Einleitung Fokale Gelenkknorpeldefekte treten in der Deutschen Bevölkerung mit einer geschätzten Inzidenz von über 300 000 jährlichen Fällen auf. In der US-amerikanischen Bevölkerung wird jährlich von über 600 000 Fällen ausgegangen. Aufgrund der Insuffizienz körpereigener Heilungskapazitäten und verfügbarer Therapieverfahren, schreitet die Erkrankung regelhaft zur post-traumatischen Arthrose fort. Neben der individuellen Lebensqualitätseinschränkung besteht eine sozioökonomische Bedeutung mit geschätzten Krankheitskosten von jährlich über 10 Milliarden US Dollar in den Vereinigten Staaten. Das Versagen zellbasierter Therapieverfahren beruht unter anderem auf einer Insuffizienz der chondrogenen Differenzierung, sowie der hypertrophen Differenzierung der Chondrozyten mit nachfolgender Osteogenese analog den Vorgängen in der Wachstumsfuge. Für die Induktion der chondrogenen Differenzierung stehen insbesondere Mitglieder der TGF-β Superfamilie, wie BMP-2, zur Verfügung. Diese sind jedoch ebenso durch eine Induktion der hypertrophen Differenzierung gekennzeichnet. Zur Induktion der Chondrogenese unter Umgehung der TGF-β-Signalwege wurde IHH in-vitro als vielversprechend beschrieben. Bislang besteht jedoch kein Nachweis der in-vivo Effektivität von IHH zur Knorpelreparation. Die Schaffung eines Wachstumsfaktor-Milieus in der Gelenkknorpelläsion in-vivo stellt ebenso eine Herausforderung dar. Diesbezüglich wurde ein vereinfachtes Verfahren zum lokalisierten in-vivo Gentransfer mittels adenoviraler Vektoren und autologen Knochenmarkskoagulaten anhand von Markergenen beschrieben. Die Effektivität jenes Verfahrens zur in-vivo Knorpelreparation wurde noch nicht gezeigt. Zweck dieses kontrollierten in-vivo Experimentes ist es, mittels des oben genannten Gentransferverfahrens die Wirksamkeit von BMP-2 und IHH zur Reparation von osteochondralen Defekten in New Zealand White Rabbits nachzuweisen. Die zentrale Hypothese lautete, dass BMP2 beziehungsweise IHH Gentransfer in einer höheren langzeit-histologischen Qualität des Reparationsgewebes resultiert. Explorativ sollten dabei Unterschiede in den einzelnen Dimensionen der Gewebequalität anhand des ICRS-II Histology Scoring Systems, sowie der Grad der Typ I (als Faserknorpelmarker), Typ II (als Marker hyalinen Gelenkknorpels) und Typ X Kollagen Deposition (als Marker hypertropher Chondrozyten) beschrieben werden. Material und Methoden Als Tiermodel wurden bilaterale 3,2 mm durchmessende osteochondrale Bohrlochdefekte in der Trochlea von New Zealand White Rabbits verwendet (n=10 unabhängige Tiere, 20 Gelenke). Die Defekte wurden mit autologen Knochenmarkkoageln gefüllt, die nach vorheriger Beckenkammaspiration gewonnen wurden. In den experimentellen Gruppen wurden die Knochenmarkkoagel beladen mit jeweils 1 x 1011 infektiösen Partikeln adenoviraler Vektoren, die cDNA codierend für BMP2 (n=3 Tiere, entsprechend 6 Gelenken) oder IHH (n=4; 8) enthielten. In der Kontrollgruppe wurde das nicht-chondrogene Markergen GFP (n=3; 6) transferiert. Beide Gelenke eines Tieres wurden der gleichen Gruppe zugeordnet. Die histologische Gewebequalität wurde nach 13 Wochen anhand des ICRS-II Scoringsystems durch 3 unabhängige, verblindete Untersucher bewertet. Als primäre Outcomes wurden der ICRS-II Parameter „Generelles Assessment“, sowie die Typ II Kollagen positive Fläche designiert. Als explorative Outcomes wurden die verbleibenden ICRS-II Parameter, sowie die Typ I und Typ X Kollagen Deposition bewertet. Die Korrelation zwischen den Untersuchern wurde nach Pearson ermittelt. Zum Test auf Signifikanz der Gruppenunterschiede wurde ein lineares gemischtes Modell verwendet, welches einer mögliche Abhängigkeit beider Gelenke eines Tieres Rechnung trägt. Ergebnisse Qualitative Bewertung des Reparationsknorpels. Dreizehn Wochen nach der Intervention zeigten die meisten der BMP-2 behandelten Gelenke (4 von 6) und alle der IHH behandelten Gelenke (8 von 8) hyalin-artigen Reparationsknorpel, während alle GFP behandelten Kontrollgelenke (6 von 6) faserknorpel-artiges Reparationsgewebe zeigten. Zwei BMP-2 behandelten Gelenke zeigten eine ausgeprägte intraläsionale Knochenformation. Primäre Outcomes - ICRS-II „Generelles Assessment“ und Typ II Kollagen positive Fläche. IHH und BMP-2 behandelte Gelenke zeigten im Vergleich zu GFP höhere Punktzahlen in dem ICRS-II „Generelles Assessment“ Parameter: +33.0 (95% Konfidenzintervall: -0.4, +66.4) Punkte für IHH und +8.5 (-26.6, +43.7) Punkte für BMP-2. Beide Effekte erreichten nicht das Level statistischer Signifikanz (p=0.052 und 0.537). IHH erhöhte die Typ II Kollagen Deposition in der Defektregion, während BMP-2 Gelenke keinen Unterschied zu GFP Kontrollen zeigten: +18.7 (-4.5, +42.0) Punkte für IHH und +0.0 (-29.7, +29.8) Punkte für BMP-2. Die erhöhte Typ II Kollagendeposition erreichte nicht das konventionelle Level statistischer Signifikanz (p=0.093). Sekundäre Outcomes - ICRS-II Parameter. In dem Vergleich von BMP-2 mit GFP Kontrollen wurde in keinem der 12 untersuchten Parameter ein signifikanter Unterschied festgestellt. IHH Gentransfer resultierte hingegen in höheren Punktzahlen in allen untersuchten Parametern, wobei der Unterschied in 5 der 12 Parameter das Niveau statistischer Signifikanz erreichte. Ein um 21.5 Punkte (+3.6, +39.4) erhöhter Score wurde für den Parameter „Gewebemorphologie“ beobachtet, sowie +21.0 (+6.4, +35.7) für „Chondrozytäres Clustering“, +31.2 (+0.8, +61.5) für „Formation der Tidemark“, +17.3 (+0.2, +34.5) für „Abnorme Kalzifikation/Ossifikation“ und +35.0 (+4.6, +65.2) für das „Assessment der mittleren und tiefen Zone“. Sekundäre Outcomes - Marker chondrozytärer Hypertrophie. Eine perizelluläre Deposition von Typ X Kollagen wurde in allen Gruppen beobachtet. Eine deutlich gesteigerte Deposition wurde nur in den Gelenken beobachtet, die nach BMP2 Gentransfer eine ausgeprägte intraläsionale Knochenformation zeigten. Diskussion Das hier beschriebene Experiment stellt die erste Veröffentlichung der Wirksamkeit von IHH zur Verbesserung der histologischen Knorpelqualität von in-vivo therapierten Gelenkknorpeldefekten dar [175]. Die Hypothese, dass IHH zu einer verbesserten histologischen Knorpelqualität führt wurde bestätigt, während die Hypothese zu den positiven Effekten von BMP-2 wiederlegt wurde. IHH führte zu besseren Ergebnissen in allen Untersuchten Parametern, das Niveau statistischer Signifikanz wurde dabei in den Parametern „Gewebemorphologie“, „Chondrozytäres Clustering“, „Formation der Tidemark“, „Abnorme Kalzifikation/Ossifikation“ und „Assessment der mittleren und tiefen Zone“ erreicht. Das primäre Ziel dieses Experimentes war es, den „Proof of concept“ zu liefern, dass IHH auch in-vivo ein attraktiver Faktor für die Induktion der Chondrogenese darstellt. Das langfristige Ziel ist die Induktion der Chondrogenese unter Umgehung des TGF-β Signalweges zu erzielen, um eine folgende hypertrophe Differenzierung der Chondrozyten und die folgende Ossifikation des reparierten Defektes zu verhindern. Die Limitationen der Studie umfassen die ausschließlich histologische und immunhistochemische durchgeführte Bewertung der Knorpelqualität und eine eingeschränkte statistische Power. Ob IHH es vermag die hypertrophe Differenzierung zu umgehen und somit eine langfristige hyaline Knorpelreparation zu ermöglichen, ist in weiteren präklinischen Studien mit biochemischer und molekulargenetischer Analyse der Hypertrophie-Marker zu untersuchen. In Bezug auf den klinischen Einsatz zur Knorpelreparation erscheint der Einsatz der Wachstumsfaktoren als Protein auf funktionalisierten Matrices vielversprechend. BMP-2 wird aufgrund der hier beobachteten intraläsionalen Knochenformation nach BMP2 Gentransfer als nicht geeignet zur Unterstützung der Knorpelreparation in-vivo bewertet. / Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. Vector-laden autologous bone marrow coagulates with 10^11 adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the International Cartilage Repair Society (ICRS) II scoring system and the type II collagen positive area. IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. In conclusion, IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects.

Hedgehog (Hh) signalling from the notochord induces the slow muscle cell fate in the adaxial cells of the developing zebrafish embryo. Slow muscle formation is disrupted in zebrafish ??you-type?? mutants resulting in U-shaped somites. In many you-type mutants, genes encoding components of the Hh signalling pathway are mutated. scube2, a gene not previously known to be involved in Hh signalling, is disrupted in the you-type mutant ??you??. you mutants are deficient in several Hh dependent cell types and show decreased expression of Hh target genes. The Scube (signal peptide-CUB domain-EGF-related) family of proteins act as secreted glycoproteins or cell-surface proteins and are thought to be involved in protein-protein interactions and ligand binding. At the protein level, the Scube family resembles the endocytic receptor Cubilin. Cubilin is known to interact with another endocytic receptor Megalin, which can function as an endocytic receptor for Sonic Hedgehog (SHH) in vitro. Megalin endocytosis of Shh may be an important part of the Hh signal transduction pathway. An anti-Scube2 antibody was developed during this work to investigate the intracellular localization pattern of Scube2 and facilitate the identification of potential Scube2 binding partner(s). In addition, this work identified and characterized two homologs of scube2 in zebrafish, scube 1 and scube 3. The high level of similarity amongst the Scube family of proteins and the weak phenotype of the you mutant suggested scube1 and scube3 might also be involved in slow muscle development. Loss of function experiments performed by antisense morpholino knockdown of scube1 and scube3 in the you mutant decreases the expression of Hh target genes to levels seen in embryos lacking Hh signalling and dramatically enhances the loss of slow muscle fibres compared to you mutants alone. Thus, injecting both scube1 and scube3 morpholinos into you blocks Hh signalling and these embryos fail to develop slow muscle. Inhibition of the three partially redundant scube genes inhibits Hh signalling in zebrafish embryos, thereby demonstrating the essential requirement for scube gene function in the Hh signalling pathway.

The hedgehog morphogenic pathway is essential for the development of numerous organs and tissues in both vertebrates and invertebrates, and dysregulation of hedgehog signalling is also associated with a broad range of mammalian cancers. While a great deal of research has been dedicated to understanding the molecular interactions of the hedgehog signalling pathway itself, much work remains in understanding the downstream transcriptional output of the pathway, and how that output modulates cellular behaviour in target tissues to produce developmental outcomes. The hedgehog pathway is activated by hedgehog proteins and repressed by patched. Downstream of these regulators, the hedgehog signalling cascade involves modification and trafficking of a series of key proteins and ultimately leads to regulation of the GLI family of transcription factors, thereby modulating the transcriptional output of the pathway. This thesis builds on previous work investigating downstream targets of one GLI protein – GLI3 – in the mouse limb (McGlinn et al., 2005). This previous study identified genes that were dysregulated in the anterior limb of the Gli3-null, extra-toes strain of mice (Gli3Xt/Xt). Amongst the identified targets of GLI3 were a number of novel genes. However, further detailed analysis of these genes was not conducted, and therefore, this thesis investigates the embryonic expression or function of three of these novel downstream targets of GLI3, to clarify their regulation by the hedgehog pathway and identify their broader role throughout development. One published work and one paper submitted for publication are contained within this thesis, describing detailed expression of two novel SHH targets, Zinc finger protein 503 (Zfp503) and Pitrolysin metallopeptidase 1 (Pitrm1). Zfp503 belongs to a family of transcription factors that regulate aspects of development across a diversity of species. However, their role in mammals and avians has been poorly described. This manuscript presents a detailed description of Zfp503 expression in the mouse and chicken and examines regulation of Zfp503 in the limb by SHH and BMP signalling. My contribution to this paper was the analysis of WT Zfp503 expression in mouse and chick by section in situ hybridisation, and as such, I am listed as a middle author. Pitrm1 is a metallopeptidase with a broad range of predicted target molecules. Comparisons with family members in mammals and plants suggest Pitrm1 has mitochondrial function and is implicated in the pathology of Alzheimers disease. It is upregulated in response to hedgehog pathway activation in the anterior limb of two mouse models of hedgehog signalling– the Gli3Xt/Xt and Ptch1:Prx-Cre mouse line, which deletes patched1 in the developing limb. It is expressed in multiple developing tissues that are patterned by SHH, suggesting that Pitrm1 may be an important regulator of developmental processes downstream of SHH. For the Pitrm1 manuscript, I contributed the majority of the experimental data and prepared the manuscript, and therefore, I am the first listed author. A third downstream hedgehog target gene described in this thesis is Tmem26. Tmem26 is an entirely novel gene with unknown cellular function, although concurrent work in the Wicking laboratory suggests that Tmem26 regulates cell migration and morphology in cell culture. Tmem26 is negatively regulated by SHH in the anterior mouse limb at 11.5dpc, as shown by use of Gli3Xt/Xt and Ptch1:Prx-Cre mice. Tmem26 expression in wild-type mice is spatially restricted and strikingly evident in the facial prominences, particularly near the point of fusion of the developing lip and in the shelves of the secondary palate. This suggests that Tmem26 may be involved in lip and palate formation and possibly play a role in the common human birth disorders of cleft lip and cleft palate. Generation of a Tmem26 conditional knockout mouse line, followed by germline inactivation of Tmem26 using a ubiquitously expressed Cre line, did not reveal a craniofacial phenotype in embryos or adults. Knockout mice appear healthy and fertile with no obvious developmental defects. This does not preclude a role for Tmem26 in facial development however, as molecular redundancy may be able to compensate for Tmem26 loss in mice. Tmem26 is also expressed in cells and organs of the adult immune system and suggests an alternative possible role for Tmem26 in regulating immune function that could be further investigated using the Tmem26 conditional knockout mouse line.

The Sonic hedgehog (Shh) signaling pathway plays a critical role in murine gastric development. When Shh is knocked out in the mouse embryonic stomach, glandular epithelial hyperplasia occurs. Furthermore, this phenotype was mimicked in Gli3−/−, but not Gli2−/− stomachs. I utilized three additional mouse models that modulate Gli3 activity to better understand the role of Gli3 in the developing stomach - the Gli3Δ699/Δ699 ,Gli3P1−4/P1−4, and Kif7−/− mice. The Gli3P1−4/P1−4 stomach displayed glandular epithelial overgrowth, as did the Kif7−/− stomach to a lesser extent; the Gli3Δ699/Δ699 stomach displayed glandular hypoplasia. Moreover, the Gli3P1−4/P1−4 and Kif7−/− stomachs have a thicker circular smooth muscle, and the Gli3Δ699/Δ699 had a thinner one relative to wild-type. It appears that altering the balance of Gli3 in favour of its activator results in gastric glandular epithelial and circular smooth muscle hyperplasia, and a balance favouring the Gli3 repressor results in hypoplasia.

The Sonic hedgehog (Shh) signaling pathway plays a critical role in murine gastric development. When Shh is knocked out in the mouse embryonic stomach, glandular epithelial hyperplasia occurs. Furthermore, this phenotype was mimicked in Gli3−/−, but not Gli2−/− stomachs. I utilized three additional mouse models that modulate Gli3 activity to better understand the role of Gli3 in the developing stomach - the Gli3Δ699/Δ699 ,Gli3P1−4/P1−4, and Kif7−/− mice. The Gli3P1−4/P1−4 stomach displayed glandular epithelial overgrowth, as did the Kif7−/− stomach to a lesser extent; the Gli3Δ699/Δ699 stomach displayed glandular hypoplasia. Moreover, the Gli3P1−4/P1−4 and Kif7−/− stomachs have a thicker circular smooth muscle, and the Gli3Δ699/Δ699 had a thinner one relative to wild-type. It appears that altering the balance of Gli3 in favour of its activator results in gastric glandular epithelial and circular smooth muscle hyperplasia, and a balance favouring the Gli3 repressor results in hypoplasia.

Small molecules play important roles in therapeutics and drug discovery. Significant progress has been made by the chemical biology community to discover small-molecule probes to explore biological processes and to treat disease. This thesis describes both the discovery of novel probes for the Hedgehog (Hh) pathway and the application of small molecules in identifying cancer dependencies. / Chemistry and Chemical Biology

During embryonic vasculogenesis, endothelial cells form in the mesoderm , assemble into cord-like structures and then undergo tube formation. Previous studies have shown that signaling by members of the hedgehog family of secreted growth factors is essential for normal development of embryonic blood vessels. Embryos lacking hedgehog function show the presence of abundant endothelial cells but the cells fail to assemble into vascular cords and lumenized endothelial tubes do not form. At present it is not known whether active hedgehog signaling is required for both cord and tube formation or only for the initial step. To address this question, we have used small molecule inhibitors and agonists to the alter activity of the hedgehog signaling pathway in the chick embryo. If development is allowed to proceed until endothelial cells of the future dorsal aortae have assembled into cords, subsequent inhibition of hedgehog signaling, using cyclopamine, does not prevent aortal cells from forming endothelial tubes, however, it does lead to a reduction in cross sectional area of the aorta and to a loss of density of the adjacent vascular plexus. In contrast, activation of the hedgehog pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. Very little, if any, of the observed effects are due to differences in number of endothelial cells in the treated embryos. Examination of endothelial cells during vascular plexus formation shows that inhibition of hedgehog signaling with cyclopamine inhibits formation of filopodia while treatment with SAG increases the number of filopodial extensions. These studies show that hedgehog signaling levels must be tightly regulated for normal vascular patterning to be achieved.

No description available.

Hedgehog (HH) signaling controls renal development. Mutations in PTC1, the HH receptor, cause cancer in non-renal tissues. We hypothesized that constitutively active HH signaling is deleterious to renal development in mice with PTC1 deficiency targeted to the metanephric mesenchyme (MM)(Rarb2-Cre;Ptc1 loxP/-, termed Ptc1 mutants). Increased HH signaling in MM of mutant mice was confirmed by qRT-PCR for Ptc1. A decrease in NCAM-positive nephrogenic precursors at E13.5 and WT1-positive glomeruli at E18.5 was found. Increased cortical expression of Foxd1 was observed. At E13.5, a cluster of ectopic cells expressing Raldh2, Ptc2 and Bmp4 accumulated at the presumptive uretero-pelvic junction (UPJ). Magnetic resonance imaging demonstrated an increase in pelvic volume. Constitutive expression of GLI3 repressor via the Gli3Δ699 allele in Ptc1 mutants increased nephron number comparable to wild type mice and decreased pelvic volume compared to Ptc1 mutants. Thus repression of HH activity is required for proper nephrogenesis and patterning of the UPJ.

The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals. Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis. In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.