RUNX2 belongs to the RUNT domain family of transcription factors of which three have been identified in humans (RUNX1, RUNX2 and RUNX3). RUNX proteins are vital for metazoan development and participate in the regulation of cellular differentiation and cell cycle progression (Coffman, 2003). RUNX2 is required for proper bone formation by driving the differentiation of osteoblasts from mesenchymal progenitors during development (Ducy et al, 1997; Komori et al, 1997; Otto et al, 1997). RUNX2 is also vital for chondrocyte maturation by promoting the differentiation of chondrocytes to the hypertrophic phenotype (Enomoto et al, 2000). The consequences of completely disrupting the RUNX2 locus in mice provided compelling and conclusive evidence for the biological importance of RUNX2 where knockout mice died shortly after birth with a complete lack of bone formation (Komori et al, 1997; Otto et al, 1997). A further indication of the requisite role of RUNX2 in skeletal development was the discovery that RUNX2 haploinsufficiency in humans and mice caused the skeletal syndrome Cleidocranial Dysplasia (CCD) (Mundlos et al, 1997; Lee et al, 1997). A unique feature of RUNX2 is the consecutive polyglutamine and polyalanine tracts (Q/A domain). Mutations causing CCD have been observed in the Q/A domain of RUNX2 (Mundlos et al, 1997). The Q/A domain is an essential part of RUNX2 and participates in transactivation function (Thirunavukkarasu et al, 1998). Previous genotyping studies conducted in our laboratory identified several rare RUNX2 Q/A variants in addition to a frequently occurring 18 base pair deletion of the polyalanine tract termed the 11Ala allele. Analysis of serum parameters in 78 Osteoarthritis patients revealed the 11Ala allele was associated with significantly decreased osteocalcin. Furthermore, analysis of 11Ala allele frequencies within a Geelong Osteoporosis Study (GOS) fracture cohort and an appropriate age matched control group revealed the 11Ala allele was significantly overrepresented in fracture cases indicating an association with increased fracture risk. To further investigate the 11Ala allele and rare Q/A variants, 747 DNA samples from the Southeast Queensland bone study were genotyped using PCR and PAGE. The experiment served two purposes: 1) to detect additional rare Q/A variants to enrich the population of already identified mutants and 2) have an independent assessment of the effect of the 11Ala allele on fracture to either support or refute our previous observation which indicated the 11Ala allele was associated with an increased risk of fracture in the GOS. From the 747 samples genotyped, 665 were WT, 76 were heterozygous for the 11Ala allele, 5 were homozygous for the 11Ala allele and 1 was heterozygous for a rare 21 bp deletion of the polyglutamine tract. Chi-square analysis of RUNX2 genotype distributions within fracture and non-fracture groups in the Southeast Queensland bone study revealed that individuals that carried at least one copy of the 11Ala allele were enriched in the fracture group (p = 0.16, OR = 1.712). The OR of 1.712 was of similar magnitude to the OR observed in the GOS case-control investigation (OR = 1.9) providing support for the original study. Monte-Carlo simulations were used to combine the results from the GOS and the Southeast Queensland bone study. The simulations were conducted with 10000 iterations and demonstrated that the maximum probability of obtaining both study results by chance was less than 5 times in two hundred (p < 0.025) suggesting that the 11Ala allele of RUNX2 was associated with an increased fracture risk. The second element of the research involved the analysis of rare RUNX2 Q/A variants identified from multiple epidemiological studies of bone. Q/A repeat variants were derived from four populations: the GOS, an Aberdeen cohort, CAIFOS and a Sydney twin study. Collectively, a total of 20 rare glutamine and one alanine variants were identified from 4361 subjects. All RUNX2 Q/A variants were heterozygous for a mutant allele and a wild type allele. Analysis of incident fracture during a five year follow up period in the CAIFOS revealed that Q-variants (n = 8) were significantly more likely to have fractured compared to non-carriers (p = 0.026, OR 4.932 95% CI 1.2 to 20.1). Bone density data as measured by quantitative ultrasound was available for CAIFOS. Analysis of BUA and SOS Z-scores revealed that Q-repeat variants had significantly lower BUA (p = 0.031, mean Z-score of -0.79) and a trend for lower SOS (p = 0.190, mean Z-score of -0.69). BMD data was available for all four populations. To normalize the data across the four studies, FN BMD data was converted into Z-scores and the effect of the Q/A variants on BMD was analysed using a one sample approach. The analysis revealed Q/A variants had significantly lower FN BMD (p = 0.0003) presenting with a 0.65 SD decrease. Quantitative transactivation analysis was conducted on RUNX2 proteins harbouring rare glutamine mutations and the 11Ala allele. RUNX2 proteins containing a glutamine deletion (16Q), a glutamine insertion (30Q) and the 11Ala allele were overexpressed in NIH3T3 and HEK293 cells and their ability to transactivate a known target promoter was assessed. The 16Q and 30Q had significantly decreased reporter activity compared to WT in NIH3T3 cells (p = 0.002 and 0.016, for 16Q and 30Q, respectively). In contrast 11Ala RUNX2 did not show significantly different promoter activation potential (p = 0.54). Similar results were obtained in HEK293 cells where both the 16Q and 30Q RUNX2 displayed decreased reporter activity (p=0.007 and 0.066 for 16Q and 30Q respectively) whereas the 11Ala allele had no material effect on RUNX2 function (p = 0.20). The RUNX2 gene target reporter assay provided evidence to suggest that variation within the glutamine tract of RUNX2 was capable of altering the ability of RUNX2 to activate a known target promoter. In contrast, the 11Ala allele showed no variation in RUNX2 activity. The third feature of the research served the purpose of identifying potential RUNX2 gene targets with particular emphasis on discovering genes cooperatively regulated by RUNX2 and the powerful bone promoting agent BMP2. The experiment was conducted by creating stably transfected NIH3T3 cells lines overexpressing RUNX2 or BMP2 or both RUNX2 and BMP2. Microarray analysis revealed very few genes were differentially regulated between standard NIH3T3 cells and cells overexpressing RUNX2. The results were confirmed via RT-PCR analysis which demonstrated that the known RUNX2 gene targets Osteocalcin and Matrix Metalloproteinase-13 were modestly induced 2.5 fold (p = 0.00017) and 2.1 fold (p = 0.002) respectively in addition to identifying only two genes (IGF-II and SCYA11) that were differentially regulated greater than 10 fold. IGF-II and SYCA11 were significantly down-regulated 27.6 fold (p = 1.95 x 10-6) and 10.1 fold (p = 0.0002) respectively. The results provided support for the notion that RUNX2 on its own was not sufficient for optimal gene expression and required the presence of additional factors. To discover genes cooperatively regulated by RUNX2 and BMP2, microarray gene expression analysis was performed on standard NIH3T3 cells and NIH3T3 cells stably transfected with both RUNX2 and BMP2. Comparison of the gene expression profiles revealed the presence of a large number of differentially regulated genes. Four genes EHOX, CCL9, CSF2 and OSF-1 were chosen to be further characterized via RT-PCR. Sequential RT-PCR analysis on cDNA derived from control cells and cells stably transfected with either RUNX2, BMP2 or both RUNX2/BMP2 revealed that EHOX and CSF2 were cooperatively induced by RUNX2 and BMP2 whereas CCL9 and OSF-1 were suppressed by BMP2. The overexpression of both RUNX2 and BMP2 in NIH3T3 fibroblasts provided a powerful model upon which to discover potential RUNX2 gene targets and also identify genes synergistically regulated by BMP2 and RUNX2. The fourth element of the research investigated the role of RUNX2 in the ascorbic acid mediated induction of MMP-13 mRNA. The study was carried out using NIH3T3 cell lines stably transfected with BMP2, RUNX2 and both BMP2 and RUNX2. The cell lines were grown to confluence and subsequently cultured for a further 12 days in standard media or in media supplemented with AA. RT-PCR analysis was used to assess MMP-13 mRNA expression. The RT-PCR results demonstrated that AA was not sufficient for inducing MMP-13 mRNA in NIH3T3 cells. In contrast RUNX2 significantly induced MMP-13 levels 85 fold in the absence of AA (p = 0.0055) and upregulated MMP-13 mRNA levels 254 fold in the presence of AA (p = 0.0017). The results demonstrated that RUNX2 was essential for the AA mediated induction of MMP-13 mRNA in NIH3T3 cells. The effect of BMP2 on MMP-13 expression was also investigated. BMP2 induced MMP-13 mRNA transcripts a modest 3.8 fold in the presence of AA (p = 0.0027). When both RUNX2 and BMP2 were overexpressed in the presence of AA, MMP-13 mRNA levels were induced a massive 4026 fold (p = 8.7 x 10-4) compared to control cells. The investigation revealed that RUNX2 was an essential factor for the AA mediated induction of MMP-13 and that RUNX2 and BMP2 functionally cooperated to regulate MMP-13 mRNA levels.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / The origin of haematopoietic stem cells (HSCs) and the relative roles of the yolk sac and the aorta-gonad-mesonephros (AGM) region in the establishment of definitive haematopoiesis are controversial. Definitive HSCs were thought to originate from the AGM, while contribution from the yolk sac was restricted to the primitive blood. However, recent mammalian studies have demonstrated a contribution of yolk sac progenitors to the formation of definitive erythroid and myeloid lineages. This is supported by the recent discovery in zebrafish of erythromyeloid progenitors (EMPs) that arise within the posterior blood island (PBI), before emergence of HSCs in the AGM. The cell type that gives rise to HSCs is also unknown, with possible contributions from a bipotential haemangioblast, the haemogenic endothelium and the mesenchyme. The Runx transcription factors are important regulators of development in a number of organ systems. Runx1 is essential for the development of definitive HSCs and is transcribed from two promoters, P1 and P2, generating two major Runx1 isoforms. To further understand the role of Runx1 in HSC biology and the roles of the two isoforms during the establishment of definitive haematopoiesis, transgenic runx1 promoter reporter zebrafish were generated. The Tg(runx1P1:EGFP) line displays fluorescence in the PBI, where EMPs develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the AGM that are shown, by lineage tracing, to later populate the pronephros and thymus. Direct tracking of marked cells from the PBI in the runx1P1 transgenic confirms the two definitive blood lineages are distinct. Time-lapse imaging of a compound Tg(runx1P2:EGFP)/ Tg(kdrl:nls-mCherry) embryo expressing the red fluorescent protein mCherry in endothelial cells shows the emergence of HSCs from endothelial cells. This is the first demonstration of the direct generation of definitive HSCs from the haemogenic endothelium in a living embryo. The differential expression and functions of Runx3 isoforms were analysed. Depletion of Runx3P2 delays the entry of primitive erythrocytes into the circulation, and blocks the initiation of definitive haematopoiesis. Runx3P1 is expressed in the gut endoderm of the embryo and may function in regulating the activity of signalling pathways controlling angiogenesis through the Hedgehog and BMP signalling pathways.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / The origin of haematopoietic stem cells (HSCs) and the relative roles of the yolk sac and the aorta-gonad-mesonephros (AGM) region in the establishment of definitive haematopoiesis are controversial. Definitive HSCs were thought to originate from the AGM, while contribution from the yolk sac was restricted to the primitive blood. However, recent mammalian studies have demonstrated a contribution of yolk sac progenitors to the formation of definitive erythroid and myeloid lineages. This is supported by the recent discovery in zebrafish of erythromyeloid progenitors (EMPs) that arise within the posterior blood island (PBI), before emergence of HSCs in the AGM. The cell type that gives rise to HSCs is also unknown, with possible contributions from a bipotential haemangioblast, the haemogenic endothelium and the mesenchyme. The Runx transcription factors are important regulators of development in a number of organ systems. Runx1 is essential for the development of definitive HSCs and is transcribed from two promoters, P1 and P2, generating two major Runx1 isoforms. To further understand the role of Runx1 in HSC biology and the roles of the two isoforms during the establishment of definitive haematopoiesis, transgenic runx1 promoter reporter zebrafish were generated. The Tg(runx1P1:EGFP) line displays fluorescence in the PBI, where EMPs develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the AGM that are shown, by lineage tracing, to later populate the pronephros and thymus. Direct tracking of marked cells from the PBI in the runx1P1 transgenic confirms the two definitive blood lineages are distinct. Time-lapse imaging of a compound Tg(runx1P2:EGFP)/ Tg(kdrl:nls-mCherry) embryo expressing the red fluorescent protein mCherry in endothelial cells shows the emergence of HSCs from endothelial cells. This is the first demonstration of the direct generation of definitive HSCs from the haemogenic endothelium in a living embryo. The differential expression and functions of Runx3 isoforms were analysed. Depletion of Runx3P2 delays the entry of primitive erythrocytes into the circulation, and blocks the initiation of definitive haematopoiesis. Runx3P1 is expressed in the gut endoderm of the embryo and may function in regulating the activity of signalling pathways controlling angiogenesis through the Hedgehog and BMP signalling pathways.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / The origin of haematopoietic stem cells (HSCs) and the relative roles of the yolk sac and the aorta-gonad-mesonephros (AGM) region in the establishment of definitive haematopoiesis are controversial. Definitive HSCs were thought to originate from the AGM, while contribution from the yolk sac was restricted to the primitive blood. However, recent mammalian studies have demonstrated a contribution of yolk sac progenitors to the formation of definitive erythroid and myeloid lineages. This is supported by the recent discovery in zebrafish of erythromyeloid progenitors (EMPs) that arise within the posterior blood island (PBI), before emergence of HSCs in the AGM. The cell type that gives rise to HSCs is also unknown, with possible contributions from a bipotential haemangioblast, the haemogenic endothelium and the mesenchyme. The Runx transcription factors are important regulators of development in a number of organ systems. Runx1 is essential for the development of definitive HSCs and is transcribed from two promoters, P1 and P2, generating two major Runx1 isoforms. To further understand the role of Runx1 in HSC biology and the roles of the two isoforms during the establishment of definitive haematopoiesis, transgenic runx1 promoter reporter zebrafish were generated. The Tg(runx1P1:EGFP) line displays fluorescence in the PBI, where EMPs develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the AGM that are shown, by lineage tracing, to later populate the pronephros and thymus. Direct tracking of marked cells from the PBI in the runx1P1 transgenic confirms the two definitive blood lineages are distinct. Time-lapse imaging of a compound Tg(runx1P2:EGFP)/ Tg(kdrl:nls-mCherry) embryo expressing the red fluorescent protein mCherry in endothelial cells shows the emergence of HSCs from endothelial cells. This is the first demonstration of the direct generation of definitive HSCs from the haemogenic endothelium in a living embryo. The differential expression and functions of Runx3 isoforms were analysed. Depletion of Runx3P2 delays the entry of primitive erythrocytes into the circulation, and blocks the initiation of definitive haematopoiesis. Runx3P1 is expressed in the gut endoderm of the embryo and may function in regulating the activity of signalling pathways controlling angiogenesis through the Hedgehog and BMP signalling pathways.

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / The origin of haematopoietic stem cells (HSCs) and the relative roles of the yolk sac and the aorta-gonad-mesonephros (AGM) region in the establishment of definitive haematopoiesis are controversial. Definitive HSCs were thought to originate from the AGM, while contribution from the yolk sac was restricted to the primitive blood. However, recent mammalian studies have demonstrated a contribution of yolk sac progenitors to the formation of definitive erythroid and myeloid lineages. This is supported by the recent discovery in zebrafish of erythromyeloid progenitors (EMPs) that arise within the posterior blood island (PBI), before emergence of HSCs in the AGM. The cell type that gives rise to HSCs is also unknown, with possible contributions from a bipotential haemangioblast, the haemogenic endothelium and the mesenchyme. The Runx transcription factors are important regulators of development in a number of organ systems. Runx1 is essential for the development of definitive HSCs and is transcribed from two promoters, P1 and P2, generating two major Runx1 isoforms. To further understand the role of Runx1 in HSC biology and the roles of the two isoforms during the establishment of definitive haematopoiesis, transgenic runx1 promoter reporter zebrafish were generated. The Tg(runx1P1:EGFP) line displays fluorescence in the PBI, where EMPs develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the AGM that are shown, by lineage tracing, to later populate the pronephros and thymus. Direct tracking of marked cells from the PBI in the runx1P1 transgenic confirms the two definitive blood lineages are distinct. Time-lapse imaging of a compound Tg(runx1P2:EGFP)/ Tg(kdrl:nls-mCherry) embryo expressing the red fluorescent protein mCherry in endothelial cells shows the emergence of HSCs from endothelial cells. This is the first demonstration of the direct generation of definitive HSCs from the haemogenic endothelium in a living embryo. The differential expression and functions of Runx3 isoforms were analysed. Depletion of Runx3P2 delays the entry of primitive erythrocytes into the circulation, and blocks the initiation of definitive haematopoiesis. Runx3P1 is expressed in the gut endoderm of the embryo and may function in regulating the activity of signalling pathways controlling angiogenesis through the Hedgehog and BMP signalling pathways.

Chez le fœtus de rat, en fin de gestation, le taux de glucocorticoïdes (GC) fœtal présente un pic de secrétion entre le 16ème et le 20ème jour de la vie embryonnaire, phénomène concomitant à l'apparition des centres d'ossification du 17ème au 19ème jour de gestation pour le calvaria et pour le septum nasal, respectivement. Dans le cartilage, les GC modulent l'effet des facteurs insulino-mimétiques (IGF-I et -II) par des mécanismes complexes et mal définis, auxquels nous nous sommes intéressés. Nos résultats montrent que les GC, en concentrations physiologiques, peuvent induire la biominéralisation des matrices extracellulaires des chondrocytes de septum nasal et des ostéoblastes du calvaria de fœtus du rat en culture primaire (augmentation de la phosphatase alcaline, du dépôt minéral, des GAG et du Co I). Dans le septum nasal, cette induction est liée à une régulation différentielle des IGFBP et de leurs fragments de dégradation. Les mécanismes moléculaires gouvernant la différenciation des structures craniofaciales par les GC, en particulier, la régulation des homéogènes de la famille Msx et Dlx par cette hormone, sont peu connus. Une partie de notre travail a donc consisté à décrypter l'effet des GC sur l'expression des gènes Msx-2, Dlx-5 et Runx-2 au cours de la minéralisation de calvaria de fœtus du rat. Nos résultats indiquent que les GC induisent, in vitro et in vivo, dans le calvaria de fœtus de rat, l'expression des homéogènes Msx-2, Dlx5 et du gène maître Runx-2, nécessaires à l'induction d'un marqueur fonctionnel de la biominéralisation, l'ostéocalcine. Dans notre laboratoire, l'implication du gène Msx-1 lors de l'amélogenèse et de la dentinogenèse ainsi que la présence d'un ARN antisens naturel de ce gène ont été montrées chez la souris. Nous avons mis en évidence un homologue de cet ARN AS chez le rat. Nos résultats montrent que ce dernier est exprimé au cours de la biominéralisation du calvaria de fœtus de rat, et permettront d'une part de poursuivre l'étude du rôle de cet ARN AS dans des modèles in vitro et in vivo, et d'autre part, d'aborder les mécanismes d'action de cet ARN AS.

L'hématopoïèse regroupe les phénomènes menant à la formation des composantes<br />cellulaires du sang. Au cours de ce processus, des cellules précurseurs vont proliférer et se<br />différencier dans les multiples types cellulaires spécialisés. Le développement du système<br />hématopoïétique de la Drosophile et des vertébrés présente de nombreuses similitudes aussi bien<br />au niveau fonctionnel et ontogénique qu'au niveau des gènes qui régulent la formation des<br />cellules sanguines. Chez la Drosophile, au stade embryonnaire, les précurseurs<br />hématopoïétiques, les prohémocytes, vont générer deux types de cellules sanguines, les<br />plasmatocytes et les cellules à cristaux. Nous avons entrepris de caractériser les mécanismes de<br />régulation de l'hématopoïèse embryonnaire chez la Drosophile.<br />Dans un premier temps, nous avons analysé la fonction et le mode d'action du facteur de<br />transcriptions de type GATA Serpent (Srp) au cours de ce processus. Nous avons mis en<br />évidence que le gène serpent code pour deux isoformes qui ont des activités différentielles au<br />cours de ce processus. D'autre part, nous avons montré que l'activité de Srp au cours de<br />l'hématopoïèse est modulée par recrutement de cofacteurs. Ainsi, nous avons montré que Srp est<br />capable de recruter U-Shaped, un cofacteur de type FOG (Friend Of GATA), mais aussi, de<br />former un complexe fonctionnel avec le facteur de transcription de type RUNX, Lozenge. La<br />caractérisation des isoformes de Srp et la mise en évidence de l'interaction de ce facteur GATA<br />avec différents partenaires a permis de mettre en évidence la versatilité des fonctions de srp au<br />cours de l'hématopoïèse.<br />Dans un second temps, nous avons entrepris de caractériser in vivo l'étape de ségrégation<br />des deux populations, plasmatocytes et cellules à cristaux. Nous avons mis en évidence que la<br />ségrégation de ces deux lignages à partir d'une population de prohémocytes bipotents est un<br />processus très dynamique, contrôlé par un mécanisme original en deux étapes. Cette régulation<br />qui fait intervenir les facteurs de transcription lignage-spécifiques Lozenge et Glial-Cell-<br />Missing (Gcm) et Gcm2, contrôle précocement la détermination des précurseurs et tardivement<br />le maintient de l'identité de ces cellules dans les phases de différenciation en cellules à cristaux<br />versus plasmatocytes. De manière intéressante, nous avons montré que la régulation de la<br />ségrégation, ne repose pas sur un antagonisme réciproque entre les facteurs de transcription<br />lignage-spécifiques. Ce mécanisme qui contrôle l'acquisition d'un destin cellulaire diffère donc<br />des processus de régulation de l'hématopoïèse mis en évidence chez les mammifères.