria during the course of regeneration? (You may need to do some searching in the literature to answer this one). What techniques and how.

Answer 1

Retinoic acid (RA) is a small Vitamin A derivative. It is important in several regenerating animals. Retinol is transported from the source, either diet or the release of dietary stores, to the tissues of interest by a series of specialized carrier proteins. Extracellularly, it is carried by a protein called retinol binding protein, RBP. RBP can also associate with a serum protein called transthyretin ,TTR, to prevent retinol from being degraded or eliminated by the kidneys. Once in the cell, it is bound to cellular retinol binding protein, CRBP. Here it can be converted to retinyl palmitate via esterification or released to be converted to RA. RA is generally restricted to the cell. It is shuttled from the cytoplasm to the nucleus via cellular retinoic acid binding protein, CRABP. Excess RA is catabolized by further oxidation by CYP26, a cytochrome P450 family member.

Conversion of  Retinol to Retinoic Acid:

Retinol is converted from an alcohol to a carboxylic acid in a two-step process. First, it is oxidized from retinol to retinaldehyde by an alcohol dehydrogenase, ADH. Then the final, rate-limiting step is the oxidation of retinaldehyde to retinoic acid by retinaldehyde dehydrogenase, RALDH.

RA affects gene expression by directly associating with targeted genes via nuclear receptors. RA is a ligand to the retinoic acid receptors, RAR that form heterodimers with RXRs. Once associated with RA, the receptors dimerize and associate with retinoic acid regulatory elements, RAREs, which are the regulatory sequences of RA-sensitive genes. Though these roles are widespread, retinoic acid is used largely in the patterning of organisms during development.

What techniques and how:

RA is known to have two routes to effecting gene expression: direct and indirect. The direct is the more canonical of the two. In this route, the dimerized RAR/RXR complex directly associates with the effected gene. This route is known to be how RA effects the expression of HOX genes. The indirect route affects another transcription factor via an intermediate. This type of control is seen in the complex relationship between RA/FGF/Wnts and certain HOX genes.

RA plays an important role in the development of many organisms. It is involved in a variety of process including patterning, organogenesis, and cell proliferation. RA is not limited to ontogenetic development, but is also frequently involved in regeneration