Expression patterns of the Ets transcription factors from the PEA3 group during early stages of mouse development.

erm, er81 and pea3 are three related genes that define a novel Ets-related subfamily of transcription factors. The expression patterns of these genes has been previously characterized in the mouse from embryonic day (E) 9.5 to birth (Oncogene 15 (1997) 937). In this study, we report differential expression patterns of the PEA3 group genes during early mouse post-implantation development. erm and pea3 expression patterns were partly overlapping. erm was activated prior to pea3 in the distal tip of the embryonic epiblast but, at primitive streak-stages, both genes were coexpressed in the posterior region of the embryo in epiblast, primitive streak and adjacent mesoderm. Similar erm and pea3 expression patterns were seen later in posterior neural plate, presomitic and lateral mesoderm, mesonephros, and tail bud. Only erm, however, was expressed in specific brain regions corresponding to prospective midbrain and ventral forebrain. erm was also strongly expressed as early as E8 in the developing branchial region, especially at the level of branchial pouches, whereas pea3 transcripts appeared later in frontonasal and first arch mesenchyme. er81 transcripts were not detected prior to E9.0-9.5, suggesting that this gene may not be involved in early developmental events.

The Ets family member Erg gene is expressed in mesodermal tissues and neural crests at fundamental steps during mouse embryogenesis.

The Erg gene belongs to the Ets family encoding a class of transcription factors. To gain new insight on the in vivo functional specificity of the Erg gene within the wide Ets family, we used in situ hybridization to determine its expression pattern during murine embryogenesis. We found that the Erg gene expression predominates in mesodermal tissues, including the endothelial, precartilaginous and urogenital areas. A specific Erg gene expression was also identified in migrating neural crest cells. A comparison with Fli-1, the most closely Erg-related gene, revealed that both gene expressions partially overlap, suggesting that they may contribute to related functions in these tissues. Like other Ets family genes, Erg seems involved in several fundamental developmental steps in murine embryogenesis, including epithelio-mesenchymal transition, cell migration, settlement and differentiation.

The expression of an Ets1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells.

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.

Differential expression patterns of the PEA3 group transcription factors through murine embryonic development.

ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.

Expression of the estrogen-related receptor 1 (ERR-1) orphan receptor during mouse development.

We studied the expression of the estrogen-related receptor 1 (ERR-1) during mouse embryonic development. ERR-1 is expressed at very early stages in ES cells and at E8.5 in the mesodermal cells of the visceral yolk sac. ERR-1 continues to be expressed later in mesodermal tissues and particularly in heart and in skeletal muscles. This expression persists during all the embryonic development and in adult stage. ERR-1 transcripts level increases during muscle differentiation. Accordingly, we show that ERR-1 expression increases during the myoblast to myotube transition in differentiating C2 myoblastic cells. ERR-1 has also been detected in the nervous system during embryonic development. At E10.5, a high level of ERR-1 transcripts can be observed in differentiated cells of the intermediate zone of the spinal cord which also suggests a role of ERR-1 in the differentiation of the nervous system. The same is observed in the telencephalon vesicules at E13.5. Later, at E15.5 and E17.5, expression persists in the spinal cord but decreases dramatically in the central nervous system. Moreover, ERR-1 expression increases during skin formation and is detected in the stratum spinosum which contains differentiated Malpighian cells. Finally, we also observed ERR-1 in endodermal derivatives such as the epithelium of intestine and urogenital system. The DNA target of ERR-1 has been identified to be the SF-1/FTZ-F1 responsive element (SFRE) and we show in this paper that SF-1/FTZ-F1 and ERR-1 bind to and activate transcription independently through the SFRE element. Our study suggests that ERR-1 may be implicated in numerous physiological or developmental functions, particularly in the muscle, the central and peripheral nervous system and the epidermis. Interestingly, in these various systems ERR-1 expression is correlated with post-mitotic cells stage, suggesting that ERR-1 may play a role in the differentiation process.

The ETS1 transcription factor is expressed during epithelial-mesenchymal transitions in the chick embryo and is activated in scatter factor-stimulated MDCK epithelial cells.

In embryos and in human tumors, the expression of the ETS1 transcription factor correlates with the occurrence of invasive processes. Although this was demonstrated in cells of mesodermal origin, the expression of ETS1 was not detected in epithelial cells. In the present study, we show that during early organogenesis in the chick embryo, ETS1 mRNA expression was transiently induced in epithelial structures, during emigration of neural crest cells and dispersion of somites into the mesenchymal sclerotome. In contrast, the expression of ETS1 was not detected in situations where epithelial layers stayed cohesive while forming a new structure, such as the dermomyotome forming the myotome. The involvement of ETS1 in epithelial cell dissociation was examined in MDCK epithelial cells stimulated by scatter factor/hepatocyte growth factor (SF/HGF), a potent inducer of cell dissociation and motility. SF/HGF was found to stimulate ETS1 mRNA and protein expressions, and these increases coincided with the dispersion of cells and the expression of protease mRNAs, such as urokinase-type plasminogen activator and collagenase, but not with the protease inhibitor, plasminogen activator inhibitor type 1. Furthermore, we showed that SF/HGF was able to induce a transcriptional response involving ETS1 by using artificial as well as cellular promoters, such as the urokinase-type plasminogen activator and collagenase 1 promoters, containing RAS-responsive elements with essential ETS-binding sites. These data demonstrate expression of ETS1 during epithelial-mesenchymal transitions in the developing embryo and show that ETS1 can act as a downstream effector of SF/HGF in MDCK epithelial cells. Taken together, these data identify ETS1 as a molecular actor of epithelia cell dissociation.

The ERR-1 orphan receptor is a transcriptional activator expressed during bone development.

We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.

Hydrocortisone modulates the expression of c-ets-1 and 72 kDa type IV collagenase in chicken dermis during early feather morphogenesis.

At the onset of chicken feather morphogenesis, dermal cells migrate along bundles of collagen fibers to colonize areas where bud outgrowth takes place. Chicken embryos treated with hydrocortisone during the critical phase of dermal rearrangement show featherless skin areas in which the dermis exhibits an increase of interstitial collagen. We had previously demonstrated that c-ets-1 is a nuclear transcription factor expressed in the dermis at the beginning of feather morphogenesis. Here we study, by in situ mRNA hybridization, the expression of c-ets-1 in the dermis of chicken embryos treated with hydrocortisone. We found that, among the two distinct products (p54 and p68) encoded by the chicken c-ets-1, the expression of the p68 product increased while expression of p54 decreased after hydrocortisone treatment. Since Ets-1 regulates matrix-metalloproteinases genes, we analyzed the expression of the 72 kDa type IV collagenase in both normal and hydrocortisone-treated embryos. We demonstrated that 72 kDa type IV collagenase mRNA expression decreased in the dermis after hydrocortisone treatment and that its expression correlated with that of p54c-ets-1. Taken together, these results indicate that hydrocortisone modulates c-ets-1 expression. In addition, they raise the interesting possibility that c-ets-1 might be involved in an altered pattern of feather development mediated by the accumulation of collagen due to a decrease in collagenase activities.

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level.

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.

Expression of c-ets-1 and uPA genes is associated with mammary epithelial cell tubulogenesis or neoplastic scattering.

Although the inductive interactions which trigger epithelial morphogenesis have been extensively described, little is known about the transcription factors involved in these processes. During mammary gland morphogenesis, we report the expression of the transcription factor c-ets-1 and one of its target genes uPA in mesenchymal cells during early stages of epithelial invasion, and later in epithelial cells themselves. In vitro studies show that both c-ets-1 and uPA mRNAs can be induced in cultured normal mammary epithelial cells in response to medium conditioned by MRC-5 fibroblasts. In contrast, invasive tumorigenic cell lines from the mammary epithelium express constitutively c-ets-1 and uPA while non-invasive tumorigenic cells do not. In three dimensional co-cultures in collagen gels, a preferential expression of these genes is detected in epithelial cells migrating through the gel either at the tips of normal ducts or in cancerous cells which are scattering. These genes are also expressed in the neighboring fibroblasts. In MRC-5 fibroblasts, conditioned media from tumorigenic epithelial cells induce more efficiently c-ets-1 and uPA mRNA accumulation than do conditioned medium from normal cells. These results suggest that epithelial-mesenchymal interactions trigger c-ets-1 and uPA expression in both compartments during mammary gland morphogenesis. The expression of the genes correlates with invasiveness of epithelial cells irrespective of their being normal or cancerous.

Experimental evidence for FGF-1 control of blastema cell proliferation during limb regeneration of the amphibian Pleurodeles waltl.

During regeneration, blastema cell proliferation depends on several different factors which are, as yet, not fully understood. Previous studies showing the presence of FGF-1 and FGF receptors in the limb blastema make FGF-1 a potentially important molecule for limb regeneration but they do not demonstrate that this factor is active during the process. In the present study, we have first of all confirmed the presence of FGF-1 in limb blastemas of the amphibian Pleurodeles waltl using immunochemistry. Second, we provide evidence in vivo that FGF-1 controls blastema cell proliferation by using different reagents which interfere with FGF activity. Sulfated polysaccharides which bind FGFs, such as heparin, iota-carrageenan and pentosan polysulfate, are able to decrease both 3H-thymidine incorporation and the mitotic index in regeneration blastemas. In addition, suramin which inhibits the binding of growth factors to their receptors, induces the same effect. The presence of receptors in blastema cells is also demonstrated by using the FGF-saporin complex which is known to bind to FGF receptors and to kill cells bearing these receptors. This complex decreases the mitotic index in mesenchyme, while saporin alone did not influence cell proliferation. Finally, results obtained using a neutralizing monoclonal antibody against FGF-1 which was able to specifically reduce blastema cell proliferation, suggests that FGF-1 plays an important function in limb regeneration.

Mesodermal expression of the chicken erg gene associated with precartilaginous condensation and cartilage differentiation.

The ets gene superfamily encodes a class of transcription factors that bind to a purine rich sequence through a 85 amino-acid ETS domain. Among them, the human erg gene has been found to be involved in Ewing's sarcoma, primitive neurectodermal tumour of childhood and acute myeloid leukaemia. Nevertheless, little is known about human erg expression. Northern blot analyses have shown a human erg expression restricted to few cell lines and thymus, but the status concerning expression during development remains unknown probably because no homologue of this gene has yet been isolated and studied in other vertebrates. We thus choose to clone the chicken erg gene (ck-erg) and to study its expression during chicken development. We obtained a bona fide clone of ck-erg and defined the transcriptional modulating properties of its product. The ck-Erg protein acts as a transcriptional activator through a conventional consensus ETS binding site. Northern blot studies on various chicken tissues, in situ analyses and comparison with the well-characterised c-ets-1 expression show that ck-erg is expressed in mesoderm- and, to a lesser extent, in ectoderm-derived tissues. During chicken development, two salient features could be observed. From stage E1 to E3.5, ck-erg expression was widely distributed in mesodermal derivatives and neural crest, resembling c-ets-1 expression. However, by E6, the expression of ck-erg exhibited, unlike c-ets-1, a drastically new and strong signal in precartilaginous condensation zones and cartilaginous skeletal primordia. These stages are the first steps of bone formation during skeletal elaboration. Our results show for the first time a possible specific involvement of ck-erg in cartilage morphogenesis.

Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis.

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.

Rev-erb beta, a new member of the nuclear receptor superfamily, is expressed in the nervous system during chicken development.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily in the chicken. This new gene, called Rev-erb beta, exhibits strong homologies with the Rev-erb alpha/ear-1 orphan receptor gene, which partially overlaps the thyroid hormone receptor alpha gene in opposite orientation. We demonstrate that both Rev-erb alpha and Rev-erb beta genes are conserved in their C and E domains. Rev-erb beta binds to DNA as a monomer and recognizes the same binding motif as the alpha gene product. The Rev-erb beta gene product does not interact with retinoid X receptors, as revealed by gel shift experiments. In situ hybridization experiments show that Rev-erb beta is expressed in the central and peripheric nervous system, spleen, and mandibular and maxillar processes, as well as in blood islands. During embryonic development, we noticed a striking specific distribution of Rev-erb beta transcripts in the notochord at 24 h and later on, in the floor plate of the neural tube. We propose that Rev-erb beta may play an important role in the complex network of inductive signals, which control neuron differentiation.

Does the transcription factor c-ets1 take part in the regulation of angiogenesis and tumor invasion?

The c-ets1 proto-oncogene encodes a transcription factor that binds a GGAA/T purine rich core DNA sequence. During normal as well as pathological development, the expression of c-ets1 is associated with the occurrence of invasive processes, either in invading cells or in the invaded tissue. Cellular regulatory sequences responsive to the c-Ets1 proteins include a urokinase-type plasminogen activator (u-PA) gene enhancer, the stromelysin-1 and the collagenase-1 gene promoters. Since invasive processes are thought to require the remodeling of the extra-cellular matrix, we investigate the relationships between c-Ets1 and the expression pattern of transcripts encoding these matrix degrading proteases, in embryos and in solid tumors.

Expression of the transcription factor c-Ets1 correlates with the occurrence of invasive processes during normal and pathological development.

The protein encoded by the c-ets1 proto-oncogene is a member of a new family of transcription factors. Cellular regulatory sequences responsive to the c-Ets1 proteins include a urokinase-type plasminogen activator (uPA) gene enhancer, the stromelysin 1 and the collagenase 1 gene promoters. During normal as well as pathological development, the expression of c-ets1 is associated with the occurrence of invasive processes, either in invading cells or in the invaded tissue. Since these invasive processes are thought to require the remodeling of the extracellular matrix, we investigate the relationships between c-Ets1 and the expression patterns of transcripts encoding the matrix-degrading proteases uPA, stromelysin 1 and collagenase 1, in embryos and in solid tumors.

Involvement of the proto-oncogene c-ets 1 and the urokinase plasminogen activator during mouse implantation and placentation.

Many of the Ets proteins have been shown to be transcription activators. In vitro, Ets 1 proteins are involved in the transcriptional induction of genes such as stromelysin 1, collagenase 1 or urokinase type plasminogen activator, which are proteases responsible for extracellular matrix degradation. In vivo, c-ets 1 is expressed in a wide variety of embryonic tissues in migrating cells, especially in endothelial cells during blood vessel formation. C-ets 1 is also expressed in stromal cells of invasive carcinomas. In the present work, we have investigated the expression of both c-ets 1 and u-PA, a putative target gene of the Ets 1 proteins, within a biological model which includes both embryonic and tumoral aspects. Implantation and placentation of the mouse embryo display migration of the trophoblastic cells, which invade the stroma of the uterine endometrium and trigger the establishment of a new vascular frame. Using in situ hybridization, we show that the overlapping of expression of c-ets 1 and u-PA is restricted to some maternal cell populations from the invasive front and to the endothelial cells of the endometrial vasculature. C-ets 1 is never expressed in trophoblasts. In contrast, u-PA expression in trophoblasts is strong and coincides with the embryo invasive phase. In the embryo proper, c-ets 1 displays a spatio-temporal expression pattern similar to that described in the chick embryo. Until E 10.5, u-PA is expressed neither in embryonic nor in extra-embryonic structures. The respective roles of c-ets 1 and u-PA and their relationship during mammalian placentation are discussed.

Hydrocortisone perturbs the cell proliferation pattern during feather morphogenesis: evidence for disturbance of cephalocaudal orientation.

In this study, we have monitored the spatial distribution of S-phase cells during successive stages of normal feather morphogenesis using the specific marker BrdU. We also disturbed the development program by administration of hydrocortisone on the chorioallantoic membrane of 6.5-day chick embryos and examined the resulting pattern of BrdU incorporation. Our results show that a specific spatio-temporal pattern of cell proliferation occurs during successive stages of feather development and that this pattern accounts for the growth of feather buds according to the cephalocaudal orientation. Our experimental analysis showed that the stage-dependent alteration of feather morphogenesis (as shown by Züst, Ann. Embryol. Morphogen. 4, 1971 and confirmed by Démarchez et al., Dev. Biol. 106, 1984), is based on a stage-dependent alteration of the proliferation pattern in the epidermis. Forty-eight hours after treatment, non-induced epidermis ceases DNA synthesis and is unable to form placodes. Induced epidermis at the placodal and dermal condensation stages fails to produce the cohorts of S-phase cells responsible for the caudal outgrowth and the slanting shape of the buds. These young buds display anarchic proliferation in the whole epidermis possibly resulting in the appearance of "curly" feathers. Together, these results show the importance of the spatial pattern of ectodermal and mesodermal cell proliferation during the normal feather morphogenesis. Moreover, they corroborate the particular role of epidermis both in the establishment of feather rudiments and in the cephalocaudal orientation of the feathers.

The relationship between cell proliferation and the transcription of the nuclear oncogenes c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo.

We have described the expression of three nuclear protooncogenes, c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo. In parallel with the expression patterns obtained by in situ hybridization, we have mapped the spatial distribution of S-phase cells by monitoring the incorporation of 5-bromodeoxyuridine. We do not detect c-myc or c-myb transcripts during the early stages when S-phase cells are scattered in the dermis and in the epidermis. Rather c-ets-1 transcripts are abundant in the dermal cells which divide and accumulate under the uniform epidermis. At the onset of the formation of the feather bud, cells within each rudiment cease DNA replicative activities and c-myc transcripts are detected both in the epidermis and in the underlying dermis. This expression precedes the reentry into the S phase. The transcription of c-myb, which has been previously tightly linked to hemopoietic cells is also detected in the developing skin. This expression is essentially located in proliferating epidermal cells on and after the beginning of feather outgrowth. As feather outgrowth proceeds, the distribution of c-myc and c-myb transcripts is restricted to the highly proliferating epidermis. In contrast c-ets-1 transcripts are never detected in the epidermis. During the later stages of skin morphogenesis, the transcription of c-ets-1 is restricted to the endothelial cells of blood vessels, as previously described. We suggest that the differential expression of these nuclear oncogenes reflects the activation of different mitotic controlling pathways during the development of the skin.

Specific effects of retinoic acid on the skeletal morphogenesis of the 11-day mouse embryo forelimb bud in vitro.

Using our improved method for culturing 11-day mouse forelimb buds in vitro, we have investigated the effects of a local application of all-trans-retinoic acid (RA) on growth, cartilaginous differentiation and skeletal patterning in the mammalian limb bud. Carrier implants of catgut impregnated with DMSO or various doses of RA in DMSO were inserted at the apex of the buds in the proximo-distal axis just beneath the apical ectodermal ridge. After 6 days of culture, cartilaginous skeletons were stained and explants were processed for morphological analysis and quantitative study using computerized optical image analysis. Buds treated with low doses of RA exhibited stimulated growth and chondrogenesis. Moreover, hypertrophied and fused metacarpals were seen within explants treated with the lowest dose. High doses strongly inhibited growth and skeletal morphogenesis. An intermediate dose sustained cartilaginous differentiation at the same level as low doses, but concomitantly disturbed the skeletal pattern. These results are discussed considering reported RA effects on other experimental systems including avian limb bud as an in vivo model or cell cultures as an in vitro simplified model.