The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be a proximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces a teratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA following genetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes in expression of proximo-distal (P–D) patterning genes, increased cell death, and delayed chondrocyte maturation. Here we show that disruption of RA receptor (RAR) gamma in a Cyp26b1−/− background is able to partially rescue limb skeletal morphology without restoring normal expression of proximo-distal patterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains of mesenchymal cell death, which are reduced in compound-null animals. This model reveals two genetically separable effects of RA in the limb: an apoptotic effect mediated by RARγ in the presence of ectopic RA, and a P–D patterning defect which is uncovered following the loss of both CYP26B1 and RARγ. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Precise regulation of the morphogen sonic hedgehog (Shh) and modulation of the Shh signaling pathway is required for proper specification of cell fate within the developing limbs and neural tube, and resultant tissue morphogenesis. Tulp3 (tubby‐like protein 3) is a protein of unknown function which has been implicated in nervous system development through gene knockout studies. We demonstrate here that mice lacking the Tulp3 gene develop abnormalities of both the neural tube and limbs consistent with improper regulation of Shh signaling. Tulp3 −/− embryos show expansion of Shh target gene expression and display a ventralization of neural progenitor cells in the caudal neural tube. We further show that Tulp3 −/− / Shh −/− compound mutant embryos resemble Tulp3 mutants, and express Shh target genes in the neural tube and limbs which are not expressed in Shh −/− embryos. This work uncovers a novel role for Tulp3 as a negative regulatory factor in the Hh pathway. Developmental Dynamics 238:1140–1149, 2009. © 2009 Wiley‐Liss, Inc.

The retinoid X receptor (RXR) participates in a multitude of nuclear receptor signaling pathways and is induced by its highly sought-after cognate ligand, 9-cis-retinoic acid (9cis-RA). In flies and moths, molting is mediated by the ecdysone receptor that consists of a heterodimer comprising the ecdysone receptor monomer (EcR) and the invertebrate RXR homolog ultraspiracle (USP); the latter, however, is believed to have diverged from its RXR origin. From the more evolutionarily-primitive insect Locusta migratoria (Lm), long and short RXR transcripts (LmRXR-L and LmRXR-S, respectively) were detected during embryogenesis. This thesis reports the immunochemical detection of RXR crossreactive material throughout Locusta embryogenesis, suggesting that the protein may have another role besides ecdysone signaling. Thus, the RXR isoforms were cloned for recombinant expression and purification in order to demonstrate retinoid specificity. Both isoforms bound 9-cis-RA and all-trans-RA with high affinity. Binding was further corroborated by the identification of endogenous retinoids during embryogenesis. Embryos were first subjected to modified “Bligh and Dyer” as well as solid phase extractions to circumvent oil precipitation that rendered whole homogenates unsuitable for retinoid assay and detection. The RA-inducible Cyp26A1-promoter reporter cell line identified the presence of endogenous RAs (5.4 nM) from insect embryo extracts. Finally, high pressure liquid chromatography followed by mass spectroscopy (HPLC/MS) confirmed the identity of all-trans-RA and the more abundant 9-cis-RA (1.3 nM). These findings suggest a functional role for 9-cis-RA in the invertebrate embryo and favour signaling through the combination of 9-cis-RA and RXR in evolutionarily early RA-driven animal development.