Genetic deletion ofCyp26b1negatively impacts limb skeletogenesis by inhibiting chondrogenesis

Abstract

SummaryCyp26b1, a retinoic acid (RA)-metabolising enzyme, is expressed in the developing limb bud, and Cyp26b1 -/-mice present with severe limb defects. These malformations might be attributable to an RA-induced patterning defect; however, recent reports suggest that RA is dispensable for limb patterning. In this study, we examined the role of endogenous retinoid signalling in skeletogenesis using Cyp26b1 -/-mice and transgenic mice in which Cyp26b1 is conditionally deleted under control of the Prrx1 promoter beginning at E9.5 (Prrx1Cre + /Cyp26b1 fl/fl ). We found that the limb phenotype in Prrx1Cre + /Cyp26b1 fl/fl mice was less severe than that observed in Cyp26b1 -/-animals and that a change in retinoid signalling contributed to the difference in phenotypes. We systematically examined the role of endogenous RA signalling in chondrogenesis and found that Cyp26b1 -/-cells and limb mesenchymal cells treated with a CYP inhibitor, are maintained in a pre-chondrogenic state, exhibit reduced chondroblast differentiation and have modestly accelerated chondrocyte hypertrophy. Furthermore, Cyp26b1 -/-mesenchyme exhibited an increase in expression of genes in a closely related tendogenic lineage, indicating that retinoid signals in the limb interfere with differentiation and maintain progenitor status. Together, these findings support an important function for RA in regulating the behaviour of mesenchymal progenitors, and their subsequent differentiation and maturation.

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos

“…Histologic analysis of phenotype for Cyp26b1 rec/rec embryos demonstrated abnormal limbs, epidermis, and whole mount photographs demonstrated a cleft of the secondary palate (Fig. 3) as have been described previously for these mutants [7,12,13]. Hematoxylin and eosin staining at E13.5 of Pitx2 rec/rec mutants showed incomplete closure of the abdominal wall with ectopic viscera including the lungs, heart, and intestine as has been reported previously for these mutants [14].…”

Mechanisms of retinoic acid signalling and its roles in organ and limb development

“…However, forelimbs and hindlimbs are truncated along the entire proximodistal axis, which is inconsistent with RA functioning as an inducer of proximal identity. Further studies revealed that teratogenic mechanisms, such as increased apoptosis and a block in chondrogenic differentiation, cause the limb defects seen in Cyp26b1 −/− mice, overshadowing the effects of disrupted patterning of molecular markers 60,62,63 . Cyp26b1 −/− Rarg −/− double-mutant embryos display a partial rescue of Cyp26b1 −/− limb truncations, indicating that RARγ is important for RA-induced teratogenicity 62 .…”

Signaling pathways regulating cartilage growth plate formation and activity

“…The genetic deletion of any single member of the RAR family ( RARα , RARβ , RARγ ) in mice results in a minor phenotype, while deletions of multiple RAR receptors (e.g. RARα/γ or RARβ/γ ) results in perinatal lethality with severe skeletal abnormalities [95] [96]. While most RAR and RXR genes are functionally redundant, RXRα -deficient mice die in utero [94].…”