Role of 6-O-endosulfatases, Sulf1 and Sulf2, during murine skeletal development

Im ersten Teil dieser Arbeit werden die Expressionsmuster der Sulfatase-Genfamilie während der Embryonalentwicklung der Maus (Mus musculus) beschrieben. Mittels in situ-Hybridisierung wurden mRNA-Transkripte für 9 der 14 murinen Sulfatasen in den Entwicklungsstadien E12,5 – E16,5 nachgewiesen. Für mehrere Sulfatasen wurden teilweise überlappende Expressionsmuster im Knorpel/Knochen, Augenanlage und Choroidplexus identifiziert. Erstmals konnte eine spezifische Expression für Arylsulfatase G (ArsG) im Choroidplexus, für ArsI in hypertrophen Chondrozyten und für ArsJ im Gelenksspalt aufgezeigt werden. Die dynamische Expression der Heparansulfat 6-O-Endosulfatasen Sulf1 und Sulf2 in den verschiedenen Skelettelementen wird eingehend beschrieben. Im zweiten Teil der Arbeit wurden zwei neue loss of function Genetrap-Allele (Sulf1gt und Sulf2gt) charakterisiert. Der Skelettphänotyp dieser Tiere wurde zusammen mit Sulf1- und Sulf2- Deletions-Mutanten vergleichend untersucht. Bei doppelt homozygoten Sulf1;Sulf2 Mausmutanten wurde ein vermindertes Körpergewicht, ein verkleinertes Skelett und verschiedene Skelettveränderungen (fusionierte Sternebrae und Schwanzwirbel, sowie Fehlbildungen: der Lendenwirbel, des 2. Halswirbels und dem Basisphenoid) festgestellt. Mit zunehmender Anzahl an mutierten Sulfatase-Allelen konnte eine höhere Penetranz und stärkere Ausprägung der Skelettfehlbildungen beobachtet werden. Am stärksten betroffen waren Sulf1-/-;Sulf2-/- Doppelmutanten. Die Untersuchung der endochondralen Ossifikation mit molekularen Markern lässt auf eine beschleunigte Chondrozyten-Differenzierung bei den Doppelmutanten, zumindest im Sternum, schließen. Insgesamt ähnelt der Phänotyp der Sulf1;Sulf2 loss of function Doppelmutanten dem von Mausmutanten mit einem aktiviertem FGF- oder reduziertem Ihh-Signalweg. Im dritten Teil der Arbeit wurde die transgene Mausmutante Col2-Sulf1tg199 untersucht, bei der keine Sulf1-Überexpression vorliegt. Die Skelettveränderungen bei diesen Tieren (verzögerte Knochenentwicklung und homeotische Transformation der Lendenwirbel) werden vermutlich durch die Transgen-Insertionsstelle auf Chromosom 15 verursacht. Es wurde ein Deletionsbereich von 2,25 Mb identifiziert, welcher 6 annotierte Gene enthält. Eine eindeutige Zuordnung einzelner Gene zum Phänotyp ist jedoch nicht möglich.The first part of the study, describes the expression patterns of several sulfatase gene family members in the developing mouse (Mus musculus) embryo. In E12.5 – E16.5 embryos transcripts, for 9 (out of 14) sulfatases could be detected by in situ hybridization. For several sulfatases an partial overlapping expression pattern in cartilage/bone, eye and choroid plexus was discoverd. For the first time, a specific expression pattern for Arylsulfatase G (ArsG) in choroid plexus, ArsI in hypertrophic chondrocytes and ArsJ in the joints could be shown. The dynamic expression patterns of the heparan sulfate 6-O-endosulfatases Sulf1 and Sulf2 were carefully investigated in various skeletal elements. The second part of the study, describes two novel loss of function gene trap alleles (Sulf1gt and Sulf2gt). The skeletal phenotype of the corresponding mouse mutants has been compared with Sulf1- and Sulf2- knock-out mutants. For both double homozygous mutants a reduced body weight, smaller skeletons and several skeletal alterations (fusions of sternebrae and tail vertebrae, malformations of lumbar vertebrae, second cervical vertebrae and basiphenoid) have been discovered. Overall penetrance and severity of the skeletal alterations is increased with increasing numbers of mutant alleles and was highest in Sulf1-/-;Sulf2-/- mutants. Characterization of endochondral ossification at a molecular level, points towards an accelerated chondrocyte differentiation at least in the sternum. In summary Sulf1;Sulf2 loss of function mutants bear similarities to mice with activated FGF or reduced Ihh signaling. The third part of the study, describes a transgenic mouse mutant Col2-Sulf1tg199, which does not overexpress Sulf1. The skeletal alterations in this mutant (delayed bone formation and a homeotic transformation of the lumbal vertebrae) are probably caused by the transgen insertion site on chromosom 15. A genomic deletion of 2.25 mb was identified in this mutants. This region contains 6 annotated genes in the wildtyp genome. However, it was not possible to connect the phenotype to a single candidate gene

FGF-2 Deficiency Does Not Influence FGF Ligand and Receptor Expression during Development of the Nigrostriatal System

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro

Analysis of the Fibroblast Growth Factor System Reveals Alterations in a Mouse Model of Spinal Muscular Atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis