Pattern formation is the process by which undifferentiated cells divide and differentiate to generate complex tissues and organs. In plants, pattern formation begins in embryogenesis and continues post-embryonically with the function of the meristems. microRNAs (miRNAs), which are small regulatory RNAs that repress gene expression, are involved in a variety of patterning processes in plants, including the formation and function of the meristems and establishment of polarity. For example, regulation of the class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors by miR165/6 is not only involved in the formation and function of the meristems, but also in polarity establishment in the leaf, and in axial patterning during embryogenesis. To gain a better understanding of the role of miRNAs in embryonic patterning, I investigated the tissue-specific functions of the miRNA biogenesis protein SERRATE (SE), which is required for the regulation of the HD-ZIP IIIs via miR165/6. By expressing SE in various domains in se-5 null mutant embryos, I revealed that although SE is expressed throughout the embryonic body, tissue-specific expression of SE from either the upper or lower tier of the embryo is sufficient for correct patterning. This observation suggests a SE-dependent non-cell autonomous and bi-directional mechanism that influences patterning in Arabidopsis embryos. Furthermore, through a suppressor screen of a se-3 loss-of-function mutant allele, I identified mutants in genes that likely function upstream of SE, and downstream or in parallel of the HD-ZIP IIIs. One of those se-3 suppressors is likely to be a mutant in the BELL homeobox gene POUND-FOOLISH (PNF).

Les voies de signalisation Wnt jouent un rôle important dans divers processus de l’embryogenèse des métazoaires. Par exemple, les signaux WNT sont requis pour l’établissement de la polarité cellulaire, les mouvements de tissus ou la mise en place de la polarité antéropostérieure. La signalisation Wnt tire son nom des protéines WNTs qui sont des protéines secrétées capables de signaler via leurs récepteurs membranaires FRIZZLED (FZD). Une interaction WNT-FZD peut déclencher au moins trois cascades de signalisation distinctes, selon le contexte cellulaire et les couples WNT-FZD impliqués. Chez l'homme, la dérégulation des voies de signalisation Wnt conduit, au cours du développement, à des maladies congénitales, ou chez l'adulte, à des cancers. La compréhension de la diversité numéraire des wnt et des fonctions biologiques qu’ils exercent représente donc un challenge contemporain qu’il est nécessaire de relever, non seulement au titre de l’acquisition de nouvelles connaissances, mais aussi en vue du développement de nouvelles approches à but de diagnostic ou de thérapie. Lors de ma thèse, je me suis dans un premier temps attaché à décrire les patrons d’expression temporels et spatiaux de l’ensemble des gènes wnt et fzd présents chez l’oursin Paracentrotus lividus. L’oursin, comme l’homme, appartient au phylum des deutérostomiens. De plus, son développement est simple et ses embryons se prêtent à l’expérimentation, du fait de multiples approches disponibles pour ce modèle. Par ailleurs, contrairement au lignage des vertébrés, celui de l’oursin n’a pas subi de duplications complète de génome, exhibant ainsi la même diversité de familles de gènes, mais avec moins de redondance au sein de ces dernières, ce qui facilite leur analyse. Etudier les mécanismes qui régissent l'embryogénèse de l’oursin permet donc d’apporter des informations quant aux mécanismes régissant celle des deuterostomes. Mes résultats mettent en évidence que les ligands wnt et les récepteurs fzd sont exprimés de façon dynamique au cours de l’embryogenèse de P. lividus. Fait intéressant, jusqu’à la gastrulation, les gènes codant les récepteurs FZD, présentent des patrons d’expressions distincts, leur somme couvrant néanmoins la très vaste majorité des territoires embryonnaires. Ainsi, cette étude a permis d’établir un catalogue des compatibilités spatio-temporelles potentielles entre les protéines WNT et FZD durant l’embryogenèse d’un deutérostomien, ainsi que des hypothèses quant aux rôles possibles jouées par ces protéines durant ce processus. Dans un second temps, je me suis intéressé à retracer l’évolution des protéines WNT, et à déterminer s’il existait une fonction intrinsèque ancestrale des WNT qui soit conservée, parmi les métazoaires. Pour cela, j’ai d’une part analysé la composition des répertoires wnt de dix-sept espèces de métazoaires, la séquence primaire des protéines qu’ils encodent, la position relative des wnt au sein de leurs génomes respectifs et, pour certaines espèces, l’environnement génomique des wnt. D’autre part, j’ai contribué à vérifier si trois ligands WNT d’éponge et une séquence ancestrale, calculée à partir d’un échantillon de séquences de métazoaires, étaient capable d’activer les voies de signalisation Wnt lorsqu’exprimées chez trois animaux planulozoaires (cnidaires et bilatériens). Les données obtenues au cours de cette étude ont permis d’établir un modèle de diversification de la famille de gènes wnt chez les métazoaires et ont révélé l’existence de fonctions intrinsèques conservées parmi les ligands WNT. / The Wnt signaling pathways play crucial roles during several processes of metazoan embryogenesis. For instance Wnt signaling is involved in morphogenetic movements as well as cell polarity and anteroposterior polarity establishment. Wnt signaling takes its name from the WNT proteins, which are secreted molecules that signal through their cognate receptors, the FRIZZLED (FZD) proteins. A WNT-FZD interaction may trigger several distinct intracellular signaling cascades, depending on the cellular context and the WNT-FZD couples involved. In humans, a deregulation of WNT signaling during embryonic development or adult life leads to congenital diseases and cancers, respectively. Understanding the great diversity of wnt genes and their biological functions is thus a modern-day challenge, which needs to be addressed, not only to foster general knowledge, but also to allow the identification of novel therapeutic targets and approaches towards future new medical applications.In the course of my Ph.D., I first aimed at describing the spatial and temporal expression patterns of all wnt and fzd genes present in the sea urchin, Paracentrotus lividus. Like humans, sea urchins belong to the deuterostome phyla. In addition, the early development of the sea urchin is relatively simple and is easy to interfere with, thanks to the experimental approaches available for this model. Moreover, the sea urchin lineage did not undergo whole genome duplications, unlike humans, exhibiting the same diversity of gene families, but with less redundancy within the subfamilies, thereby facilitating functional analyses. Thus, studying the mechanisms that drive sea urchin embryogenesis represent a valuable asset to provide insights into the conserved mechanisms controlling the development of deuterostomes. My results show that wnt and fzd genes are expressed in a dynamic fashion throughout P. lividus embryogenesis. Interestingly, until the gastrulation, the genes encoding FZD receptors are expressed in distinct territories that, taken together, cover almost the entire embryo. Accordingly, this work allowed me to establish a catalog of possible WNT-FZD couples likely to form during the embryogenesis of a deuterostome animal, based on the compatibility of their respective spatiotemporal distribution, and to further provide insights into the potential biological functions of these WNT and FZD proteins during this process. The second goal of my Ph.D. was to reconstruct the evolution of the WNT proteins and to determine whether an ancestral intrinsic function of WNTs is conserved within metazoans. To this end, I analyzed the composition of the wnt repertoires of seventeen metazoan species, the primary sequences of the WNT proteins they encode, the relative positions of the wnts within their respective genomes, and, for selected species, the genomic environment of their wnts. In addition, I investigated whether three sponge WNT ligands and an ancestral WNT sequence, which was calculated from a sample of metazoan WNT sequences, are able to trigger Wnt signaling pathways, when expressed in three planulozoan (cnidarian and bilaterian) species. The data obtained during this study allows me to propose a model for the diversification of wnt genes in metazoans and revealed conserved intrinsic functional capabilities for the WNT ligands.

Le développement des plantes repose sur le fonctionnement des méristèmes qui sont à la base de la production des organes durant toute la vie post-embryonnaire de la plante. Ce développement repose également sur la définition de frontières d'une part entre les méristèmes et les organes et d'autre part entre les organes. Les gènes NAM/CUC3 de la famille de facteur de transcription à domaine NAC et leur microARN régulateur miR164, jouent un rôle important dans de tels mécanismes chez les eudicotylédones. En plus de leur rôle dans la définition de frontières, ces gènes sont nécessaires à la formation du méristème apical durant l'embryogenèse, et au contrôle de l'architecture de la plante et de ses organes. Sur la base des connaissances acquises chez les eudicotylédones, se posait la question de la conservation de ces gènes et de leur régulation ainsi que leur implication dans le contrôle du développement chez les palmiers (Arecales, Arecaceae) et, de façon plus large, chez les monocotylédones. Dans ce contexte, trois gènes similaires aux gènes NAM/CUC3 ont été isolés chez le palmier à huile (Elaeis guineensis L.), EgNAM1, EgNAM2 et EgCUC3. Ces gènes sont exprimés dans les tissus méristématiques végétatifs et reproducteurs. Notre analyse a révélée une conservation du module de régulation NAM-miR164 chez cette espèce et une divergence en terme de domaine d'expression entre monocotylédones et eudicotylédones, qui pourrait être associée à des différences majeures des régions cis-régulatrices des gènes NAM. L'analyse des profils d'expression des gènes NAM/CUC3 au cours de l'embryogenèse somatique précoce indique des similarités entre le palmier à huile, le maïs et le riz, ainsi qu'une conservation de la régulation via l'auxine comme observé chez Arabidopsis thaliana. Cependant, même si une régulation post-transcriptionnelle via miR164 a été détectée au cours de l'embryogenèse somatique, la répression par l'auxine semble essentiellement transcriptionelle. / Plant development depends on functioning of meristems, which are at the base of organ production during the post-embryonic phase. The development depends also on the definition of boundaries between meristem and primordia but also between organs. NAM/CUC3 genes belonging to the NAC domain transcription factor family and their microRNA regulator miR164 play an important role in these mechanisms in eudicot species. In addition to their role in boundary definition, they are involved in the establishment of the shoot apical meristem during embryogenesis and in the control of the plant and organ architectures. On the basis of data from eudicot species, the conservation of these genes and their regulation, and their involvement in meristem functioning in palm species (Arecales, Arecaceae) and more generally in monocot species was still an open question. In this context, three NAM/CUC3-related genes have been isolated in oil palm (Elaeis guineensis L.), EgNAM1, EgNAM2 and EgCUC3. Theses genes are expressed in both vegetative and reproductive meristematic tissues. Our analysis revealed the conservation of the NAM-miR164 regulatory module in this species and a divergence in term of expression pattern between monocots and eudicots, which may be related to differences in cis-regulatory regions of NAM genes. In contrast the expression pattern of NAM/CUC3 during somatic embryogenesis indicates similarity in the timing of expression between oil palm, maize and rice and also a conservation of the auxin-dependent regulation of NAM genes during this developmental phase as observed in Arabidopsis thaliana. However, even if miR164-dependent post-transcriptional regulation of NAM genes was detected during somatic embryogenesis, auxin-dependent repression seems to be essentially through transcriptional regulation.

Protea cynaroides L. (King Protea) is a well known cutflower. Seeds and stem cuttings are commonly used to propagate P. cynaroides. However, the success rate and rooting rate of seeds and cuttings, are inconsistent and slow. The potential of in vitro propagation as an alternative method to produce P. cynaroides plantlets was investigated. In vitro studies consisted of in vitro germination of mature zygotic embryos, micrografting and direct somatic embryogenesis of zygotic embryos and excised cotyledons. In the germination study, temperature was the most important factor in obtaining a high germination percentage. Alternating temperatures of 21±2ºC/12±2ºC (light/dark) was suitable for germination and over 90% of embryos germinated, while the germination percentage of embryos at 25±2ºC was poor. Plantlets were successfully established in ex vitro conditions when planted in a peat/coir/sand mixture. Micrografting of P. cynaroides was done by grafting microshoots (microscion), which was taken from in-vitro-established nodal explants, onto roots of decapitated in-vitro-germinated seedlings. After the graft union formed, buds on the microscion sprouted. A protocol to induce direct somatic embryogenesis was developed. Direct somatic embryogenesis was achieved on both P. cynaroides mature zygotic embryos and excised cotyledons. The addition of auxins such as NAA and 2,4-D singly or in combination with TDZ, BAP or kinetin suppressed the formation of somatic embryos. Formation of somatic embryos was observed in medium lacking growth regulators. Germination of somatic embryos was highest in medium containing GA3. The roles of starch and phenolic compounds in the rooting of P. cynaroides cuttings were also studied. Starch and total soluble phenol analyses results revealed a positive correlation between high root formation and increased starch and phenolic content. NMR and MS analyses identified high amounts of 3,4-dihydroxybenzoic acid in stems of P. cynaroides. In vitro bioassay showed that 3,4-dihydroxybenzoic acid stimulated and inhibited root growth of P. cynaroides explants, depending on the concentration. A link was made between the endogenous concentration levels of 3,4-dihydroxybenzoic acid and rooting of P. cynaroides stem cuttings. Findings of this study contribute towards a better understanding of the roles starch and phenolic compounds play in the rooting of P. cynaroides. / Thesis (PhD (Horticulture))--University of Pretoria, 2006. / Plant Production and Soil Science / unrestricted

L’adénosine extracellulaire appartient à la voie de signalisation purinergique et réguledivers processus physiologiques à travers l’activation de ses récepteurs spécifiques (adora).La disponibilité de cette purine dans l’espace extracellulaire est régulée par plusieurs ectoenzymesassurant sa production ou sa dégradation, mais également par des transporteurs denucléosides permettant son passage à travers la membrane. Chez l’adulte, le rôle del’adénosine est assez bien connu. Cependant, l’implication de cette purine au cours del’embryogenèse reste très peu étudiée. Pourtant, un excès d’adénosine dans les phasesprécoces du développement est létal chez la souris et l’oursin, démontrant l’importance de larégulation des concentrations de cette molécule de signalisation lors de l’embryogénèse. Lebut de ma thèse est de comprendre le rôle de l’adénosine au cours de l’embryogenèse enutilisant l’amphibien xénope. En effet, ce modèle a permis de mettre en évidence in vivol’implication de l’ADP au cours du développement de l’oeil chez les vertébrés. La premièrepartie de ce projet a permis de caractériser les acteurs de la voie de signalisation del’adénosine chez le xénope afin d’établir la première carte comparative de leur profild’expression embryonnaire. Cette partie a également permis de mettre en avant laphosphatase alcaline alpl pour son profil d’expression particulier, dans le rein et la rétine. Laseconde partie s’est focalisée sur l’étude fonctionnelle de cette enzyme. Les expériences deperte de fonction montrent son implication lors de la formation de ces deux tissus. / Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development.

Experiments were carried out with the objectives of developing propagation protocols for Uapaca kirkiana and Pappea capensis tree species of southern Africa, and evaluating the graft compatibility within U. Kirkiana tree clones, provenances and species. Reverse phase high performance liquid chromatography (RP-HPLC), Folin-Ciocalteau reagent, fluorescence microscopy and callus fusion methodologies were used to diagnose graft compatibility. Results indicated that U. Kirkiana culture asepsis was achieved with 0.1% w/v mercuric chloride HgCl2) and using pre-conditioned grafted trees. Sodium hypochlorite (NaOCl) improved P. Capensis seed asepsis and germination, and discarding floating seeds improved germination. Murashige and Skoog (MS) medium with 2.0 mg l-1 benzylaminopurine (BAP) and 0.3 mg l-1 casein hydrolysate (CH) was superior in shoot multiplication and 0.5 mg l-1 indole-3-butyric acid (IBA) for rooting of P. Capensis microshoots. For somatic embryogenesis, three quarter strength MS medium with 0.05 mg l-1 thidiazuron (TDZ) and 0.3 mg l-1 CH, or 0.2 mg l-1 BAP with 0.3 mg l-1 CH, were effective in germination of P. Capensis somatic embryos. For U. Kirkiana lateral shoot explants, shoot multiplication was superior on three quarter strength MS medium with 0.1 mg l-1 BAP and 0.3 mg l-1 CH. Rooting of micro-cuttings (36%) was achieved on ½ MS with 2.5 mg l-1 IBA. RP-HPLC, fluorescence microscopy and callus fusion studies showed that phenolic compounds play a major role in U. Kirkiana graft incompatibility. Less graft compatible combinations showed an increase in phenol deposits above the union and graft incompatibility was more pronounced above the union than below the union. Proliferation of parenchymatous tissues was better below the union than above the union. Fluorescence microscopy showed presence of flavonoids and polymers above the union of less graft compatible combinations. The chromatograms showed that ferulic acid was abundant and responsible for wood discolouration. The chromatograms also isolated ρara-coumaric acids which were predominant above the union of the less compatible combinations. Therefore, ρara-coumaric acids, flavonoids and polymers were implicated in graft incompatibility of U. kirkiana trees. Copyright / Thesis (PhD)--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted

Nous proposons un modèle de l'embryogenèse de la moelle épinière considérée comme un système dynamique constitué d'unités comportementales (les cellules). Pour que cet organe soit fonctionnel, il faut que les neurones des différents types soient mis en place au cours de l'embryogenèse, avec le bon nombre de neurones du bon type au bon endroit. Cette mise en place résulte d'un processus dynamique au cours duquel les progéniteurs neuronaux prolifèrent dans les proportions adéquates avant de se différencier en neurones spécialisés. Les données expérimentales disponibles donnent l'évolution des populations de progéniteurs et de neurones, et la balance globale entre prolifération et différenciation. Un premier modèle est énoncé à l'échelle collective pour rendre compte de la dynamique observée sur toute la durée du processus, avec une évolution de la balance prolifération / différenciation ajustée sur les données expérimentales. Un second modèle introduit une différenciation comportementale des progéniteurs sous la forme d'une perte de leur capacité proliférative. Ce changement comportemental, de type tout-ou-rien à l'échelle cellulaire, se traduit à l'échelle collective par une transition continue de la balance prolifération / différenciation telle qu'observée expérimentalement. Enfin, nous explorons un raffinement de ce modèle où ce changement comportemental est gouverné par l'état du système (rétro-contrôle). Nous examinons les différentes possibilités de rétro-contrôles et nous retenons celle qui rend le mieux compte de la dynamique collective observée. En perspective, nous proposons des pistes pour intégrer la dimension spatiale du phénomène, et pour la prolongation de ce travail vers la modélisation du développement du cortex cérébral. / We consider the developing spinal cord as a dynamical system made of behavioral units (cells). For the adult spinal cord to be functional, different neurons must be of the right kind at the right place. They are issued from a dynamical process of proliferative and differenciating neural progenitors, with a fine control of the balance between proliferation and differentiation. Some experimental data are available for the evolution of progenitors and neurons population and for how the balance progresses with time. Based on these data, we propose a first model at the collective scale to account for these dynamics all along the process. A second model is proposed at the cell scale which includes a loss of proliferative capacity that progressively concerns more and more progenitors. This behavioural switch at cell scale is reflected by a continous progression of the balance proliferation / differentiation at population scale, as it is experimentally observed. Finally, we introduce a feedback control process so that this progression is under the control of the progenitors and neurons populations. Among the multiple possibilities for this feedback, we point out to the most relevant process. We discuss these findings, and how they can be extended to spatialized dynamics and neocortex development.

La régénération est un mode de développement, qui suite à un stresse physique permet de reformer à l’identique des structures biologiques initialement développer au cours de l’embryogénèse. De plus ce phénomène, plus ou moins important en fonction des organismes, est néanmoins répandu chez les métazoaires, suggérant ainsi une origine monophylogénique. D’où l’hypothèse d’un lien étroit entre la régénération et l’embryogénèse. En me basant sur cette hypothèse j’ai employé comme modèle pendant ma thèse, l’anémone de mer Nematostella vectensis. Ce modèle cnidaire offre effectivement l’opportunité unique de comparer la régénération d’un corps entier, dite extrême, à l’embryogénèse et ainsi étudier leurs liens au niveau moléculaire. Initialement établie entant que modèle d’embryologie permettant d’étudier l’évolution des réseaux de régulation génétique (RRG) orchestrant les moments clé de l’embryogénèse et s’imposer en tant que modèle d’étude de la régénération extrême. Tout d’abord, au cours de ma thèse j’ai participé à caractérisation tissulaire et cellulaire de la régénération de ce model afin d’en établir un répertoire de référence des étapes clés. En employant ce répertoire et le criblage de 80 d’inhibiteur de kinase, j’ai pu identifier plusieurs voies de signalisation régissant différente étape de la régénération, impliquant les MAPKs, JNK et ERK ainsi que plusieurs récepteurs de facteurs de croissances. Notamment ERK a également été décrit dans le processus de gastrulation chez Nematostella, dont j’ai contribué à l’établissement du RRG associé. C’est donc en me basant sur ce RRG et une base de donnée transcriptomic complète de la régénération de ce modèle, que j’ai pu établir le RRG en aval de ERK associé à la régénération. Par cette approche j’ai pu démontrer la relation au niveau moléculaire entre ces processus développementaux et surtout identifier des aspects spécifiques à la régénération. / Regeneration is a developmental process, which allow to regrow missing structures initially develop during embryogenesis, in response to an injury. Although, this ability to regenerate can be more or less dramatic depending on the organism, it is widely spread among metazoan. As such, suggesting a monophyletic origin and a tight link with embryogenesis has also been hypothesized. Based on this hypothesis, I used during my thesis the sea anemone Nematostella vectensis, a cnidarian model offers the unique opportunity to compare, whole body regeneration and embryogenesis to investigate their molecular links. In fact, Nematostella was established as an embryonic model to investigate evolution of gene regulatory network (GRN) underlying key stages e.g. gastrulation, but recently it has been a rising model to study whole body regeneration. I started to my thesis by carefully characterizing hallmarks of Nematostella regeneration starting from tissular to molecular level, establishing a comprehensive regeneration time line. By taking advantage of this tool, in association to the screening of 80 kinases inhibitors, I have identify several signaling pathways regulating various steps of regeneration in Nematostella, including the MAPK ERK, JNK and growth factor receptors. In parallel I participated to the study of ERK role during Nematostella gastrulation and the underpinning (GRN). Which offered a solid groundwork for the comparison with regeneration at the GRN level. Combining a candidate approach based on the embryonic GRN and a global transcriptomic analysis of regeneration, I have been able to bring evidence on the relationship between embryogenesis and regeneration and additionally to identify regeneration specific aspects.

The TALE factors, comprising the pbx and prep/meis gene families, are transcription factors (TFs) vital to the proper formation of anterior anatomical structures during embryonic development. Although best understood as essential cofactors for tissue-specific TFs such as the hox genes during segmentation, the TALE factors also form complexes with nuclear factor Y (NFY) in the early zygote. In zebrafish, Pbx4, Prep1, and NFY are maternally deposited and can access their DNA binding sites in compact chromatin. Our results suggest that TALE/NFY complexes have a unique role in early embryonic development which is distinct from each factor’s independent functions at later stages. To characterize these TALE/NFY complexes, we employed high-throughput transcriptomic and genomic techniques in zebrafish embryos. Using dominant negatives to disrupt the function of each factor, we find that they display similar, but not identical, loss-of-function phenotypes and co-regulate genes involved in transcription regulation and embryonic development. Independently, the TALE factors regulate homeobox genes and NFY governs cilia-related genes. ChIP-seq analysis at zygotic genome activation reveals that the TALE factors occupy DECA sites adjacent to CCAAT boxes near genes expressed early in development and involved with transcription regulation. Finally, DNA elements containing TALE and NFY binding sites drive reporter gene expression in transgenic zebrafish, and disruption of TALE/NFY binding via mutation or dominant negatives eliminates this expression. Taken together, this data suggests that the TALE factors and NFY cooperate to regulate a set of development and transcription control genes in early zygotic development but also have independent roles after gastrulation.

Student Number : 9502128Y - MSc dissertation - School of Anatomical Sciences - Faculty of Science / Retinoic acid, the active metabolite of Vitamin A is known to play a major role in embryonic growth and differentiation during development. It has been shown that either excess or deficiency of retinoic acid during embryogenesis can be teratogenic. In order to study the teratogenic effects of retinoic acid, the aim of the present study was therefore to investigate the effect of all-trans retinoic acid on the migration and fate of neural crest cells in vitro and in vivo. In addition, the study investigated the effect of retinoic acid on the cytoskeletal elements of neural crest cells and on Rac and Rho, two members of the Rho family of GTPases. The neural tubes containing neural crest cells of quail embryos were removed at cranial levels and cultured on fibronectin as a substrate. The neural tubes were cultured in either Dulbecco’s minimal essential medium (DMEM) or in DMEM+Dimethylsulphoxide (DMSO) as controls. In order to test the effect of retinoic acid, the neural tubes were cultured in 10-5M all-trans retinoic acid (RA) which was reconstituted in DMSO. The distance of migration of the cultured quail neural crest cells was measured and compared between the controls and the experimentals. To study the effect of RA on the cell actin cytoskeleton in vitro, cultured neural crest cells were stained with rhodamine phalloidin. In addition, following 24 hours of culture, the quail neural crest cells were brought into suspension and micro-injected into 36 hour-old chick hosts. While the migration of neural crest cells was extensive in the control cultures in vitro, migration was inhibited in the retinoic acid-treated neural crest cells. In addition, retinoic-acid treated neural crest cells showed pigmentation and neuronal processes earlier than did the control neural crest cells. Retinoic acid-treated neural crest cells showed a disarray of the cytoskeletal elements as they were devoid of stress fibres and focal adhesions. In addition, retinoic acid appears to decrease the expression of Rac and Rho of cultured quail neural crest cells. Following micro-injection of cultured control and RA-treated quail neural crest into the cranial region of chick hosts, the control cells populated the beak area, whereas the retinoic acid-treated quail neural crest cells migrated to the retina of the eye, a region they normally do not populate. These results suggest that retinoic acid disturbs the migration of neural crest cells. It appears to do this by affecting the cytoskeletal elements of neural crest cells and the genes that are involved in forming these elements.