Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas.

Sox9, (SRY-type HMG box), has been shown to play a critical role throughout chondrogenesis. Haploinsufficiency of Sox9 in humans leads to a skeletal malformation syndrome known as campomelic dysplasia. To understand the regulation of Sox9 during chondrogenesis, the developing mouse limb was used to identify and characterize regulatory regions within the Sox9 promoter. Luciferase-based reporter assays in mouse revealed a proximal promoter spanning – 2 kb from the transcriptional start site, while mobility shift assays demonstrated that a CCAAT motif is involved in the transactivation of Sox9. Moreover, luciferase-based reporter assays revealed a proximal promoter spanning – 4 kb in Fugu rubripes, and potential regulatory regions spanning the remainder of the promoter. Comparison of mammalian Sox9 upstream intergenic sequences to that of Fugu has identified 5 conserved regions that are contained within 18 kb of upstream Fugu sequence. Analysis of the transcriptional activity of these sequences has led to the identification of regulatory elements within the Sox9 promoter. Several studies also provide evidence of a role for wingless (WNT) and bone morphogenetic (BMP) signaling molecules in the regulation of chondrogenesis. TCF/LEF-LacZ reporter mice show activated canonical WNT signaling distributed throughout the embryonic age (E) 9.5 forelimb. At later stages, LacZ expression becomes confined to distal regions of the limb bud. Previous studies have demonstrated that canonical WNTs inhibit chondrogenesis. Our studies demonstrate that treatment of cultures derived from E11.5 proximal limb buds with the canonical WNT, WNT3a, inhibits chondrogenesis. However, treatment of cultures derived from E9.5 and distal E11.5 limb buds with WNT3a stimulates chondrogenesis. Quantitative PCR (qPCR) also demonstrates that WNT3a modulates a number of genes expressed throughout chondrogenesis. To gain insights into BMP function in the early limb, we have characterized BMP action in sub-populations of cells from the E10.5 limb. Surprisingly, BMPs were found to inhibit cartilage formation in immature cells, while promoting cartilage formation in more mature cells. Transcriptional profiling coupled with qPCR and time course analyses revealed that the extent of induction of Gatas by BMPs was associated with its stimulatory versus inhibitory activity. Further, SOX9 activity was inhibited following over-expression of Gatas. / Medicine, Faculty of / Graduate

Many developmental genes are located in gene-poor genomic regions and are activated by long-range enhancers located up to 1Mb away. Modification and reorganisation of chromatin structure is pivotal to such long-range gene regulation. A prerequisite for enhancer activity is the binding of transcription factors and co-factors with the interplay between activating and repressive factors determining tissue, spatial and temporal specificity. Spatiotemporal control of sonic hedgehog (Shh) and the 5′ Hoxd genes (especially Hoxd13) is crucial for vertebrate limb anterior-posterior (A-P) axis and autopod patterning. Shh tissue specificity is controlled by multiple enhancers throughout an adjacent gene desert. The ~0.8Mb-distant limb enhancer (ZRS) bypasses nearby genes to activate only Shh. In contrast, limb-specific HoxD expression is regulated by multiple enhancers, with the ~200kb-distant global control region (GCR) regulatory element the most characterised. In this thesis I investigated the mechanisms of ZRS and GCR regulation of Shh and Hoxd13 respectively. The model system used was immortalised cell lines derived from the anterior and posterior distal forelimb buds of E10.5 and E11.5 mouse embryos. Cell line data were confirmed in dissected limb tissue. Increased expression of the 5′ Hoxd genes, particularly Hoxd13, correlated with the loss of the repressive, polycomb catalysed, histone modification H3K27me3 and decompaction of chromatin structure over the HoxD locus at the distal posterior forelimb bud at stage E10.5. Moreover, I show that the GCR spatially co-localises with the 5′ HoxD locus at the distal posterior region of E10.5-11 embryos. These data are consistent with the formation of a chromatin loop between Hoxd13 and the GCR at the time and place of distal limb bud development when the GCR is required to initiate 5′ Hoxd gene expression. This is the first example of A-P differences in chromatin compaction and local folding in the limb. Point mutations within the ZRS cause ectopic (anterior) Shh expression, which results in preaxial polydactyly (PPD). The ZRS contains multiple canonical ETS transcription factor binding motifs, and point mutations in two families with PPD results in the formation of additional ETS binding sites. The point mutations cause the loss or reduction of ETV4/5 transcription factor binding at a non-canonical ETS binding site and enable additional binding instead of ETS1. I show that ETV4/5, ETS1 and another ETS protein GABPα all bind to the ZRS. This work has revealed the differential effect on Shh expression of two groups of ETS factors mediated through the ZRS. The binding of ETS1/ GABPα determines the posterior Shh expression domain while ETV4/5 restricts anterior Shh expression. Two point mutations alter the ETS-binding profile, creating an additional ETS1/ GABPα site that is sufficient to drive ectopic Shh expression. DNA FISH on E11.5 forelimb and floorplate tissue sections revealed that the Shh-ZRS genomic locus is in a compact chromatin conformation in both Shhexpressing and non-expressing cells. However, I show that the ZRS co-localises with Shh to a significantly greater extent in the distal posterior limb bud and the floorplate compared with cells where Shh is not expressed. This thesis presents novel research into long-range gene regulation during limb development, elucidating the role of chromatin re-organisation and how spatial-specific enhancer activity is determined by opposing sets of binding factors.

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This study examined the generalization of the Dynamic Dominance Hypothesis (DDH) in regard to limb dominance, limb selection, and limb action. This study was inspired by the finding that limb selection changes from dominant-arm to nondominant-arm occur around an object position of 80° for right-handers and 100° for left-handers after passing the body midline (90°) into contralateral hemispace. For Study 1 and Study 2, 10 right-handed and 10 left-handed adults participated and reaching with the right and left arms of right- and left-handers was made to each of nine targets using free-choice and forced-choice paradigms. The purpose of Study 1 was to determine the relationship between limb selection and the DDH among both handedness groups. Thus, Study 1 addressed the following questions: Can the DDH explain why people select their nondominant hand for reaching into their contalateral hemispace? Do predictions of the DDH hold for right- and left-handers? Our results suggest that control efficiency with regard to a reduction in degrees of freedom in reaching movements seems to be a more fundamental cause for the limb selection phenomenon rather than the DDH. Also, our data reveal that kinematic differences between right- and left-handers with regard to utilization of joints for reaching explain limb selection differences between both handedness groups. The aim of Study 2 was to extend generalization of the DDH using a wide range of movement speed. Thus, Study 2 addressed the following question: Do propositions of the DDH hold for a wide range of speeds? Our data indicate the DDH does not hold for either slow or fast speed in reaching movements. Rather, a change in kinematics with regard to utilization of joints in reaching movements is associated with movement speed. Considered together, our data indicate that the DDH is an inadequate explanation of differences in limb selection, limb dominance (handedness), and limb action (speed). Rather, our findings with regard to control efficiency seem to be more fundamental and justified explanations for limb differences in the control of reaching based on the context of our task.