BTB/POZ-domain C 2 H 2 zinc(Zn)-finger proteins are encoded by a subfamily of genes related to the Drosophila gap gene krüppel . To date, two such proteins, PLZF and LAZ-3/BCL-6, have been implicated in oncogenesis. We have now identified a new member of this gene subfamily which encodes a 62 kDa Zn-finger protein, termed LRF , with a BTB/POZ domain highly similar to that of PLZF. Both human and mouse LRF genes, which localized to syntenic chromosomal regions (19p13.3 and 10B5.3, respectively), were widely expressed in adult tissues and cell lines. At approximately 9.5 – 10.0 days of embryonic development, the mouse LRF gene was expressed in the limb buds, pharyngeal arches, tail bud, placenta and neural tube. The LRF protein associated in vivo with LAZ-3/BCL-6, but not with PLZF to which it was more related. Although the LRF, or LAZ-3/BCL-6, BTB/POZ domain could readily homodimerize, no heterodimerization was detected in vivo between the LRF and LAZ-3/BCL-6 BTB/POZ domains and interaction between full length LRF and LAZ-3/BCL-6 required the presence of both the BTB/POZ domain and Zn-fingers in each partner protein. As expected from the above results, LRF and LAZ-3/BCL-6 also colocalized with each other in the nucleus. Taken together, our findings suggest that BTB/POZ-domain Zn-finger proteins may function as homo and heterodimeric complexes whose formation, and hence the resultant effect on transcription of their downstream target genes, is determined by the levels and expression domains of a given partner protein.

The retinoids form a family of compounds structurally and functionally related to vitamin A (all-trans-retinol). Their name derives from the role of vitamin A in visual function in the vertebrate retina. The natural retinoids are isoprenoid compounds with a six-membered carbocyclic ring and an eleven-carbon side chain (Fig. 1), although many of the synthetic compounds with biological retinoid activity differ somewhat from this basic plan (Sporn and Roberts 1985).

During mammalian neurulation regional differences are evident between the cranial region, in which neurulation is most complex, the trunk as far as the caudal neuropore and the secondary neurulation region of the caudal trunk plus tail. Differences among these three regions are characterized by specific patterns of morphogenesis and by specific patterns of gene expression. During cranial neurulation distinct regions develop in the brain and the presomitic hindbrain forms seven rhombomeric divisions. The first clear morphological boundary is the preotic sulcus (later transformed into the gyrus between rhombomeres 2 and 3), which may limit cell movement as neuroepithelial cells rostral to it flow towards and into the rapidly expanding forebrain region. The formation of rhombomeres as morphological entities and the development of a normal rhombomere-specific pattern of homeobox and other gene expression domains depend on relatively low levels of retinoic acid. Retinoic acid receptors, which are retinoic acid-activated transcription factors, and retinoid binding proteins, which control the availability of retinoic acid to the receptors, show regional patterns of expression in the cranial, trunk and caudal regions of the neuroepithelium during neurulation. These patterns suggest a possible mechanism for region-specific gene expression during neurulation.

ABSTRACT We have compared the relationship between the patterns of altered morphogenesis and of altered gene expression in mouse embryos exposed to excess retinoic acid (RA) (a) just before and (b) just after the onset of somitic segmentation (day 7.75 to day 8.25). Exposure to RA prior to the onset of somitic segmentation results in suppression of rhombomeric (but not somitic) segmentation, and conversion of the genetic identity of the whole preotic hindbrain to that of rhombomere 4. In contrast, exposure to RA at early somite stages results in near-normal rhombomeric segmentation; rhombomeric gene expression domains indicate that only rhombomere 2 has changed its genetic identity to that of rhombomere 4, the other preotic segments showing normal expression patterns for HoxB genes and Krox-20. The results indicate that RA has separable effects (1) on the genes mediating the process of rhombomeric segmentation per se, such as Krox-20, and (2) on the genes that influence the nature of the structures that subsequently develop from the individual rhombomeres, such as the Hox genes.

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.

In the mammalian embryonic trunk, neural crest cells emigrate from the closed neural tube in a cranio-caudal sequence and appear to have similar migration pathways and derivatives to those of avian embryos. In the cranial region, however, there are mammalian-specific features, which are related to the mammalian-specific pattern of cranial neurulation. Midbrain and rostral hindbrain neural crest cells emigrate from widely open neural folds; caudal hindbrain crest emigrates in a caudo-rostral sequence, following the sequence of neural tube closure in this region. The forebrain is also a source of neural crest cells at early stages of neurulation; both forebrain and midbrain crest cells contribute to the frontonasal mesenchyme, although their relative contributions have not been analysed. Few studies have provided direct information about mammalian neural crest cell derivatives. Studies on the effects of retinoid excess on craniofacial development provide indirect evidence that mammalian cranial neural crest, like that of avian embryos, includes two populations whose differentiated phenotype and morphological tissue structure are determined prior to emigration. Retinoid-induced shortening of the preotic hindbrain leads to abnormal migration pathways of the neural crest cells that normally migrate into the mandibular arch to form Meckel's cartilage, so that an ectopic Meckel's cartilage-like structure forms in the maxillary region of the face. Slow descent of the heart in retinoid-exposed embryos enables the "wrong" crest cell population to populate the wall of the truncus arteriosus. These observations correlate well with observations of retinoid-induced craniofacial and heart abnormalities in human infants.

We have compared the transcript distribution of cellular retinoic acid binding protein (CRABP) I and II genes in mouse embryos at various stages of development. Both CRABP transcripts are present in embryonic structures from the earliest stages studied and exhibit specific patterns of distribution, suggesting that the two retinoic acid (RA) binding proteins perform different functions during mouse embryogenesis. The CRABP I transcript distribution correlates well with structures known to be targets of excess retinoid-induced teratogenesis (e.g. neural crest cells and hindbrain), suggesting that cells expressing CRABP I are those that cannot tolerate high levels of RA for their normal developmental function. The embryonic structures expressing CRABP II transcripts include those structures that have been shown to be adversely affected by excess of retinoids, such as limbs and hindbrain, but CRABP II transcripts are also found in structures not known to be specifically vulnerable to raised RA levels. The CRABP II gene is coexpressed with retinoic acid receptor (RAR)-beta and cellular retinol binding protein (CRBP) I genes in a number of tissues such as the gut endoderm, hypophysis and interdigital mesenchyme, all of which are devoid of CRABP I transcripts. Interestingly, the expression of the three genes, RAR-beta, CRABP II and CRBP I, is induced by retinoic acid, which suggests a link between the synthesis of RA from retinol and the control of expression of subsets of RA-responsive genes. The transcript distribution of CRABP I and II is discussed in relation to the teratogenic effects of RA, and compared to the RA-sensitive pattern of expression of other important developmental genes.

Review Articles| October 07 2008 Retinoic Acid and Development Subject Area: Further Areas , Oncology , Pathology and Cell Biology Gillian Morriss-Kay Gillian Morriss-Kay Department of Human Anatomy, Oxford, UK Search for other works by this author on: This Site PubMed Google Scholar Pathobiology (1992) 60 (5): 264–270. https://doi.org/10.1159/000163733 Article history Received: August 12 1991 Accepted: August 12 1991 Published Online: October 07 2008 Content Tools Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Gillian Morriss-Kay; Retinoic Acid and Development. Pathobiology 1 May 1992; 60 (5): 264–270. https://doi.org/10.1159/000163733 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsPathobiology Search Advanced Search Article PDF first page preview Close Modal Keywords: Retinoic acid, Mouse embryo, Human embryo, Cell death, Retinoic acid receptors, Retinoid binding proteins, Gene expression, Teratogenesis, Vitamin A deficiency, Cell membrane This content is only available via PDF. 1992Copyright / Drug Dosage / DisclaimerCopyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. You do not currently have access to this content.

Retinoic acid receptors are ligand activated transcription factors that have a variety of important roles in normal development, through regulating the transcription of other developmental control genes. The great diversity of RA mediated developmental events is made possible through the existence of three receptor subtypes, each of which has a number of isoforms. The mechanism of gene regulation is thought to involve binding to an RA response element on the target gene, although in practice, few genes known to be activated by RA have so far been demonstrated to include such an element in the promoter region. Differential spatiotemporal expression patterns of the various subtypes have been described in mouse embryos, including very fine differences within different subpopulations of cells of the same tissue. In addition to qualitative differences in the gene activation responses to RA through spatiotemporal differences in RAR expression patterns, local differences in RAR activation may be modulated by cytoplasmic binding proteins for retinol and RA, through quantitative control of ligand availability. The developmental control genes known to be activated by RA include the 3' members of each of the homoeogene clusters, and it is likely that others will be identified in the near future.

Head-trunk differences are well established in the most primitive vertebrates, and are clear from early developmental stages of all modern forms. The boundary between the two regions is not constant in all vertebrate classes in terms of the number of occipital somites. The occipital region is in some respects a transitional zone, giving rise to trunk-like somitic derivatives in the head. It is also highly specialised, providing a unique population of neural crest cells that are essential for formation of the aorticopulmonary septum (which divides the outflow tract of the heart) in mammals and birds. In the preoccipital hindbrain, rhombomeres represent a segmental structural pattern that is quite distinct from that of the somites, with a segment-specific pattern of gene expression. Expression of some of these genes in mesenchyme close to the primitive streak at earlier stages suggests that this pattern may be established at the time of neural induction. Mammalian embryos have taken cranial specialization further than other classes of vertebrate, particularly in relation to the pattern of development and eventual structural complexity of the forebrain. Mammalian specialisations of craniofacial development are described through references to studies on cranial neurulation, on cranial neural crest cell migration, and on the possible morphogenetic roles of extracellular matrix components.

Retinoic acid (RA) is a signalling molecule important for pattern formation during development. There are three known types of nuclear receptors for RA in mammals, RAR-alpha, RAR-beta and RAR-gamma, which transduce the RA signal by inducing or repressing the transcription of target genes. Here we describe the developmental expression pattern of the mouse RAR-beta-2 promoter. Independent lines of transgenic animals expressing RAR-beta-2 promoter sequences fused to the E. coli beta-galactosidase gene were examined throughout the course of embryogenesis and found to exhibit reproducible and specific patterns of beta-galactosidase expression in a majority of sites that have been shown previously to contain mRAR-beta transcripts. In the limbs, mRAR-beta-2 promoter activity and mRAR-beta transcripts were both excluded from precartilagenous condensations; interestingly, mRAR-beta-2 promoter activity was observed in the apical ectodermal ridge (AER) where mRAR-beta transcripts could not be detected, while no mRAR-beta-2 promoter activity or mRAR-beta transcripts were associated with the limb region that contains the zone of polarizing activity (ZPA). Analysis of the lacZ expression pattern in embryos from mothers treated with teratogenic doses of RA, indicated that mRAR-beta-2 promoter is selectively induced in a manner suggesting that overexpression of the mRAR-beta-2 isoform is involved in RA-generated malformations. The normal and induced expression pattern of the mRAR-beta-2 promoter suggests several possible roles for mRAR-beta-2 in development of the limbs, as an inhibitor of cartilage formation, in programmed cell death and in the formation of loose connective tissue.

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.

Retinoic acid (RA), a putative morphogen in vertebrates, has profound effects on development during embryogenesis, chondrogenesis and differentiation of squamous epithelia. The distribution of the transcripts of the retinoic acid receptor gamma (RAR-gamma) gene has been studied here by in situ hybridization during mouse development from days 6.5 to 15.5 post-coïtum (p.c.). RAR-gamma transcripts are detected as early as day 8 p.c. in the presomitic posterior region. Between days 9.5 and 11.5 p.c., the transcripts are uniformly distributed in the mesenchyme of the frontonasal region, pharyngeal arches, limb buds and sclerotomes. At day 12.5 p.c., RAR-gamma transcripts are found in all precartilaginous mesenchymal condensations. From day 13.5 p.c., the transcripts are specifically localized in all cartilages and differentiating squamous keratinizing epithelia, irrespective of their embryological origin. RAR-gamma transcripts are also found in the developing teeth and whisker follicles. The developmental pattern of expression of the RAR-gamma gene suggests that RAR-gamma plays a crucial role for transducing RA signals at the level of gene expression during morphogenesis, chondrogenesis and differentiation of squamous epithelia.

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.

Studies on cell behaviour in vitro have indicated that the chondroitin sulphate proteoglycan (CSPG) family of molecules can participate in the control of cell proliferation, differentiation and adhesion, but its morphogenetic functions had not been investigated in intact embryos. Chondroitin/chondroitin sulphates have been identified in rat embryos at low levels at the start of neurulation (day 9) and at much higher levels on day 10. In this study we have sought evidence for the morphogenetic functions of CSPGs in rat embryos during the period of neurulation and neural crest cell migration by a combination of two approaches: immunocytochemical localization of CSPG by means of an antibody, CS-56, to the chondroitin sulphate component of CSPG, and exposure of embryos to the enzyme chondroitinase ABC. Staining of the CS-56 epitope was poor at the beginning of cranial neurulation; bright staining was at first confined to the primary mesenchyme under the convex neural folds late on day 9. In day 10 embryos, all mesenchyme cells were stained, but at different levels of intensity, so that primary mesenchyme, neural crest and sclerotomal cells could be distinguished from each other. Basement membranes were also stained, particularly bright staining being present where two epithelial were basally apposed, e.g., neural/surface ectoderms, dorsal aorta/neural tube, prior to migration of a population of cells between them. Staining within the neural epithelium was first confined to the dorsolateral edge region, and associated with the onset of neural crest cell emigration; after neural tube closure, neuroepithelial staining was more general. Neural crest cells were stained during migration, but the reaction was absent in areas associated with migration end-points (trigeminal ganglion anlagen, frontonasal mesenchyme). Embryos exposed to chondroitinase ABC in culture showed no abnormalities until early day 10, when cranial neural crest cell emigration from the neural epithelium was inhibited and neural tube closure was retarded. Sclerotomal cells failed to take their normal pathway between the dorsal aorta and neural tube. Correlation of the results of these two methods suggests: (1) that by decreasing adhesiveness within the neural epithelium at specific stages, CSPG facilitates the emigration of neural crest cells and the migratory movement of neuroblasts, and may also provide increased flexibility during the generation of epithelial curvatures; (2) that by decreasing the adhesiveness of fibronectin-containing extracellular matrices, CSPG facilitates the migration of neural crest and sclerotomal cells. This second function is particularly important when migrating cells take pathways between previously apposed tissues.

Studies on avian and amphibian embryos have revealed a wealth of information about neural crest cell pathways and differentiated fates. While it may be valid to extrapolate this work to interpreting trunk crest cell behaviour in mammals, differences in the cranial region are considerable and direct experimentation on mammalian embryos is essential. This has now begun, by the microinjection of labelled cells into rat embryos in vitro; the new generation of genetic and in situ labelling techniques hold potential for further progress.