Ulm, Univ., Diss., 2008.

Universiẗat, Diss., 2004--Köln.

Universiẗat, Diss., 2004--Köln.

Zugl.: Berlin, Freie Universiẗat, Diss., 2008.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

Tauopathien wie u.a. die Alzheimer-Demenz sind eine Gruppe neurodegenerativer Erkrankungen mit intrazellulärer Ablagerung des hyperphosphorylierten Tau-Proteins. Perineuronale Netze sind eine spezialisierte Form der extrazellulären Matrix im Zentralen Nervensystem. Sie bestehen u.a. aus Chondroitinsulfat-Proteoglykanen und Tenascin-R. Ziel der vorliegenden Arbeit war der Nachweis einer Protektion perineuronaler Netze vor Tau-Protein-Einlagerung. Zu Beginn wurde die Verteilung des Tau-Proteins und die verschiedenen Arten perineuonaler Netze in der Mauslinie C57Bl6 (Wildtyp) und deren Tenascin-R-Knockout beschrieben. Die Darstellung der Komponenten erfolgte mithilfe von Gefrierschnitten und Immunhistochemie. Um die Ausbreitung von Tau-Protein zu untersuchen, wurden organotypische Hirnschnittkulturen angelegt und markiertes Tau-Protein zugesetzt. Es wurden verschiedene Antikörper genutzt, welche bestimmte Seitenketten von Chondroitinsulfat-Proteoglykanen darstellen. Die Chondroitinsulfat-Seitenketten wurden mit dem Enzym Chondroitinase ABC entfernt, um deren Funktion zu untersuchen. Die Ergebnisse der Arbeit zeigten deutliche Unterschiede in der Verteilung von Tau-Protein. Es reicherte sich bei den Wildtypen und den Tenascin-R-Knockout-Mäusen diffus extrazellulär um Neuronen mit perineuronalen Netzen an. Im Unterschied dazu verteilte sich Tau-Protein nach der Abspaltung der Chondroitinsulfat-Seitenketten vom perineuronalen Netz gleichmäßig über die Schnittkultur ohne sich an perineuronalen Netzen anzureichern. Die vorliegende Arbeit stellt die neuroprotektive Funktion der perineuronalen Netze und insbesondere ihrer Chondroitinsulfat-Seitenketten gegenüber zugegebenem Tau-Protein heraus. Weiterhin weist sie den Verlust der neuroprotektiven Funktion gegenüber zugegebenem Tau-Protein nach Behandlung mit Chondroitinase ABC nach.