Retinoic Acid Signaling During Development

Retinoic acid (RA) functions as a ligand controlling a nuclear receptor signaling pathway involved in growth and development of vertebrate organisms including humans. RA action requires enzymatic conversion of retinol (vitamin A) to an active ligand (retinoic acid) which can then bind RA receptors in the nucleus. Synthesis of RA from retinol is a two-step process in which alcohol/retinol dehydrogenases (ADH/RDH) perform oxidation of retinol to retinaldehyde, and retinaldehyde dehydrogenases (RALDH) perform oxidation of retinaldehyde to RA. Among the various secreted cell-cell signaling factors that direct developmental processes, RA is unique in that it is a small molecule (M.W. 300) that directly regulates gene transcription by entering the nucleus of target cells and binding to target genes via nuclear receptors. This is in stark contrast to other secreted cell-cell signaling factors such as fibroblast growth factor (FGF), WNT, transforming growth factor-beta (TGFb), and sonic hedgehog (SHH) which all bind cell-surface receptors and initiate intracellular signaling pathways that regulate transcription in the nucleus. RA signaling appears to be primarily a paracrine pathway as the cells that synthesize RA are not the targets of RA action (Fig. 1).

One major challenge in the study of vitamin A function is to discover when and where RA synthesis occurs during embryogenesis as this provides the ligand that initiates RA signaling. Another challenge is to determine exactly where RA acts when it is released from cells and functions as a signaling molecule. Our studies are also aimed at determining what genes are regulated by RA during formation of specific structures in target tissues, and exploring how RA signaling cooperates with other developmental signaling molecules encoded by Fgf, Shh, TGFb, and Wnt to control morphogenesis in target tissues. Our laboratory has generated RA-deficient mouse mutants that have allowed us to undertake a detailed investigation into the mechanism of RA signaling in order to more fully understand intercellular signaling pathways during development.

Some milestones made by our laboratory:

• Genetic Identification of Enzymes Controlling Retinoic Acid Synthesis Raldh1, Raldh2, and Raldh3 encode retinaldehyde dehydrogenases essential for oxidation of retinaldehyde to RA. Knockout mice for Raldh1, Raldh2, and Raldh3 lose RA synthesis in specific tissues during embryogenesis resulting in abnormal development. Raldh1-/-, Raldh2-/-, and Raldh3-/- mice as well as compound knockout mice are being used to learn more about the mechanism of RA signaling during development of specific tissues.
  

• Retinoic Acid Signaling Does Not Require 9-cis-Retinoic Acid Two isomers of RA were originally thought to function as receptor ligands: all-trans-RA for retinoic acid receptors (RAR) and 9-cis-RA for retinoid X receptors (RXR). However, our Raldh2-/- rescue studies show that 9-cis-RA is not required to correct a lethal defect in RA synthesis as an RAR-specific synthetic ligand can rescue Raldh2-/- embryos. Also, HPLC analyses demonstrate that 9-cis-RA is not detectable in mouse embryos unless treated with high doses of retinoids.
  

• Retinoic Acid Acts as an Instructive Signal for Neural Development During gastrulation, RA synthesized in the somitic mesoderm travels to the adjacent neuroectoderm where it acts as an instructive signal to induce Hoxb1, Hnf1b, and Olig2 needed for development of the hindbrain and spinal cord. This pathway leads to development of hindbrain rhombomeres and facial motor neurons as well as motor neuron differentiation along the spinal cord.
  

• Retinoic Acid Represses FGF8 Expression During Early Organogenesis to Allow Proper Differentiation of Trunk Mesoderm: Limb Buds, Somites and Heart During gastrulation, RA acts as a permissive signal for differentiation of mesoderm by limiting the size of the primitive streak and cardiac Fgf8 expression domains, thus creating an FGF-free zone in between where the trunk develops. RA thus sets the anterior boundary of the primitive streak to allow proper somitogenesis and the posterior boundary of the heart to allow proper heart and forelimb bud development. We hypothesize that failure of this mechanism generates excessive FGF8 signaling to adjacent mesoderm resulting in smaller somites (vertebra precursors) displaying left-right asymmetry, a larger heart domain, and a failure to initiate forelimb budding (Fig. 2).
  

• Retinoic Acid Initiates Limb Budding But is Not Required for Limb Patterning Our findings show that RA signaling is not required for limb proximodistal or anteroposterior patterning as originally postulated, but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

Fig. 1. Paracrine mechanism of RA signaling. Retinol carried by retinol-binding protein (RBP4) enters cells by the receptor STRA6, and cellular retinol-binding protein (CRBP) converts retinol to retinyl esters for storage. In an RA-generating tissue, retinol is reversibly oxidized to retinaldehyde by either alcohol dehydrogenase (ADH) or retinol dehydrogenase (RDH), and retinaldehyde is irreversibly oxidized to RA by retinaldehyde dehydrogenase (RALDH). RA is then released and taken up by surrounding cells. Cells that express cytochrome P450 (CYP26) initiate degradative oxidation of RA and are not RA target cells. Some RA target cells express cellular-RA binding protein-2 (CRABP2) that facilitates RA transport to the nucleus where RA binds the RA receptor (RAR). RAR-RXR heterodimers bind retinoic acid response elements (RARE) and regulate transcription by altering the binding of cofactors. RXR also binds other nuclear receptors, so its function is not limited to RA signaling.




















Fig. 2. Tbx5 mRNA detected by whole-mount in situ hybridization in wild-type (WT) and Raldh2-/- mutant mouse embryos. The mutant lacks a forelimb (f) domain, has an altered heart (h) domain that fails to loop, and fails to undergo embryonic turning needed to achieve the fetal position due to a failure in somitogenesis.