L’ostéosarcome (OS), plus fréquente des tumeurs osseuses primitives malignes, se caractérise par une croissance ostéoïde parfois associée à une ostéolyse. Malgré les avancées thérapeutiques, le taux de survie reste faible (30 % à 5 ans si métastases ou chimiorésis-tances). De nouvelles approches thérapeutiques ciblant la cellule tumorale et son environnement sont néces-saires. Les travaux présentés se sont intéressés aux poten-tiels facteurs pro-tumoraux que sont les gènes Dlx et à une signalisation clé de l’environnement osseux (RANKL/RANK) susceptible d’influer l’agressivité tumo-rale. L’OS étant une tumeur ostéoblastique, la famille Dlx a été choisie, car impliquée dans l’ostéoblasto-genèse, et la signalisation RANKL/RANK, car voie car-dinale du couplage entre ostéoblastes et ostéoclastes. De plus un lien entre Dlx et signalisation RANK était suspecté. Les gènes Dlx1, Dlx4 et Rank non-exprimés dans l’ostéoblaste sain le sont dans les lignées d’OS. Des modulations d’expression des Dlx et de Rank ont été réalisées afin d’en évaluer l’impact sur les cellules tumo-rales. L’implication de la signalisation RANK/RANKL dans le microenvironnement tumoral a été analysée. La perturbation du remodelage est en faveur de la tumeur en participant à l’établissement d’un cercle vicieux entre la tumeur et l’environnement. Les travaux ont établi l’implication des Dlx, surtout Dlx4 pour lequel un nouveau transcrit codant a été ca-ractérisé. Cependant des études supplémentaires sont nécessaires. Concernant la signalisation RANK/RANKL, il s’avère qu’au-delà du cercle vicieux, important au stade d’initiation tumorale, l’expression de RANK par la tumeur s’avère être un facteur pro-métastatique. / Osteosarcoma (OS), the most common malignant primary bone tumor, is characterized by an osteoid formation occasionally associated with osteolysis. De-spite therapeutic advances, the 5-years survival rate remains low (30% in case of metastasis or drug-resistance). New therapeutic approaches targeting the tumor cell and its environment are needed. Presented studies focused on potential pro-tumor factors namely Dlx genes and a key signaling pathway of the bone environment (RANKL / RANK) that may influence tumor aggressiveness. The OS is an osteo-blastic tumor and Dlx family was chosen due to its in-volvement in osteoblastogenesis. RANKL / RANK path-way was selected as it constitutes a main element in the coupling between osteoblasts and osteoclasts. A link between Dlx genes and RANK signaling was suspected. Dlx1, Dlx4 and Rank genes are not normally ex-pressed in osteoblasts but are present in the OS cell lines. Dlx and Rank expression modulations were real-ized to assess the impact on tumor cells. RANK / RANKL signaling involvement in the tumor microenvi-ronment was analyzed. Disruption of remodeling is in favor of the tumor taking part in the establishment of a vicious circle between tumor and environment. This work established the involvement of Dlx, espe-cially DLX4 to which a new coding transcript has been characterized. However, additional studies are needed. Regarding the RANK / RANKL signaling, it turns out that beyond the vicious circle, leading to tumor initiation stage, the RANK expression by the tumor proves to be pro-metastatic elements.

DLX transcriptional targets have been implicated during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 mutant mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data suggests that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.

Introduction: The Dlx1/Dlx2 double knockout mouse has reduced numbers (33% fewer) of retinal ganglion cells (RGC), due to enhanced apoptosis. Brn3a and Brn3b are closely related members of the Class IV POU-domain gene family, and play functionally interchangeable roles in retinal development. We hypothesized that Brn3a and/or Brn3b are direct DLX transcriptional targets during retinal development. Methods: Chromatin immunoprecipitation (ChIP) assays were performed on E16.5 retinas to identify DLX proteins bound to the Brn3a or Brn3b promoters. Electrophoretic mobility shift assays (EMSA) were used to confirm the specificity of this binding. Luciferase reporter gene assays were performed to confirm the functional significance of DLX binding to the Brn3 or Brn3ba promoters in vitro. In utero retinal electroporation was used to study the effect of DLX gain-of-function on Brn3a/Brn3b expression in vivo. Compound Dlx1/Dlx2/Brn3a and Dlx1/Dlx2/Brn3b knockout mice were generated for analysis of retinal phenotypes. Results: Both DLX1 and DLX2 proteins bound to the Brn3b promoter, but only DLX2 bound to the Brn3a promoter in vivo. Using EMSA, recombinant DLX1 and DLX2 bound to Brn3b and specific supershifted bands resulted from the addition of specific DLX1 or DLX2 antibodies; only recombinant DLX2 bound to Brn3a and the supershifted band resulted from the addition of DLX2 antibody. Both DLX1 and DLX2 binding to the Brn3b promoter activated transcription of a luciferase reporter gene in vitro. Only Dlx2, but not Dlx1, co-transfection with Brn3a activated luciferase reporter gene expression. In utero retinal electroporation showed that ectopic DLX2 expression promoted both Brn3b and Brn3a expression in vivo. Loss of Dlx1/Dlx2 and Brn3b resulted in loss of 90% of RGC and increased cholinergic amacrine cell differentiation. Conclusion: Brn3b is transcriptionally regulated by both DLX1 and DLX2, whereas Brn3a is only regulated by DLX2 in vitro and in vivo. Dlx1/Dlx2 and Brn3b play combinatorial roles in retinogenesis.

DLX transcriptional targets have been implicated during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 mutant mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data suggests that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.

Introduction: The Dlx1/Dlx2 double knockout mouse has reduced numbers (33% fewer) of retinal ganglion cells (RGC), due to enhanced apoptosis. Brn3a and Brn3b are closely related members of the Class IV POU-domain gene family, and play functionally interchangeable roles in retinal development. We hypothesized that Brn3a and/or Brn3b are direct DLX transcriptional targets during retinal development. Methods: Chromatin immunoprecipitation (ChIP) assays were performed on E16.5 retinas to identify DLX proteins bound to the Brn3a or Brn3b promoters. Electrophoretic mobility shift assays (EMSA) were used to confirm the specificity of this binding. Luciferase reporter gene assays were performed to confirm the functional significance of DLX binding to the Brn3 or Brn3ba promoters in vitro. In utero retinal electroporation was used to study the effect of DLX gain-of-function on Brn3a/Brn3b expression in vivo. Compound Dlx1/Dlx2/Brn3a and Dlx1/Dlx2/Brn3b knockout mice were generated for analysis of retinal phenotypes. Results: Both DLX1 and DLX2 proteins bound to the Brn3b promoter, but only DLX2 bound to the Brn3a promoter in vivo. Using EMSA, recombinant DLX1 and DLX2 bound to Brn3b and specific supershifted bands resulted from the addition of specific DLX1 or DLX2 antibodies; only recombinant DLX2 bound to Brn3a and the supershifted band resulted from the addition of DLX2 antibody. Both DLX1 and DLX2 binding to the Brn3b promoter activated transcription of a luciferase reporter gene in vitro. Only Dlx2, but not Dlx1, co-transfection with Brn3a activated luciferase reporter gene expression. In utero retinal electroporation showed that ectopic DLX2 expression promoted both Brn3b and Brn3a expression in vivo. Loss of Dlx1/Dlx2 and Brn3b resulted in loss of 90% of RGC and increased cholinergic amacrine cell differentiation. Conclusion: Brn3b is transcriptionally regulated by both DLX1 and DLX2, whereas Brn3a is only regulated by DLX2 in vitro and in vivo. Dlx1/Dlx2 and Brn3b play combinatorial roles in retinogenesis.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain. In the forebrain, four Dlx genes, Dlx1, Dlx2, Dlx5, Dlx6, play essential role in the differentiation and proper migration of GABAergic interneurons to the cortex. Dlx genes are organized in convergently transcribed bigene clusters and each cluster includes a short intergenic region harboring cis-regulatory elements (CREs): specifically, the Dlx1/2 cluster includes I12b and I12a CREs, while Dlx5/6 harbors I56i and I56ii. In an effort to determine the regulatory role of the CREs on Dlx expression and forebrain development, I characterized mice with an I56i deletion and both I12b and I56i deletions. At late embryonic stage (E18.5) and the adult stage (P35), both mutants had similar expression levels of Dlx2 and Gad2 gene, encoding enzyme glutamic acid decarboxylase that is responsible for synthesis of GABA. Mutant mice showed impaired expression levels of Dlx5. The expression levels of Gad1 were decreased in ΔI56i mutants but increased in ΔI12b/I56i mutants at E18.5, and both adult mutants had comparable expression of Gad1 as wildtype mice. Together with previous in situ hybridization results of mice at earlier stages (E11.5, E14.5), my data show that Dlx CREs have different levels of activity in regulating the expression of Dlx genes at different developmental stages. The mutations of I56i and I12b CREs did not affect the development of two subtypes of GABAergic neurons (calbindin and calretinin expressing neurons) in the forebrain. Compared to wildtype mice, both mutants had hypersociability and deficits of memory and learning ability. This opens the possibility that the deletions of Dlx intergenic CREs caused some developmental abnormalities and, therefore affected the behavior of the mice. Through studying the mice lacking Dlx intergenic CREs, I gained a better understanding of the role that I56i and I12b play in the regulation of the expression of Dlx genes, the development of GABAergic neurons, and the social and cognitive behavior.

The Distal-less (Dll) gene has an ancient evolutionary origin. Chordates have retained duplicated Dll genes; vertebrates have six distinct paralogues (Dlx1 through Dlx6 in mammals) arranged in three cis-linked pairs that are co-expressed. Dlx genes are expressed in a conserved nested pattern that defines a proximal-distal axis in the pharyngeal arch tissue of vertebrates. Dlx5-/- and Dlx6-/- mouse neonates have similar phenotypic variations in the lower jaw and inner ear bones, with the Dlx6-/- phenotype being a less perturbed version of the Dlx5-/- phenotype. Conversely, Dlx5/6-/- double mutants have a homeotic transformation of the lower jaw into a second set of maxillary structures. The combination of expression patterns and null phenotypes has led to the proposal of a “Dlx code” that patterns the craniofacial tissue. However, the nature of this code, whether individual Dlx transcription factors supply unique functions, or whether they make a quantitative contribution to a more generic and shared Dlx function, is not well understood. One prediction of a quantitative model for Dlx function in the pharyngeal arches is the functional equivalency of the proteins encoded by divergent cis-linked Dlx paralogues. To address this aspect of the model, three core functions of Dlx5 and Dlx6 were compared quantitatively: suppression of cell growth, transcription activity and DNA binding affinity. In most respects both proteins behaved very similarly.

The Distal-less (Dlx) homeobox genes of mammals are expressed in many tissues of the developing organism including the limbs, craniofacial skeleton and the forebrain. In the forebrain, Dlx1, Dlx2, Dlx5 and Dlx6 play a critical role in driving tangential migration of GABAergic progenitors from the ventral telencephalon to their final destinations, notably the neocortex and the striatum. These Dlx genes are organised into convergently transcribed clusters with short intergenic regions that contain notable cis regulators elements (CREs) that drive Dlx expression in unique subdomains of the developing ventral telencephalon. Previous studies have characterised Dlx regulation including but not limited to the direct activation of these CREs by effector proteins. However, to date very little work has been done to examine how the forebrain Dlx genes may be regulated at the level of the chromatin. To explore this, I used in silico and in vivo methods to examine some key histone modifications of the Dlx1/2 and Dlx5/6 bigene clusters in the developing forebrain; namely H3K27Ac, H3K4me3, H3K4me1 and H3K27me3. I found that within the Dlx expressing ganglionic eminences (GE), at midgestation, the Dlx loci are marked by bivalent chromatin which is enriched in both permissive H3K4me3 and repressive H3K27me3 marks. By performing ChIP-qPCR on the GE tissue of embryonic mice with targeted deletions of enhancer CREs, I found that these CREs play unique roles in shaping the chromatin. Removal of one of these CREs has widespread effects on the chromatin at both loci. Since these changes in chromatin signatures do not accompany significant changes in expression of histone modifying genes, we believe these CREs play yet-to-be determined roles in recruiting the modifying proteins to the loci, thereby establishing bivalent chromatin to fine-tune Dlx expression.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.