Regulation of Retinoic Acid Distribution Is Required for Proximodistal Patterning and Outgrowth of the Developing Mouse Limb

Abstract

Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.

Expression of key retinoic acid modulating genes suggests active regulation during development and regeneration of the amphibian limb

“…1B-G). This is in sharp contrast to mouse limb buds, which have no intrinsic Raldh activity and can only receive RA by diffusion from the adjacent body wall lateral plate mesoderm (Yashiro et al, 2004). Between st. 51 and 52, two new patches of cells began expressing Raldh2 in the anterior/proximal quadrant, on either side of the limb, within the future stylopod (Fig.…”

“…Cyp26b is also expressed distally in chick and mouse limb buds, where it is thought to remove RA produced by Raldh2-expressing cells in the body wall lateral plate (reviewed in Niederreither and Dolle, 2008). Modulation of RA levels by Cyp26b is essential for normally developing limbs in amniotes, as shown by the phenotype of the Cyp26b knockout mouse (Yashiro et al, 2004). Cyp26b À/À mice have meromelic limbs, with 2-3 digits complete with nails but more proximal elements shortened and fused.…”

“…The no-fin mutant, which lacks pectoral fins, is due to loss of functional Raldh2 (Grandel et al, 2002). While Raldh2 is not expressed in the mouse limb at all E9.5-E11 (Yashiro et al, 2004), mice lacking Raldh2 have no forelimb buds (Niederreither et al, 2002).…”

“…No limb phenotypes have been reported in these mice even though proximal-distal pattern is established and digits are apparent well before this stage (Martin, 1990). Compelling evidence for RA's involvement in the proximal distal patterning of the limb comes from examination of transgenic mice harbouring a reporter driven by retinoic acid response elements (RAREs), which show the existence of a gradient, and from cyp26b knockout mice, which have truncated limbs, a phenotype that can be reproduced with exposure to exogenous RA (Yashiro et al, 2004). Recently, however, experiments using RA supplementation in mice lacking both Raldh2 and Raldh3 have suggested an alternative role for RA in positioning the limbs (Zhao et al, 2009).…”

Fate of retinoic acid–activated embryonic cell lineages

“…The rationale for these studies is that most, if not all, tissues express at least one of the RARs (Dolle, 2009 for a review), that should activate the reporter upon binding of endogenous RA. The RARE-hsp68-lacZ transgene has been widely used in subsequent studies (e.g., Malpel et al, 2000;Mic et al, 2002;Niederreither et al, 2002b;Fan et al, 2003;Matt et al, 2003Matt et al, , 2005Yashiro et al, 2004;Sirbu et al, 2005;Molotkov et al, 2006;Sirbu and Duester, 2006;Ribes et al, 2009;Zhao et al, 2009). Three lines of evidence have validated this transgene as a reliable indicator of RA activity: (1) there is a close match between the patterns of lacZ activity and the expression patterns of RA-synthesizing enzymes (Rdh10, Raldhs) at early embryonic stages, (2) activity of the lacZ reporter is severely down-regulated in knockout mice for the above enzymes (Niederreither et al, 1999;Mic et al, 2002;Matt et al, 2005;Molotkov et al, 2006;Sandell et al, 2007), (3) on the other hand, the reporter is almost ubiquitously activated within 6 h after administration of a bolus dose of RA to the pregnant mothers (Rossant et al, 1991), and is ectopically activated when RA-metabolizing enzymes are absent Sakai et al, 2001;Uehara et al, 2009).…”