INTRODUCTION Retinoic acid (RA), a hormonal signal derived from vitamin A (retinol), is essential in vertebrates for controlling processes involved in embryonic patterning and organogenesis, cell proliferation, differentiation and apoptosis, and homeostasis

Retinoic acid (RA), a hormonal signal derived from vitamin A (retinol), is essential in vertebrates for controlling processes involved in embryonic patterning and organogenesis, cell proliferation, differentiation and apoptosis, and homeostasis (Sporn et al., 1994; Blomhoff, 1994; Chambon, 1996). RA levels are controlled locally, through the combined action of retinaldehyde dehydrogenases (RALDH1, RALDH2 and RALDH3) that catalyze the last step in its synthesis, and of the RA-metabolizing cytochrome P450 (CYP26) (McCaffery and Dräger, 2000; Duester, 2000; Abu-Abed et al., 2001). RA binds to nuclear receptors, the RARs (α, β and γ isotypes, that bind both all-trans and 9-cis RA) and the RXRs (α, β and γ isotypes, that bind 9-cis RA only), which act as transcriptional regulatory proteins mainly in the form of RAR/RXR heterodimers (Kastner et al., 1995; Chambon, 1996). The highly pleiotropic effects of RA during mammalian development have been established from the analysis of embryos and fetuses (1) obtained from females that were raised on vitamin A-deficient diets (Wilson et al., 1953; Dickman et al., 1997; White et al., 2000; Zile et al., 2000; and references therein); (2) carrying loss-of-function mutations of RARs and/or RXRs (Kastner et al., 1995; Mascrez et al., 1998; and references therein); (3) lacking the RA-generating enzyme RALDH2 (Niederreither et al., 1999); (4) lacking the RAmetabolizing enzyme CYP26 (Abu-Abed et al., 2001); (5) treated with antagonists of RARs (Wendling et al., 2000; Chazaud et al., 1999); or (6) expressing dominant-negative RARs (van der Wees et al., 1998). An extensive functional redundancy between RARs accounts for the observation that RAR (α, β or γ)-null mutants exhibit only few developmental defects, whereas altogether the phenotypes of mutants that lack both RARα and RARβ (Aα/Aβ mutants), RARα and RARγ (Aα/Aγ mutants) and RARβ and RARγ (Aβ/Aγ mutants) recapitulate all the abnormalities characteristic of the fetal vitamin A-deficiency (VAD) syndrome (Wilson et al., 1953; Kastner et al., 1995). Of the three types of RAR double-null mutants, those that lack RARα and RARγ are overall the most severely affected. Many Aα/Aγ mutants die in utero in contrast to Aα/Aβ and Aβ/Aγ mutants, which survive until birth. Moreover, near-term (embryonic day (E)18.5) Aα/Aγ fetuses are markedly growth deficient and exhibit evident external malformations, whereas E18.5 Aα/Aβ and Aβ/Aγ fetuses are externally undistinguishable from their wild-type littermates (Lohnes et al., 1994; Mendelsohn et al., 1994; Ghyselinck et al., 1997; Luo et al., 1996). In the first part of this work, we have established the timing of the appearance of defects previously observed in Aα/Aγ 2031 Development 128, 2031-2038 (2001) Printed in Great Britain © The Company of Biologists Limited 2001 DEV2705