This project aimed to provide a greater understanding of limb development through the characterisation of Mendelian disorders. The more specific aim was to identify the developmental basis of the Post Axial Longitudinal Limb Reduction Deformity (PALLRD) seen in the autosomal recessive Miller syndrome caused by mutations in Dihydroorotate Dehydrogenase (DHODH)[1]. In addition whole exome sequence analysis was used to identify further causative variants in a group of individuals with Non Classical Miller syndrome. These individuals were negative for mutations in DHODH although they had a clinically overlapping PALLRD. A single novel variant was discovered in Fibroblast Growth Factor Receptor 1 gene (FGF1) in one individual in this cohort. Due to the known vital role of FGF signaling in limb bud development the functional significance of this variant was investigated further[2]. In vitro data suggested that this variant has a dominant negative effect. Finally I compared the differential gene expression profile of embryonic mouse forelimb and hindlimb at a later stage of development. Digital Gene Expression Serial Analysis of Gene Expression (DGE-SAGE) produced gene-expression profiles of the forelimbs and hind limbs from 14.5 days post conception (d.p.c) murine embryos. This data included known differentially expressed genes as well as novel candidate genes that are putative regulators of limb growth. Whole mount In Situ Hybrisation (WISH) and Quantitative Real Time Polymerase Chain Reaction (qRTPCR) provided corroborating evidence for the differential expression of a subset of these genes between the forelimbs and hind limbs. This project suggests a role for DHODH in limb bud cell proliferation. It also demonstrates a novel potentially dominant negative mutation within FGFR1 in an individual with a limb deformity. Finally a subset of genes involved in regulating the differential growth between the forelimb and hindlimb were presented.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24189号 / 医博第4883号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小林 恭, 教授 羽賀 博典, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.

Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.

No description available.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The endocrine-like factor FGF21 is a potent regulator of nutrient metabolism. Systemic FGF21 administration to obese animals improves glucose tolerance, lowers blood glucose and triglycerides, and decreases fasting insulin levels. Although FGF21 improves the survival and function of islet β-cells, the mechanisms are currently unknown. This thesis examines mechanisms of FGF21 in the regulation of pancreatic islet metabolism. Biochemistry studies showed FGF21 decreased Acetyl-CoA carboxylase (ACC) and Uncoupling protein-2 (UCP2) protein expression in mouse islets. Autofluorescence microscopy showed difference in NAD(P)H responses when challenged with TCA cycle intermediate citrate. FGF21-treated islets showed significant decreased mitochondrial energetics when acutely stimulated with high concentrations of glucose and palmitate. This decrease in energetics correlated with increased generation of NADPH. Importantly, insulin secretion was lowered but not abolished in this state. These data confirm that FGF21 alters pancreatic islets metabolism during high glucose and high fat loading and reduces insulin during nutrient stress.

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.