Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, which is characterized by cleft palate and severe defects of the skin, is an autosomal dominant disorder caused by mutations in the gene encoding transcription factor p63. Here, we report the generation of a knock-in mouse model for AEC syndrome (p63(+/L514F) ) that recapitulates the human disorder. The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment. These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes. In parallel, a defective stem cell compartment is observed in humans affected by AEC syndrome and in Fgfr2b(-/-) mice. Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation. These findings establish a functional link between FGF signalling and p63 in the expansion of epithelial progenitor cells and provide mechanistic insights into the pathogenesis of AEC syndrome.

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.

Control of pancreatic β-cell mass and function by gluco-incretin hormones : identification of novel regulatory mechanisms for the treatment of diabetes

Summary : Control of pancreatic ��-cell mass and function by gluco-incretin hormones: Identification of novel regulatory mechanisms for the treatment of diabetes The ��-cells of islets of Langerhans secrete insulin to reduce hyperglycemia. The number of pancreatic islet ��-cells and their capacity to secrete insulin is modulated in normal physiological conditions to respond to the metabolic demand of the organism. A failure of the endocrine pancreas to maintain an adequate insulin secretory capacity due to a reduced ��-cell number and function underlies the pathogenesis of both type 1 and type 2 diabetes. The molecular mechanisms controlling the glucose competence of mature ��-cells, i.e., the magnitude of their insulin secretion response to glucose, ��-cell replication, their differentiation from precursor cells and protection against apoptosis are poorly understood. To investigate these mechanisms, we studied the effects on ��-cells of the gluco-incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) which are secreted by intestinal endocrine cells after food intake. Besides acutely potentiating glucose-stimulated insulin secretion, these hormones induce ��-cell differentiation from precursor cells, stimulate mature ��-cell replication, and protect them against apoptosis. Therefore, understanding the molecular basis for gluco-incretin action may lead to the uncovering of novel ��-cell regulatory events with potential application for the treatment or prevention of diabetes. Islets from mice with inactivation of both GIP and GLP-1 receptor genes (dK0) present a defect in glucose-induced insulin secretion and are more sensitive than control islets to cytokine-induced apoptosis. To search for regulatory genes, that may control both glucose competence and protection against apoptosis, we performed comparative transcriptomic analysis of islets from control and dK0 mice. We found a strong down-regulation of the IGF1 Rexpression in dK0 islets. We demonstrated in both a mouse insulin-secreting cell line and primary islets, that GLP-1 stimulated IGF-1R expression and signaling. Importantly, GLP-1induced IGF-1R-dependent Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism. We further showed that activation of IGF-1R signaling was dependent on the secretion of IGF-2 and IGF-2 expression was regulated by nutrients. Finally, we demonstrated that the IGF-Z/IGF-1R autocrine loop was required for GLP-1 i) to protect ��-cells against cytokine-induced apoptosis, ii) to enhance their glucose competence and iii) to increase ��-cell proliferation. R��sum�� : Contr��le de la masse des cellules �� pancr��atiques et de leur fonction par les hormones glucoincr��tines: Identification de nouveaux m��canismes r��gulateurs pour le traitement du diab��te Les cellules �� des ��lots de Langerhans s��cr��tent l'insuline pour diminuer l'hyperglyc��mie. Le nombre de cellules �� et leur capacit�� �� s��cr��ter l'insuline sont modul��s dans les conditions physiologiques normales pour r��pondre �� la demande m��tabolique de l'organisme. Un ��chec du pancr��as endocrine �� maintenir sa capacit�� s��cr��toire d'insuline d�� �� une diminution du nombre et de la fonction des cellules �� conduit au diab��te de type 1 et de type 2. Les m��canismes mol��culaires contr��lant la comp��tence au glucose des cellules �� matures, tels que, l'augmentation de la s��cr��tion d'insuline en r��ponse au glucose, la r��plication des cellules ��, leur diff��rentiation �� partir de cellules pr��curseurs et la protection contre l'apoptose sont encore peu connus. Afin d'examiner ces m��canismes, nous avons ��tudi�� les effets sur les cellules �� des hormones gluco-incr��tines, glucose-d��pendent insulinotropic polypeptide (G1P) et glucagon-like peptide-1 (GLP-1) qui sont s��cr��t��es par les cellules endocrines de l'intestin apr��s la prise alimentaire. En plus de potentialiser la s��cr��tion d'insuline induite par le glucose, ces hormones induisent la diff��rentiation de cellules �� �� partir de cellules pr��curseurs, stimulent leur prolif��ration et les prot��gent contre l'apoptose. Par cons��quent, comprendre les m��canismes d'action des gluco-incr��tines permettrait de d��couvrir de nouveaux processus r��gulant les cellules �� avec d'��ventuelles applications dans le traitement ou la pr��vention du diab��te. Les ��lots de souris ayant une double inactivation des g��nes pour les r��cepteurs du GIP et du GLP-1 (dK0) pr��sentent un d��faut de s��cr��tion d'insuline stimul��e par le glucose et une sensibilit�� accrue �� l'apoptose induite par les cytokines. Afin de d��terminer les g��nes r��gul��s, qui pourraient contr��ler �� la fois la comp��tence au glucose et la protection contre l'apoptose, nous avons effectu�� une analyse comparative transcriptomique sur des ��lots de souris contr��les et dKO. Nous avons constat�� une forte diminution de l'expression d'IGF-1R dans les ��lots dKO. Nous avons d��montr��, �� la fois dans une lign��e cellulaire murine s��cr��tant l'insuline et dans ��lots primaires, que le GLP-1 stimulait l'expression d'IGF-1R et sa voie de signalisation. Par ailleurs, la phosphorylation d'Akt d��pendante d'IGF1-R induite parle GLP-1 n��cessite une s��cr��tion active, indiquant la pr��sence d'un m��canisme d'activation autocrine. Nous avons ensuite montr�� que l'activation de la voie de signalisation d'IGF-1R ��tait d��pendante de la s��cr��tion d'IGF-2, dont l'expression est r��gul��e par les nutriments. Finalement, nous avons d��montr�� que la boucle autocrine IGF-2/IGF-1R est n��cessaire pour le GLP-1 i) pour prot��ger les cellules �� contre l'apoptose induite par les cytokines, ii) pour am��liorer la comp��tence au glucose et iii) pour augmenter la prolif��ration des cellules ��. R��sum�� tout public : Contr��le de la masse des cellules �� pancr��atiques et de leur fonction par les hormones gluco-incr��tines: Identification de nouveaux m��canismes r��gulateurs pour le traitement du diab��te Chez les mammif��res, la concentration de glucose sanguine (glyc��mie) est r��gul��e et maintenue �� une valeur relativement constante d'environ 5 mM. Cette r��gulation est principalement contr��l��e par 2 hormones produites par les ��lots pancr��atiques de Langerhans: l'insuline s��cr��t��e par les cellules �� et le glucagon s��cr��t�� par les cellules a. A la suite d'un repas, l'augmentation de la glyc��mie entra��ne la s��cr��tion d'insuline ce qui permet le stockage du glucose dans le foie, les muscles et le tissu adipeux afin de diminuer le taux de glucose circulant. Lors d'un je��ne, la diminution de la glyc��mie permet la s��cr��tion de glucagon favorisant alors la production de glucose par le foie, normalisant ainsi la glyc��mie. Le nombre de cellules �� et leur capacit�� s��cr��toire s'adaptent aux variations de la demande m��tabolique pour assurer une normoglyc��mie. Une destruction compl��te ou partielle des cellules �� conduit respectivement au diab��te de type 1 et de type 2. Bien que l'augmentation de la glyc��mie soit le facteur stimulant de la s��cr��tion d'insuline, des hormones gluco-incr��tines, principalement le GLP-1 (glucagon-like peptide-1) et le GIP (glucose-dependent insulinotropic polypeptide) sont lib��r��es par l'intestin en r��ponse aux nutriments (glucose, acides gras) et agissent au niveau des cellules ��, potentialisant la s��cr��tion d'insuline induite par le glucose, stimulant leur prolif��ration, induisant la diff��rentiation de cellules pr��curseurs en cellules �� matures et les prot��gent contre la mort cellulaire (apoptose). Afin d'��tudier plus en d��tail ces m��canismes, nous avons g��n��r�� des souris d��ficientes pour les r��cepteurs du GIP et du GLP-l. Les ��lots pancr��atiques de ces souris pr��sentent un d��faut de s��cr��tion d'insuline stimul��e par le glucose et une sensibilit�� accrue �� l'apoptose par rapport aux ��lots de souris contr��les. Nous avons donc cherch�� les g��nes r��gul��s pas ces hormones contr��lant la s��cr��tion d'insuline et la protection contre l'apoptose. Nous avons constat�� une forte diminution de l'expression du r��cepteur �� l'IGF-1 (IGF-1R) dans les ��lots de souris d��ficientes pour les r��cepteurs des gluco-incr��tines. Nous avons d��montr�� dans un model de cellules �� en culture et d'��lots que le GLP-1 augmentait l'expression d'IGF-1R et la s��cr��tion de son ligand (IGF-2) permettant l'activation de la voie de signalisation. Finalement, nous avons montr�� que l'activation de la boucle IGF-2/IGF-1R induite par le GLP-1 ��tait n��cessaire pour la protection contre l'apoptose, l'augmentation de la s��cr��tion et la prolif��ration des cellules ��.

Energy dispersive X-Ray fluorescence : measuring elements in solid and liquid matrices

The subject of this project is about ���Energy Dispersive X-Ray Fluorescence ��� (EDXRF).This technique can be used for a tremendous variety of elemental analysis applications.It provides one of the simplest, most accurate and most economic analytical methods for thedetermination of the chemical composition of many types of materials.The purposes of this project are:- To give some basic information about Energy Dispersive X-ray Fluorescence.- To perform qualitative and quantitative analysis of different samples (water-dissolutions,powders, oils,..) in order to define the sensitivity and detection limits of the equipment.- To make a comprehensive and easy-to-use manual of the ���ARL QUANT���X EnergyDispersive X-Ray Fluorescence��� apparatus

Disseny dels elements constitutius per adaptar un centre de mecanitzat de control num��ric a la tecnologia Incremental Sheet Forming (ISF)

Un dels processos tradicionals de conformaci�� de xapa m��s utilitzats a nivell industrial, degut a la seva rapidesa d���operaci�� i la seva maduresa del proc��s, ��s l���embotici��. Per a dur a terme aquest proc��s ��s necess��ria la construcci�� d���uns utillatges (matriu i punx��) per a cada tipus de producte. Aquests utillatges estan fabricats amb materials altament resistents ja que han de poder suportar c��rregues molt importants durant la deformaci��. A m��s, s���han de garantir unes precisions dimensionals molt bones per tal d���evitar el xoc entre les dues parts de l���utillatge i per a assegurar que la pe��a obtinguda tingui les dimensions desitjades. Aquest fet implica que el proc��s d���obtenci�� de les matrius i punxons tingui un cost molt elevat i per a amortitzar-lo ��s necessari que la producci�� sigui en massa.L���objecte d���aquest projecte ��s adaptar el centre de mecanitzat Kondia HS1000 per tal de poder dur a terme recerca b��sica de la tecnologia de conformat incremental de xapa (ISF) en el Grup de Recerca en Enginyeria del Producte, Proc��s i Producci�� (GREP)

A critical lineage-nonspecific role for pTalpha in mediating allelic and isotypic exclusion in TCRbeta-transgenic mice.

Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.

Mls: a link between immunology and retrovirology.

The nature of the mysterious minor lymphocyte stimulating (Mls) antigens has recently been clarified. These molecules which were key elements for our current understanding of immune tolerance, have a strong influence on the mouse immune system and are encoded by the open reading frame (orf) of endogenous and exogenous mouse mammary tumor viruses (MMTV's). The knowledge that these antigens are encoded by cancerogenic retroviruses opens an interdisciplinary approach for understanding the mechanisms of immune responses and immune tolerance, retroviral carcinogenesis, and retroviral strategies for infection.

Increased frequency of regulatory T cells and selection of highly potent CD62L+ cells during treatment of human lung transplant recipients with rapamycin.

The currently available immunosuppressive agents applied in human transplantation medicine are highly potent in the protection from acute allograft rejection. However, long-term allograft survival is still poor as these drugs fail to sufficiently prevent chronic allograft rejection. Naturally occurring regulatory T cells have been postulated as the key players to establish long-lasting transplantation tolerance. Thus, the development of immunosuppressive regimens which shift the pathological balance of cytopathic versus regulatory T cells of human allograft recipients towards a protective T-cell composition is a promising approach to overcome limitations of current transplantation medicine. Thirty-three patients that received rapamycin (RPM) or calcineurin inhibitor treatment following lung transplantation were included and their T-cell compartments analysed. Twelve healthy volunteers without history of lung disease served as controls. In this article, we show that treatment of human lung transplant recipients with RPM is associated with an increased frequency of regulatory T cells, as compared with treatment with calcineurin inhibitors or to healthy controls. Moreover, regulatory T cells during treatment with RPM were CD62Lhigh, a phenotype that displayed an enhanced immunosuppressive capacity ex vivo. Our data support the use of RPM in human lung transplant recipients and undertaking of further prospective studies evaluating its impact on allograft and patient survival.

Iowa Utilities Board Fiscal Year 2002 Regulatory Plan Prepared Pursuant to Executive Order Nine, August 1, 2001

Report for the Iowa Utilities Board

Iowa Utilities Board Fiscal Year 2004 Regulatory Plans Pursuant to Executive Order Nine, August 1, 2003

Report for Iowa Utilities Board

Anti-CD154 mAb and rapamycin induce T regulatory cell mediated tolerance in rat-to-mouse islet transplantation.

BACKGROUND: Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0-28 d) or late (100-128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. CONCLUSIONS/SIGNIFICANCES: These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

SOURCE, FATE AND FUNCTION OF EARLY GLIAL CELLS EXPRESSING NKX2.1 IN MOUSE EMBRYONIC BRAINS

The brain tissue is made of neuronal and glial cells generated in the germinal layer bordering the ventricles. These cells divide, differentiate and migrate following specific pathways. The specification of GABAergic interneurons and glutamatergic neurons has been broadly studied but little is known about the origin, the fate and the function of early glial cells in the embryonic telencephalon. It has been commonly accepted since long that the glial cells and more particularly the astrocytes were generated after neurogenesis from the dorsal telencephalon. However, our work shows that, unlike what was previously thought, numerous glial cells (astroglia and polydendrocytes) are generated during neurogenesis in the early embryonic stages from E14.5 to E16.5, and originate from the ventral Nkx2.1-expressing precursors instead. NK2 homeobox 1 (Nkx2.1) is a member of the NK2 family of homeodomaincontaining transcription factors. The specification of the MGE precursors requires the expression of the Nkx2.1 homeobox gene. Moreover, Nkx2.1 is previously known to regulate the specification of GABAergic interneurons and early oligodendrocytes in the ventral telencephalon. Here, in my thesis work, I have discovered that, in addition, Nkx2.1 also regulates astroglia and polydendrocytes differentiation. The use of Nkx2.1 antibody and Nkx2.1 riboprobe have revealed the presence of numerous Nkx2.1-positive cells that express astroglial markers (like GLAST and GFAP) in the entire embryonic brain. Thus, to selectively fate map MGE-derived GABAergic interneurons and glia, we crossed Nkx2.1-Cre mice, Glast-Cre ERT+/- inducible mice and NG2-Cre mice with the Cre reporter Rosa26-lox-STOP-lox-YFP (Rosa26-YFP) mice. The precise origin of Nkx2.1-positive astroglia has been directly ascertained by combining glial immunostaining and focal electroporation of the pCAG-GS-EGFP plasmids into the subpallial domains of organotypic slices, as well as, by using in vitro neurosphere experiments and in utero electroporation of the pCAG-GS-tomato plasmid into the ventral pallium of E14.5 Nkx2.1-Cre+/Rosa-YFP+/- embryos. We have, thus, confirmed that the three germinal regions of the ventral telencephalon i.e. the MGE, the AEP/POA and the triangular septal nucleus are able to generate early astroglial cells. Moreover, immunohistochemistry for several astroglial cells and polydendrocyte markers, both in the Nkx2.1-/- and control embryos and in the neurospheres, has revealed a severe loss of both glial cell types in the Nkx2.1 mutants. We found that the loss of glia corresponded to a decrease of Nkx2.1-derived precursor division capacity and glial differentiation. There was a drastic decrease of BrdU+ dividing cells labeled for Nkx2.1 in the MGE*, the POA* and the septal nucleus* of Nkx2.1 mutants. In addition, we noticed that while some remaining Nkx2.1+ precursors still succeeded to give rise to post-mitotic neurons in vitro and in vivo in the Nkx2.1-/-, they completely lost the capacity to differentiate in astrocytes. Altogether, these observations indicate for the first time that the transcription factor Nkx2.1 regulates the proliferation and differentiation of precursors in three subpallial domains that generate early embryonic astroglia and polydendrocytes. Furthermore, in order to investigate the potential function of these early Nkx2.1- derived glia, we have performed multiple immunohistochemical stainings on Nkx2.1-/- and wild-type animals, and Nkx2.1-Cre mice that were crossed to Rosa-DTA+/- mice in which the highly toxic diphtheria toxin aided to selectively deplete a majority of the Nkx2.1-derived cells. Interestingly, in these two mutants, we observed a drastic and significant loss of GFAP+, GLAST+, NG2+ and S100��+ astroglial cells at the telencephalic midline and in the medial cortical areas. This cells loss could be directly correlated with severe axonal guidance defects observed in the corpus callosum (CC), the hippocampal commissure (HIC), the fornix (F) and the anterior commissure (AC). Axonal guidance is a key step allowing neurons to form specific connections and to become organized in a functional network. The contribution of guidepost cells inside the CC and the AC in mediating the growth of commissural axons have until now been attributed to specialized midline guidepost astroglia. Previous published results in our group have unravelled that, during embryonic development, the CC is populated in addition to astroglia by numerous glutamatergic and GABAergic guidepost neurons that are essential for the correct midline crossing of callosal axons. Therefore, the relative contribution of individual neuronal or glial populations towards the guidance of commissural axons remains largely to be investigated to understand guidance mechanisms further. Thus, we crossed Nkx2.1-Cre mice with NSE-DTA+/- mice that express the diphtheria toxin only in neurons and allowed us to selectively deplete Nkx2.1-derived GABAergic neurons. Interestingly, in the Nkx2.1-/- mice, the CC midline was totally disorganized and the callosal axons partly lost their orientation, whereas in the Nkx2.1Cre+/Rosa-DTA+/- and the Nkx2.1Cre+/NSE-DTA+/- mice, the axonal organization of the CC was not affected. In the three types of mice, hippocampal axons of the fornix were not properly fasciculated and formed disoriented bundles through the septum. Additionally, the AC formation was completely absent in Nkx2.1-/- mice and the AC was divided into two/three separate paths in the Nkx2.1Cre+/Rosa-DTA+/- mice that project in wrong territories. On the other hand, the AC didn't form or was reduced to a relatively narrower tract in the Nkx2.1Cre+/NSE-DTA+/- mice as compared to wild-type AC. These results clearly indicate that midline Nkx2.1-derived cells play a major role in commissural axons pathfinding and that both Nkx2.1-derived guidepost neurons and glia are necessary elements for the correct development of these commissures. Furthermore, during our investigations on Nkx2.1-/- and Nkx2.1Cre+/Rosa-DTA+/- mice, we noticed similar and severe defects in the erythrocytes distribution and the blood vessels network morphology in the embryonic brain of both mutants. As the Cre-mediated recombination was never observed to occur in the blood vessels of Nkx2.1-Cre mice, we inferred that the vessels defects observed were due to the loss of Nkx2.1-derived cells and not to the cells autonomous effects of Nkx2.1 in regulating endothelial cell precursors. Thereafter, the respective contribution of individual Nkx2.1-regulated neuronal or glial populations in the blood vessels network building were studied with the use of transgenic mice strains. Indeed, the use of Nkx2.1Cre+/NSE-DTA+/- mice indicated that the Nkx2.1-derived neurons were not implicated in this process. Finally, to discriminate between the two Nkx2.1-derived glial cell populations, the GLAST+ astroglia and the NG2+ polydendrocytes, an NG2-Cre mouse strain crossed to the Rosa-DTA+/- mice was used. In that mutant, the blood vessel network and the erythrocytes distribution were similarly affected as observed in Nkx2.1Cre+/Rosa-DTA+/- animals. Therefore, this result indicates that most probably, the NG2+ polydendrocytes are involved in helping to build the vessels network in the brain. Taken altogether, these observations show that during brain development, Nkx2.1- derived embryonic glial cells act as guidepost cells on the guidance of axons as well as forming vessels. Both Nkx2.1-regulated guidepost GABAergic neurons and glia collaborate to guide growing commissural axons, while polydendrocytes are implicated in regulating brain angiogenesis. - Le tissu c��r��bral est compos�� de cellules neuronales et gliales g��n��r��es dans les couches germinales qui bordent les ventricules. Ces cellules se divisent, se diff��rencient et migrent selon des voies particuli��res. La sp��cification des interneurones GABAergiques et des neurones glutamatergiques a ��t�� largement ��tudi��e, par contre, l'origine, le destin et la fonction des cellules gliales pr��coces du t��lenc��phale embryonnaire restent peu ��lucid��es. Depuis longtemps, il ��tait commun��ment accept�� que les cellules gliales, et plus particuli��rement les astrocytes, sont g��n��r��s apr��s la neurog��n��se �� partir du t��lenc��phale dorsal. Toutefois, notre travail montre que de nombreuses cellules gliales sont g��n��r��es �� partir de pr��curseurs ventraux qui expriment le g��ne Nkx2.1, entre E14.5 et E16.5, c'est-�� dire,�� des stades embryonnaires tr��s pr��coces. Le g��ne NK2 hom��obox 1 (Nkx2.1) appartient �� une famille de facteurs de transcription appel��e NK2. Il s'agit de prot��ines qui contiennent un hom��o-domaine. La sp��cification des pr��curseurs de la MGE requiert l'expression du g��ne hom��obox Nkx2.1. De plus, la fonction du g��ne Nkx2.1 dans la r��gulation de la sp��cification des interneurones GABAergiques et des oligodendrocytes dans le t��lenc��phale ventral ��tait d��j�� connue. Au cours de mon travail de th��se, j'ai ��galement mis en ��vidence que, Nkx2.1 r��gule aussi les ��tapes de prolif��ration et de diff��renciation de divers sous-types de cellules gliales soit de type astrocytes ou bien polydendrocytes. L'utilisation d'un anticorps contre la prot��ine Nkx2.1 ainsi qu'une sonde �� ribonucl��otides contre l'ARN messager du g��ne Nkx2.1 ont r��v��l�� la pr��sence de nombreuses cellules positives pour Nkx2.1 qui exprimaient des marqueurs astrocytaires (comme GLAST et GFAP) dans le t��lenc��phale embryonnaire. Afin de d��terminer de mani��re s��lective le sort des interneurones GABAergiques, des polydendrocytes et des astrocytes d��riv��s de la MGE, nous avons crois�� soit des souris Nkx2.1-Cre, des souris Glast-Cre ERT+/- inductibles ou bien des souris NG2-Cre avec des souris Rosa26-lox-STOP-lox-YFP (Rosa26-YFP) Cre rapportrices. L'origine pr��cise des astroglies positives pour Nkx2.1 a ��t�� directement ��tablie en combinant une coloration immunologique pour les glies et une ��lectroporation focale d'un plasmide pCAG-GS-EGFP dans les domaines subpalliaux de tranches organotypiques, puis ��galement, par des cultures de neurosph��res in vitro et des exp��riences d'��lectroporation in utero d'un plasmide pCAG-GS-tomato dans le pallium ventral d'embryons Nkx2.1-Cre+/Rosa- YFP+/- au stade E14.5. Nous avons donc confirm�� que les trois r��gions germinales du t��lenc��phale ventral, c'est-��-dire, la MGE, l'AEP/POA et le noyau triangulaire septal sont capables de g��n��rer des cellules astrogliales. D'autre part, l'immunohistochimie pour plusieurs marqueurs d'astrocytes ou de polydendrocytes, dans les embryons Nkx2.1-/- et contr��les ainsi que dans les neurosph��res, a r��v��l�� une s��v��re perte de ces deux types gliaux chez les mutants. Nous avons trouv�� que la perte de glies correspondait �� une diminution de la capacit�� de division des pr��curseurs d��riv��s de Nkx2.1, ainsi que l'incapacit�� de ces pr��curseurs de se diff��rencier en cellules gliales. Nous avons en effet observ�� une diminution importante des cellules BrdU+ en division exprimant Nkx2.1dans la MGE*, la POA* et le noyau septal* des mutants pour Nkx2.1. D'autre part, nous avons pu mettre en ��vidence aussi bien in vitro, qu'in vivo, que certains pr��curseurs Nkx2.1+ chez le mutant gardent la capacit�� �� se diff��rencier en neurones tandis qu'ils perdent celle de se diff��rencier en cellules gliales. Prises dans leur ensemble, ces observations indiquent pour la premi��re fois que le facteur de transcription Nkx2.1 r��gule les ��tapes de prolif��ration et de diff��rentiation des pr��curseurs des trois domaines subpalliaux qui g��n��rent les astroglies et polydendrocytes embryonnaires pr��coces. Par la suite, dans le but de comprendre la fonction potentielle de ces glies pr��coces, nous avons proc��d�� �� de multiples colorations immunohistochimiques sur des animaux Nkx2.1-/- et sauvages, ainsi que sur des souris Nkx2.1-Cre crois��es �� des souris Rosa-DTA+/- dans lesquelles la toxine diphth��rique hautement toxique a permis de supprimer s��lectivement la majorit�� des cellules d��riv��es de Nkx2.1. De mani��re int��ressante, nous avons observ�� dans ces deux mutants, une perte drastique et significative de cellules astrogliales GFAP+, GLAST+ et polydendrocytaires NG2+ et S100��+ dans le t��lenc��phale, �� la midline et dans les aires corticales m��dianes. Ces pertes ont pu ��tre directement corr��l��es avec des d��fauts de guidage axonal observ��s dans le corps calleux (CC), la commissure hippocampique (HIC), le fornix (F) et la commissure ant��rieure (AC). Le guidage axonal est une ��tape cl�� permettant aux neurones de former des connections sp��cifiques et de s'organiser dans un r��seau fonctionnel. La contribution des cellules �� guidepost �� dans le CC et dans la AC comme m��diateurs de la croissance des axones commissuraux �� jusqu'�� aujourd'hui ��t�� attribu��e sp��cifiquement �� des astroglies �� guidepost �� de la midline. Des r��sultats publi��s pr��c��demment dans notre groupe, ont permis de montrer que, pendant le d��veloppement embryonnaire, le CC est peupl�� en plus de la glie par de nombreux neurones �� guidepost �� glutamatergiques et GABAergiques qui sont essentiels pour le croisement correct des axones callosaux �� la midline. Ainsi, la contribution relative des populations individuelles neuronales ou gliales pour le guidage des axones commissuraux demande �� ��tre approfondie afin de mieux comprendre les m��canismes de guidage. A ces fins, nous avons crois�� des souris Nkx2.1-Cre avec des souris NSE-DTA+/- qui expriment la toxine diphth��rique uniquement dans les neurones et ainsi, nous avons pu s��lectivement supprimer les neurones d��riv��s de domaines Nkx2.1+. Dans les souris Nkx2.1-/-,nous avons d��couvert que le CC ��tait d��sorganis�� avec des axones callosaux perdant partiellement leur orientation, alors que dans les souris Nkx2.1Cre+/Rosa-DTA+/- et Nkx2.1Cre+/NSE-DTA+/-, l'organisation axonale n'��tait pas affect��e. De plus, les faisceaux hippocampiques du fornix ��taient d��fascicul��s dans les trois types de mutants. Par ailleurs, la formation de la commissure ant��rieure (AC) ��tait compl��tement absente dans les souris Nkx2.1-/- d'une part, et d'autre part, celle-ci ��tait divis��e en deux �� trois voies s��par��es dans les souris Nkx2.1Cre+/Rosa-DTA+/-. Finalement, la AC ��tait soit absente, soit r��duite de mani��re ne former plus qu'un faisceau relativement plus ��troit dans les souris Nkx2.1Cre+/NSE-DTA+/- en comparaison avec la AC sauvage. Ces derniers r��sultats indiquent clairement que les cellules d��riv��es de Nkx2.1 �� la midline, jouent un r��le majeur dans le guidage des axones commissuraux et que, autant les neurones, que les astrocytes �� guidepost �� d��riv��s de Nkx2.1, sont des ��l��ments n��cessaires au d��veloppement correct de ces commissures. En outre, lors de nos investigations sur les souris Nkx2.1-/- et Nkx2.1Cre+/Rosa-DTA+/-, nous avons remarqu��s des d��fauts s��v��res et similaires dans la distribution des erythrocytes et dans la morphologie du r��seau de vaisseaux sanguins dans le cerveau embryonnaire des deux mutants pr��cit��s. Puisque nous n'avons jamais observ�� de recombinaison de la Cre recombinase dans les vaisseaux sanguins des souris Nkx2.1Cre, nous en avons d��duit que les d��fauts de vaisseaux observ��s ��taient dus �� la perte de cellules d��riv��es de Nkx2.1. Il existerait donc en plus de la fonction cellulaire autonome de Nkx2.1 reconnue pour r��gul��e directement la sp��cification des cellules endoth��liales, une fonction indirecte de Nkx2.1. Afin de d��terminer la contribution respective des populations individuelles neuronales ou gliales r��gul��es par Nkx2.1 dans la construction du r��seau de vaisseaux sanguins, nous avons utilis�� diverses lign��es de souris transg��niques. L'utilisation de souris Nkx2.1Cre+/NSE-DTA+/- a indiqu�� que les neurones d��riv��s de Nkx2.1 n'��taient pas impliqu��s dans ce processus. Finalement, afin de discriminer entre les deux populations de cellules gliales d��riv��es de Nkx2.1, les astroglies et les polydendrocytes, nous avons crois�� une lign��e de souris NG2-Cre avec des souris Rosa-DTA+/-. Dans ce dernier mutant, le r��seau de vaisseaux sanguins du cortex ainsi que la distribution des erythrocytes ��taient affect��s de la m��me mani��re que dans le cortex des souris Nkx2.1Cre+/Rosa-DTA+/-. Par cons��quent, ce r��sultat indique que tr��s probablement, les polydendrocytes NG2+ sont impliqu��s dans la mise en place du r��seau de vaisseaux dans le cerveau. Prises dans leur ensemble, ces observations montrent que durant le d��veloppement embryonnaire du cerveau, des sous-populations de glies r��gul��es par Nkx2.1 jouent un r��le de cellules �� guidepost �� dans le guidage des axones, ainsi que des vaisseaux. Les polydendrocytes sont impliqu��es dans la r��gulation de l'angiogen��se tandis que, autant les neurones GABAergiques que les astrocytes collaborent dans le guidage des axones commissuraux en croissance.