Don Cameron

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis

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.

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect.

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC50 = 61.2–107.7 nM; Kd = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.

Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.

Peripheral nerve contains heterogeneous growth factors that support sensory neurons in vitro.

The availability of dissociated cultures of embryonic neurons has given impetus to a number of studies which have detected trophic factors in many mammalian and avian tissues maintained in vivo and in vitro [1, 2, 4-8, 10, 11, 13, 16, 18-21]. Using a single sensory neuron biological assay for mouse nerve growth factor (NGF) [17] as a reference standard, we report here that neurite promoting activity is highly enriched in segments of adult mouse peripheral nerve. Neurite outgrowth at 30 h, kinetics of neurite appearance and inhibition with rabbit antiserum to mouse NGF indicate that the trophic activities are heterogeneous and support both NGF-dependent and NGF-independent neuron populations of chick embryo sensory ganglia.

Tulp3 is a critical repressor of mouse hedgehog signaling

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.

Focal facial dermal dysplasia, type IV, is caused by mutations in CYP26C1

Focal facial dermal dysplasia (FFDD) Type IV is a rare syndrome characterized by facial lesions resembling aplasia cutis in a preauricular distribution along the line of fusion of the maxillary and mandibular prominences. To identify the causative gene(s), exome sequencing was performed in a family with two affected siblings. Assuming autosomal recessive inheritance, two novel sequence variants were identified in both siblings in CYP26C1-a duplication of seven base pairs, which was maternally inherited, c.844_851dupCCATGCA, predicting p.Glu284fsX128 and a missense mutation, c.1433G>A, predicting p.Arg478His, that was paternally inherited. The duplication predicted a frameshift mutation that led to a premature stop codon and premature chain termination, whereas the missense mutation was not functional based on its in vitro expression in mammalian cells. The FFDD skin lesions arise along the sites of fusion of the maxillary and mandibular prominences early in facial development, and Cyp26c1 was expressed exactly along the fusion line for these facial prominences in the first branchial arch in mice. Sequencing of four additional, unrelated Type IV FFDD patients and eight Type II or III TWIST2-negative FFDD patients revealed that three of the Type IV patients were homozygous for the duplication, whereas none of the Type II or III patients had CYP26C1 mutations. The seven base pairs duplication was present in 0.3% of healthy controls and 0.3% of patients with other birth defects. These findings suggest that the phenotypic manifestations of FFDD Type IV can be non-penetrant or underascertained. Thus, FFDD Type IV results from the loss of function mutations in CYP26C1.

Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth

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 RARgamma in the presence of ectopic RA, and a P-D patterning defect which is uncovered following the loss of both CYP26B1 and RARgamma. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells.

INTRODUCTION The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA. METHODS We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. RESULTS We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC(50) values of 16, 27, and 15nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC(50) of 62nM, and was sensitive to ketoconazole inhibition. DISCUSSION CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.

Regulation of murine embryonic patterning and morphogenesis by retinoic acid signaling

Publisher Summary This chapter discusses the regulation of murine embryonic patterning and morphogenesis by retinoic acid (RA) signaling. RA participates in the continuum of morphogenetic events that give rise to pattern at all stages of development. The systematic analyses of single and compound retinoid receptor mutants have clearly demonstrated that RA action through these receptors is essential for proper embryogenesis. The unexpected functional redundancy that has been observed among the retinoic acid receptors (RARs), as compared to the spectrum of defects observed in the compound mutants, suggests that the overlapping spatial organization of RARs (and RXRs) within the developing organism establishes a complex and intricate control platform to mediate RA signaling. The role of RA is to synchronously activate genetic algorithms within nascent cell fields, triggering the initiation of morphogenetic events that give rise to pattern. As development proceeds, the coupling of retinoic acid synthesis (source) and catabolism (sink) provides the requisite amount of RA at precise times within various tissue primordia. This triggers morphogenetic events in systems as diverse as hindbrain, limb, eyes, teeth, and tailbud.

Chapter 25:Retinoic Acid in Development

Evidence indicates that vitamin A and its active derivatives (retinoids) are critical throughout mammalian development. Retinoids exert biological effects on cell differentiation, proliferation, and morphogenesis by binding to their cognate receptors, the retinoic acid receptors (RARs) and retinoid ...