Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and bone defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding protein (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day 10 post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the morphogenetic field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.
Two cDNAs encoding two human receptors for retinoic acid (RA), RAR-alpha and RAR-beta, have been characterized recently. Synthetic peptides corresponding to the cDNA-deduced amino acid sequences unique to RAR-alpha and RAR-beta were used to generate anti-RAR-alpha antiserum (SP171) and anti-RAR-beta antisera (SP172 and SP248). The specificity of these antisera was confirmed both by immunocytochemical detection of these receptors in COS-1 cells transfected with RAR-alpha and RAR-beta expression vectors and by immunoblot analyses performed with whole extracts of these cells. We also demonstrate that these antisera recognize RAR-alpha and RAR-beta endogenously expressed in the RA-responsive human promyelocytic leukemia cell line HL-60.
In situ hybridization with 35S-labelled RNA probes was used to study the distribution of transcripts of genes coding for the retinoic acid receptors, RAR-alpha, -beta and -gamma, and the cellular binding proteins for retinoic acid (CRABP I) and retinol (CRBP I), in mouse embryos during the period of early morphogenesis. Primary mesenchyme formation was associated with CRBP I labelling of both epiblast and mesenchyme of the primitive streak, while the CRABP probe labelled the migrating primary mesenchyme cells. Neural crest cell emigration and migration were associated with CRABP labelling of both neural epithelium (excluding the floor plate) and neural crest cells, while CRBP I expression was restricted to basal and apical regions of the epithelium (excluding the floor plate). The strongest neuroepithelial signal for CRABP was in the preoptic hindbrain. RAR-beta was present in presomitic stage embryos, being expressed at highest levels in the lateral regions. RAR-alpha was associated with crest cell emigration and migration, while RAR-gamma was present in the primitive streak region throughout the period of neurulation. There was a change from RAR-beta to RAR-gamma expression at the junction between closed and open neural epithelium at the caudal neuropore. RAR-alpha and RAR-beta were expressed at specific levels of the hindbrain and in the spinal cord. These distribution patterns are discussed in relation to segmental expression patterns of other genes, and to maturational changes in the caudal neuropore region. The CRABP transcript distribution patterns correlated well with known target tissues of excess retinoid-induced teratogenesis (migrating primary mesenchyme and neural crest cells, preoptic hindbrain), providing further support for our hypothesis that cells expressing CRABP are those that cannot tolerate high levels of RA for their normal developmental function.
We report here the gene expression patterns, as revealed by in situ hybridisation, of the retinoic acid receptors alpha, beta and gamma (RAR-alpha, -beta and -gamma), and the cellular binding proteins for retinol and retinoic acid (CRBP, CRABP) in non-neural tissues of mouse embryos during the period of organogenesis. At all stages, RAR-alpha transcripts were almost ubiquitous, whereas the distribution of transcripts of the other four genes was distinctive in all systems. At early stages in the formation of an organ, the expression patterns were different in the epithelium, the adjacent mesenchyme, and in mesenchyme more distant from the epithelium, suggesting a role for RA and RA receptors in epithelial-mesenchymal tissue interactions. In the developing face, limb bud and genital tubercle, where large expanses of mesenchyme are present, differential patterns of expression were established before the onset of overt tissue differentiation, suggesting some significance for pattern formation in these regions. The distribution of RAR-beta transcripts in tracheobronchial, intestinal and genital tract epithelial is consistent with the possibility that RAR-beta plays a role in mediating retinoid effects on the differentiated stage of these epithelia. Possible developmental roles of RARs in relation to the expression patterns of other genes are discussed. CRBP expression domains showed a high degree of overlap with RAR-beta and RAR-gamma, and a mutual exclusivity with CRABP expression domains. Correlation of these expression patterns with the morphogenetic effects of vitamin A deficiency and retinoid excess lead us to propose that the function of CRBP is to store and release retinol where high levels of RA are required for specific morphogenetic processes, while CRABP serves to sequester RA in regions where normal developmental functions require RA levels to be low. Where both binding protein genes are expressed in a non-overlapping pattern within a large area of mesenchyme, a gradient of free RA may be created between them by release of retinol-derived RA from CRBP-expressing cells, with binding to CRABP enhancing the steepness of the decline in concentration distant to the source.
We have studied the transcript distribution of the retinoic acid receptors (RARs) and the cytoplasmic retinoid binding proteins during embryonic development of the mouse nervous system. Of the three retinoic acid receptors, only RAR-gamma was not expressed in developing neural structures. RAR-beta and RAR-alpha both showed rostral limits of expression in the medulla oblongata equivalent to their patterns of expression in the neuroepithelium of the early hindbrain neural tube. Within their expression domains in the spinal cord and brain, RAR-alpha was ubiquitously expressed, whereas RAR-beta transcripts showed very specific patterns of expression, suggesting that this receptor is involved in mediating retinoic acid-induced gene expression in relation to the development of specific neural structures or pathways. The cytoplasmic binding proteins, cellular retinoic acid binding proteins type I and II (CRABP I and CRABP II) and cellular retinol binding protein type I (CRBP I), were widely distributed in developing neural structures. Their differential spatiotemporal patterns of expression suggest that fine regional control of availability of retinoic acid (RA) to the nuclear receptors plays an important role in organization and differentiation of the nervous system. For instance, expression of CRABP I in the migrating cells that give rise to the olivary and pontine nuclei, which develop abnormally in conditions of retinoid excess, is consistent with observations from a variety of other systems indicating that CRABP I limits the access of RA to the nuclear receptors in normal physiological conditions. Similarly, expression of CRBP I in the choroid plexuses, which develop abnormally in conditions of vitamin A deficiency, is consistent with observations indicating that this binding protein mediates the synthesis of RA in tissues requiring high levels of RA for their normal developmental programme. RAR-beta and CRABP II, which are both RA-inducible, were coexpressed with CRBP I in the choroid plexus and in many other sites, perhaps reflecting the fact that all three genes are RA-inducible. The function of CRABP II is not well understood; its domains of expression showed overlaps with both CRABP I and CRBP I.
This study provides a detailed description of the anatomical defects in the Hoxa-1−/− mutant mice previously generated in our laboratory (T. Lufkin, A. Dierich, M. LeMeur, M. Mark and P. Chambon, 1991; Cell 66, 1105–1119). Three-dimensional reconstructions of the Hoxa-1−/− rhombencephalon reveals that it bears only five rhombomeric structures (ie. morphological segments) instead of the normal seven. The first three of these rhombomeres appear normal as judged from the distribution pattern of CRABPI transcripts in the neurectoderm and from the histological analysis of the cranial nerve components derived from these structures. In contrast, the neural-crest-cell-free region normally located opposite rhombomere 5 is lacking in Hoxa-1−/− embryos, and motor neurons of the facial and abducens nerves, which normally differentiate within rhombomeres 4, 5 and 6, are missing in Hoxa-1−/− fetuses. These morphological data, combined with the determination of the molecular positional identities of the rhombomeres 4 and 5 (P. Dolle, T. Lufkin, R. Krumlauf, M. Mark, D. Duboule and P. Chambon, 1993; Proc. Natl. Acad. Sci. USA, in press), suggest that rhombomere 4 is markedly reduced, whereas rhombomere 5 is almost absent. Thus, the remnants of rhombomeres 4 and 5 appear to be fused caudally with rhombomere 6 to form a single fourth rhombomeric structure. Moreover, the migration of neural crest cells contributing to the glossopharyngeal and vagus nerves occurs in a more rostral position, resulting in abnormalities of these cranial nerves, which were visualized by whole-mount anti-neurofilament immunostaining. The mutual relationship along the rostrocaudal axis between the otic pit and the neuroepithelial site of int-2 protein secretion (a putative otogenic cue) is not significantly changed in Hoxa-1−/− embryos. However, the abnormal relationship between the rhombencephalon and the epithelial inner ear may account for the aplasia and faulty differentiation of the membranous labyrinth, the disruption of the cartilaginous otic capsule and the disorganisation of some middle ear structures. This phenotype is compared with that of the Hoxa-1−/− mutants generated by O. Chisaka, T. S. Musci and M. R. Capecchi, 1992 (Nature 335, 516–520) and with that of the mice homozygous for the kreisler mutation.
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