Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning.

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RAR gamma (A alpha/A gamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 A alpha/A gamma embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 A alpha/A gamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RAR alpha and RAR beta (A alpha/A beta mutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an A alpha/A beta-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an A alpha/A gamma-like phenotype. Altogether, our data suggest that RAR alpha and/or RAR gamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RAR beta is subsequently involved in setting up the caudal boundary of this territory.

Early embryonic lethality in PARP-1 Atm double-mutant mice suggests a functional synergy in cell proliferation during development.

PARP-1 and ATM are both involved in the response to DNA strand breaks, resulting in induction of a signaling network responsible for DNA surveillance, cellular recovery, and cell survival. ATM interacts with double-strand break repair pathways and induces signals resulting in the control of the cell cycle-coupled checkpoints. PARP-1 acts as a DNA break sensor in the base excision repair pathway of DNA. Mice with mutations inactivating either protein show radiosensitivity and high radiation-induced chromosomal aberration frequencies. Embryos carrying double mutations of both PARP-1 and Atm genes were generated. These mutant embryos show apoptosis in the embryo but not in extraembryonic tissues and die at embryonic day 8.0, although extraembryonic tissues appear normal for up to 10.5 days of gestation. These results reveal a functional synergy between PARP-1 and ATM during a period of embryogenesis when cell cycle checkpoints are not active and the embryo is particularly sensitive to DNA damage. These results suggest that ATM and PARP-1 have synergistic phenotypes due to the effects of these proteins on signaling DNA damage and/or on distinct pathways of DNA repair.

Retinoid signaling is essential for patterning the endoderm of the third and fourth pharyngeal arches.

The requirement of retinoic acid (RA) in the initial formation of the pharyngeal arches was investigated by treating headfold-stage mouse embryos with a pan-RAR antagonist in vitro and in vivo. This results in a complete absence of mesenchyme, arteries, nerves and epibranchial placodes of the 3rd and 4th pharyngeal arches, complete agenesis of the 3rd and 4th pouches and consistent lack of the 6th arch artery. Mesodermally derived endothelial cells are absent from the 3rd and 4th pharyngeal arch region and the distribution domain of EphA2 transcripts in mesodermal cells is shifted caudally. In situ hybridization with CRABPI, kreisler and EphA4 probes and the pattern of expression of a Wnt1-lacZ transgene show that neural crest cells (NCC) normally destined to the 3rd and 4th arches migrate ectopically. Most interestingly, the appearance of the 3rd and 4th arches is prevented by the antagonist only during a very narrow window of time, which does not correspond to the period of post-otic NCC migration. Both the timing of appearance and the nature of the defects in RAR antagonist-treated embryos indicate that migrating NCC and mesodermal cells destined to the caudal pharyngeal arches do not represent primary targets of RA action. Alterations in the endodermal expression pattern of Hoxa1, Hoxb1, Pax1, Pax9, Fgf3 and Fgf8 in response to the antagonist-induced block in RA signal transduction demonstrate for the first time that RA signaling is indispensable for the specification of the pharyngeal endoderm and suggest that this signaling is necessary to provide a permissive environment locally for the migration of NCC and mesodermal cells. Our study also indicates that the formation of the 2nd pharyngeal arch and that of the 3rd and 4th pharyngeal arches probably involve distinct RA-dependent developmental processes.

Key roles of retinoic acid receptors alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches and otocyst in the mouse.

Mouse fetuses carrying targeted inactivations of both the RAR(&agr;) and the RARbeta genes display a variety of malformations in structures known to be partially derived from the mesenchymal neural crest originating from post-otic rhombomeres (e.g. thymus and great cephalic arteries) (Ghyselinck, N., Dupé, V., Dierich, A., Messaddeq, N., Garnier, J.M., Rochette-Egly, C., Chambon, P. and Mark M. (1997). Int. J. Dev. Biol. 41, 425-447). In a search for neural crest defects, we have analysed the rhombomeres, cranial nerves and pharyngeal arches of these double null mutants at early embryonic stages. The mutant post-otic cranial nerves are disorganized, indicating that RARs are involved in the patterning of structures derived from neurogenic neural crest, even though the lack of RARalpha and RARbeta has no detectable effect on the number and migration path of neural crest cells. Interestingly, the double null mutation impairs early developmental processes known to be independent of the neural crest e.g., the initial formation of the 3rd and 4th branchial pouches and of the 3rd, 4th and 6th arch arteries. The double mutation also results in an enlargement of rhombomere 5, which is likely to be responsible for the induction of supernumerary otic vesicles, in a disappearance of the rhombomere 5/6 boundary, and in profound alterations of rhombomere identities. In the mutant hindbrain, the expression domain of kreisler is twice its normal size and the caudal stripe of Krox-20 extends into the presumptive rhombomeres 6 and 7 region. In this region, Hoxb-1 is ectopically expressed, Hoxb-3 is ectopically up-regulated and Hoxd-4 expression is abolished. These data, which indicate that retinoic acid signaling through RARalpha and/or RARbeta is essential for the specification of rhombomere identities and for the control of caudal hindbrain segmentation by restricting the expression domains of kreisler and of Krox-20, also strongly suggest that this signaling plays a crucial role in the posteriorization of the hindbrain neurectoderm.

Spatio-temporally controlled site-specific somatic mutagenesis in the mouse.

The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type will facilitate studies of gene function and the generation of animal models for human diseases. We have shown previously that conditional recombination-excision between two loxP sites can be achieved in mice by using the Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (Cre-ERT), which binds tamoxifen but not estrogens. DNA excision was induced in a number of tissues after administration of tamoxifen to transgenic mice expressing Cre-ERT under the control of the cytomegalovirus promoter. However, the efficiency of excision varied between tissues, and the highest level ( approximately 40%) was obtained in the skin. To determine the efficiency of excision mediated by Cre-ERT in a given cell type, we have now crossed Cre-ERT-expressing mice with reporter mice in which expression of Escherichia coli beta-galactosidase can be induced through Cre-mediated recombination. The efficiency and kinetics of this recombination were analyzed at the cellular level in the epidermis of 6- to 8-week-old double transgenic mice. We show that site-specific excision occurred within a few days of tamoxifen treatment in essentially all epidermis cells expressing Cre-ERT. These results indicate that cell-specific expression of Cre-ERT in transgenic mice can be used for efficient tamoxifen-dependent, Cre-mediated recombination at loci containing loxP sites to generate site-specific somatic mutations in a spatio-temporally controlled manner.