Retinoic acid itself is thought to be regulated by other proteins such as the cellular retinoic acid-binding proteins I and II (CRABPs I and II), which are cytoplasmic RA binding proteins thought to prevent retinoic acid from entering the nucleus by binding to it. Support for the regulation of retinoic acid function by CRABP proteins includes the finding that CRABP-I protein reduces the amount of RA available in the nucleus to regulate gene expression (Gudas, 1994). Because all cells with retinoic acid also have CRABP, the presence of CRABP is an indication that the cell requires retinoic acid for some aspect of development and cell differentiation (Maden et al., 1989). CRABPs may also direct the metabolism of retinoic acid into other important molecules with important biological functions. Maden et al. studied four stages of chick development from stage 16 to stage 24 while looking for the presence of CRABPs. CRABP antibody was stained in nine different cell populations:
The presence of CRABP cells in a wide variety of ectodermal and mesodermal cells demonstrate that retinoic acid may be expressed in the area as well. Maden et al. (1989) thus conclude that retinoic acid take up by the cells is concentrated in the neural crest region, an area in which retinoic acid was not thought to be present at the time. RA is likely being secreted from the dorsal blastopore lip. Organizer proteins emitted from the Nieuwkoop center in the dorsal blastopore lip may be indirectly affecting how much RA is being made by regulating retinoic acid expression. Maden et al. (1989) suggest that the teratogenicity of retinoids to the nervous system may occur because those tissues contain CRABP which may facilitate the uptake of retinoic acid into the cell and thus change normal gene expression.
Some of these genes that retinoic acid directly regulates are ones which function in both embryos and adults and encode other transcription factors. Other genes regulated by retinoic acid encode peptide growth factors (Means and Gudas, 1995). One of the key functions of retinoic acid is the regulation of homeobox genes. The homeobox genes are transcription factors which affect embryonic development. RA was initially associated with the regulation of Hox genes from studies examining the differentiation of teratocarcinoma cells. It was found that the responsiveness of a Hox gene to retinoic acid corresponds to its location in the gene cluster. Low levels of RA were found to induce the transcription of genes near the 3' end of the cluster, while genes farther away from the 3' end of the cluster required a longer time period and higher concentration of retinoic acid was required to activate the transcription of the Hox gene (Langston and Gudas, 1994). Although each gene in the cluster is being transcribed in the 5' to 3' direction, activation by retinoic acid occurs in the 3' to 5' direction in mammalian development. The expression patterns of Hox genes thus correspond linearly with the order of genes along the DNA.