Introduction: Colorectal cancer (CRC) is the fourth-most common cancer in Canada with a high mortality rate. Familial adenomatous polyposis (FAP) is a hereditary form of CRC; FAP patients carry germline mutations of the tumour suppressor gene adenomatous polyposis coli (APC). The function of Dlx genes in the gastrointestinal tract (GIT) has not been previously explored. Methods: Immunofluorescence (IF) was performed to identify Dlx2+ intestinal cells. Chromatin immunoprecipitation (ChIP) was performed to identify DLX2-Apc promoter interaction. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on mouse small and large intestines (normal and Dlx1/Dlx2 mutant mice). Electrophoretic mobility shift assays (EMSA) and reporter assays were carried out to investigate direct binding and activity, respectively, of DLX2 on the Apc promoter in-vitro. Dlx2 expression was explored in ApcMIN mice and human CRC tumor specimens. Results: Dlx2 is highly expressed in mouse embryonic and adult intestinal epithelia. Moreover, Dlx2 is expressed in the ApcMIN mice GIT as well as in some human CRC tumor specimens. ChIP, EMSA and reporter gene assays demonstrated that DLX2 protein specifically interacts with the Apc promoter in-situ and activates its expression in vitro. In-vivo and in-vitro, β-catenin protein levels are increased when DLX2 is absent or reduced by shRNA to Dlx2. Conclusions: Regulation of APC expression during development is poorly understood. We have evidence that DLX2 interacts with the Apc promoter in-vivo. We have shown that DLX2 induces Apc transcription by directly binding to the Apc promoter in-vitro. We also showed that β-catenin expression is altered in the Dlx1/Dlx2 mutant GIT. This finding implicates the involvement of DLX2 in the canonical Wnt signalling pathway. Ultimately, restoring APC expression may be a novel strategy towards preventing progression of intestinal polyps to adenocarcinoma. This research will contribute to our knowledge of the genetic and epigenetic regulatory pathways that control intestinal development, mucosal self-renewal and CRC.

Introduction: Colorectal cancer (CRC) is the fourth-most common cancer in Canada with a high mortality rate. Familial adenomatous polyposis (FAP) is a hereditary form of CRC; FAP patients carry germline mutations of the tumour suppressor gene adenomatous polyposis coli (APC). The function of Dlx genes in the gastrointestinal tract (GIT) has not been previously explored. Methods: Immunofluorescence (IF) was performed to identify Dlx2+ intestinal cells. Chromatin immunoprecipitation (ChIP) was performed to identify DLX2-Apc promoter interaction. Quantitative real time polymerase chain reaction (qRT-PCR) was performed on mouse small and large intestines (normal and Dlx1/Dlx2 mutant mice). Electrophoretic mobility shift assays (EMSA) and reporter assays were carried out to investigate direct binding and activity, respectively, of DLX2 on the Apc promoter in-vitro. Dlx2 expression was explored in ApcMIN mice and human CRC tumor specimens. Results: Dlx2 is highly expressed in mouse embryonic and adult intestinal epithelia. Moreover, Dlx2 is expressed in the ApcMIN mice GIT as well as in some human CRC tumor specimens. ChIP, EMSA and reporter gene assays demonstrated that DLX2 protein specifically interacts with the Apc promoter in-situ and activates its expression in vitro. In-vivo and in-vitro, β-catenin protein levels are increased when DLX2 is absent or reduced by shRNA to Dlx2. Conclusions: Regulation of APC expression during development is poorly understood. We have evidence that DLX2 interacts with the Apc promoter in-vivo. We have shown that DLX2 induces Apc transcription by directly binding to the Apc promoter in-vitro. We also showed that β-catenin expression is altered in the Dlx1/Dlx2 mutant GIT. This finding implicates the involvement of DLX2 in the canonical Wnt signalling pathway. Ultimately, restoring APC expression may be a novel strategy towards preventing progression of intestinal polyps to adenocarcinoma. This research will contribute to our knowledge of the genetic and epigenetic regulatory pathways that control intestinal development, mucosal self-renewal and CRC.

The Distal-less (Dll) gene has an ancient evolutionary origin. Chordates have retained duplicated Dll genes; vertebrates have six distinct paralogues (Dlx1 through Dlx6 in mammals) arranged in three cis-linked pairs that are co-expressed. Dlx genes are expressed in a conserved nested pattern that defines a proximal-distal axis in the pharyngeal arch tissue of vertebrates. Dlx5-/- and Dlx6-/- mouse neonates have similar phenotypic variations in the lower jaw and inner ear bones, with the Dlx6-/- phenotype being a less perturbed version of the Dlx5-/- phenotype. Conversely, Dlx5/6-/- double mutants have a homeotic transformation of the lower jaw into a second set of maxillary structures. The combination of expression patterns and null phenotypes has led to the proposal of a “Dlx code” that patterns the craniofacial tissue. However, the nature of this code, whether individual Dlx transcription factors supply unique functions, or whether they make a quantitative contribution to a more generic and shared Dlx function, is not well understood. One prediction of a quantitative model for Dlx function in the pharyngeal arches is the functional equivalency of the proteins encoded by divergent cis-linked Dlx paralogues. To address this aspect of the model, three core functions of Dlx5 and Dlx6 were compared quantitatively: suppression of cell growth, transcription activity and DNA binding affinity. In most respects both proteins behaved very similarly.

Malkostrake Krebse zeigen im postnauplialen Keimstreif ein invariantes Zellteilungsmuster und eine Zelllinie. Embryonen eines Isopoden (Porcellio scaber) und eines Amphipoden (Orchestia cavimana) wurden bezüglich ihrer Zelllinie vergleichend Untersucht. Mittels immunhistochemischer Färbungen wurde das Expressionsmuster der Gene engrailed und Distal-less in Hinblick auf die Zelllinie und die Morphogenese der Segmente und der Beinentwicklung analysiert. Die Zelllinie wurde bei Porcellio scaber mit der Methode der 4D-Mikroskopie untersucht. Mit Zellablationsexperimenten wurden Abhängigkeiten der Zellen untereinander aufgezeigt. Die Ergebnisse dieser Untersuchungen zeigen: I. Die Genexpression von Distal-less und engrailed wird unabhängig von der Zelllinie reguliert. II. Die Bildung der konvergenten einästigen Beine im Thoraxbereich beider Krebse erfolgt durch unterschiedliche Regulation des Gens Distal-less - bei Orchestia cavimana wird die Expression des Gens in den lateralen Zellen abgeschaltet, während diese Zellen bei Porcellio scaber erst gar nicht Distal-less exprimieren. III. Die Anwendung des Systems der 4D-Mikroskopie zeigte unter anderem, dass in den vorderen Reihen nichtektoteloblastischen Ursprungs bei Porcellio scaber die Zelllinie variabel ist und eine Zellsortierung und Zellimmigration stattfindet. IV. Die Ähnlichkeit der Bildung dieser Reihen bei Porcellio scaber mit der der Tanaidaceen lässt auf ein Schwestergruppenverhältnis der Taxa Isopoda und Tanaidacea schliessen. Die Dissertation enthält fünf Videoaufnahmen als separate AVI-Dateien. / Malacostracan crustaceans show in their postnaupliar germ-band an invariant cleavage pattern and a cell-lineage. A comparative analysis of this cell-lineage in an Isopod (Porcellio scaber) and an amphipod (Orchestia cavimana) was done in this thesis. Immunohistochemical stainings of the gene products Distal-less and Engrailed were used, to show the relation of these genes to the morphogenesis of segments and legs. Further, the cell-lineage of Porcellio scaber was analyzed with a 4D-microscope system. Cell-ablation experiments were used to show regulational networks in the development of the germ-band. The results of this work show: I. The regulation of the genes Distal-less and engrailed is independent of the cell-lineage. II. The morphogenesis of the convergent monoramous limbs in the thorax is reflected by different expression patterns of the gene Distal-less - in Orchestia cavimana the expression of Distal-less is switched off in the lateral cells, in Porcellio scaber these cells do not start the Distal-less expression. III. The 4D-microscopy analysis show, that the cell-lineage in the cellrows wich have a non-ectoteloblastic origin is not invariant. In these rows of cells show cell sorting. IV. The formation of these rows in the isopod Porcellio scaber shows similarity to the formation in tanaidaceans. A sister group relationship of Tanaidacea and Isopoda is strongly supported. This dissertation contains five video recordings as separate AVI files.

Die vorliegende Arbeit beinhaltet verschiedene Aspekte der Embryogenese des Pfeilschwanzkrebses Limulus polyphemus (Chelicerata, Xiphosura), darunter die frühe Neurogenese, die Axogenese, eine Analyse der "Kopf"segmentierung bei Cheliceraten und anderen Arthropoden, sowie das Expressionsmuster des Homöoboxgens Distal-less insbesondere in neuronalen Zusammenhängen. Darüber hinaus wurde eine neue Embryonalstadieneinteilung geleistet. Markierungen mit Phalloidin sowie weiterer neurospezifischer Marker ergaben, daß die frühe Neurogenese bei Limulus polyphemus durch die Immigration von Zellclustern erfolgt. Die Zellen nehmen eine flaschenförmige Gestalt annehmen, bevor sie sich aus dem ventralen Neuroektoderm lösen. Die Anzahl der Zellen pro Zellcluster steigt mit fortschreitender Entwicklung. Die Zellcluster konzentrieren sich in in Zentrum jedes Hemisegmentes, und in ihrem dorsalen Bereich beginnt die rasch voranschreitende Axogenese. Die Untersuchung der "Kopf"segmentierung mittels alpha-Tubulin-Markierungen bei Limulus polyphemus, Triops cancriformis (Crustacea) und Lepisma saccharina (Hexapoda) ergab sowohl bei der Entwicklung des circumoesophagealen Neuropilringes und der Innervierung der dazugehörigen Anhangspaare als auch hinsichtlich des Verlaufs des Stomatogastrischen Nervensystems beachtliche Übereinstimmungen, die entgegen der klassischen Auffassung den Schluß zulassen, daß das Deutocerebrum der Cheliceraten keineswegs reduziert wurde oder mit dem Tritocerebrum verschmolzen ist, sondern die Chelicere innerviert. Somit wäre das Chelicerenneuromer homolog zum Deutocerebrum der Crustacea und Hexapoda (1. Antenne). Der Vergleich des Expressionsmuster des Homöoboxgens Distal-less bei Limulus und Lepisma saccharina ergab neben den typischen Expressionen in auswachsenden Extremitäten- und andern Anhangsknospen bei beiden Vertretern Expressionen in neuronalen Zusammenhängen (im Lobus opticus, Ganglien bei Limulus oder in das ZNS umgebende Zellen bei Lepisma), an den verschiedensten Positionen späterer Sinnesorgane wie Mechano- oder Chemorezeptoren. Doppelmarkierungen mit Synorf-1 deuten darauf hin, daß es sich bei den Dll-positiven Zellen zum größten Anteil um Glia-Zellen handelt. / The following study contains different aspects of the embryology of the horseshoe crab Limulus polyphemus (Chelicerata, Xiphosura) with the main focus on early neurogenesis, axogenesis and the "head"segmentation in chelicerates and other arthropods. The expression pattern of the homeobox gene Distal-less was examined with main focus on neuronal correlations. In addition, a new staging was provided. Phalloidin stainings and other markers showed that the early neurogenesis in Limulus polyphemus happens via immigration of cell clusters. Cellclusters in the prosoma contain cells that become bottle shaped before they immigrate from the ventral neuroectoderm. The number of these cells increases during further development, and the cells concentrate in the middle of each hemisegment. Axogenesis starts at the dorsal edge of these concentrated cellclusters and progresses quite fast building the typical ladder like CNS of arthropods. The investigation of the "head"segmentation using alpha-tubulin stainings in Limulus polyphemus, Triops cancriformis (Crustacea), and Lepisma saccharina (Hexapoda) showed remarkable similarities in the development of the circumesophageal neuropil ring, the related appendages, and the course of the stomatogastric nerves. These results lead to the thesis that the deutocerebrum of chelicerates is neither completely reduced nor totally merged into the tritocerebrum but innervates the chelicerae which contradicts the classical view. According to these results the neuromer of the chelicerae would be homologous to the deutocerebrum of Crustaceans and Hexapods (first antennae). The expression pattern of the homeobox gene Distal-less was examined and compared in Limulus polyphemus and Lepisma saccharina. Beside the typical expression pattern in the developing appendages a participation of the gene in development of the nervous system was observed. Dll positve cells were found in or at least in direct contact with the CNS (optical lobe, ganglia in Limulus or surrounding the entire CNS including the brain of Lepisma), at different positions of later mechano- and chemoreceptors (lateral spines, bristles, flabellum, Johnstons organ etc.). Double stainings using Dll and Synorf-1 showed that at least most of these Dll-postive cells are most likely glia cells.

Die Notwendigkeit von Bx42 für Drosophila Entwicklung und seine Beteiligung an unterschiedlichen zellulären Prozessen wurde mit Hilfe von RNA Interference (RNAi) demonstriert. Das ubiquitäre Ausschalten oder die Reduktion der Bx42 Expression mittels RNAi führte dabei zu embryonaler Letalität. Weiterhin führte eine gewebespezifische Induktion von Bx42 in Abhängigkeit der verwendeten Treiberlinien bei unterschiedlichen Temperaturen zu mehreren verschiedenen adulten Phänotypen. Diese Phänotypen waren die Grundlage für die Annahme, dass Bx42 eine Rolle in der Regulation mehrerer verschiedener Zellsignalwege spielt. In der Tat interagiert Bx42 mit den Proteinen des Notch-Signalweges Suppressor of hairless [Su(H)] und Notch intracellular domain (N-IC). Zusätzlich werden bei einer Verminderung von Bx42 die Notch Zielgene cut (ct) und enhancer of split m8 [e(Spl)m8] reprimiert (Negeri et al., 2002). In dieser Arbeit wurde die Beteiligung von Bx42 am TGF-ß/Dpp Signalweg untersucht. Es wurde gezeigt, dass Bx42 mit den TGF-ß/Dpp-Signalweg Proteinen Mad und Medea sowohl in vitro als auch in vivo interagiert. Die dabei verwendeten Methoden waren das Hefe-Zwei Hybrid-Sytem und Ni-NTA-Pulldown-Assays. Domänen der Smad Proteine (Mad und Medea), die für die Interaktion mit Bx42 notwendig sind, wurden mit Hilfe von Deletionskonstrukten untersucht. Es konnte gezeigt werden, dass die stark konservierte MH2-Domäne dieser Proteine für die Interaktion notwendig ist. Zudem belegten Versuche die genetische Interaktion zwischen Bx42 und Medea, in denen ein Bx42-RNAi-Phänotyp durch die gleichzeitige Überexpression von Medea gerettet werden konnte. Es ist bekannt, dass das humane Bx42-Homolog Skip sowohl mit den Proteinen Smad2 und 3 des TGF-ß/Activin Signalweg, als auch mit den Onkogenen Sno und Ski interagiert. Skip wirkt hier als Antagonist der Ski/Sno-Wirkung auf den TGF-ß/Activin-Signalweg und fungiert als Koaktivator (Leong et al., 2001). Die Interaktion zwischen Bx42 und der TGF-ß/Activin-Signalweg Komponente dSmad2, sowie mit dem Onkogen dSno konnte in dieser Arbeit auch für Drosophila bewiesen werden. Die Bedeutung dieser Wechselwirkung muss noch in weiteren Arbeiten analysiert werden. Der Einfluss der Bx42-RNAi-Induktion auf die TGF-ß/Dpp Zielgene distal-less (dll), optomotor blind (omb) und spalt (sal) wurde anhand von Reportergen Untersuchungen mit enhancer-trap-Linien und RNA in situ Hybridisierung untersucht. Es konnte gezeigt werden, dass das Ausschalten von Bx42 die Expression dieser Gene in ähnlicher Weise reprimiert, wie eine Elimination des TGF-ß/Dpp-Signals. Diese Ergebnisse unterstützen die Annahme, dass Bx42 in der Lage ist, TGF-ß/Dpp Zielgene durch eine Wechselwirkung mit Mad und Medea zu aktivieren. / The importance of Bx42 in Drosophila development was demonstrated using Bx42-RNA interference. The ubiquitous downregulation of Bx42 generated embryonic lethality, indicating the importance of this protein in early development. The tissue specific induction of Bx42-RNAi resulted in several different phenotypes depending on the driver line and the temperature at which animals were raised. The phenotypes obtained were the key point for the assumption that Bx42 may play a role in the regulation of a number of different cellular signalling pathways. Indeed, within the Notch signalling pathway Bx42 interacts genetically with Suppressor of hairless [Su(H)] and Notch intracellular domain (N-IC). Additionally, the reduction of Bx42 negatively affected the expression of the Notch target Genes cut (ct) and enhancer of split m8 [e(Spl)m8] (Negeri et al., 2002). In this work, the involvement of Bx42 in the Dpp signalling pathway was investigated. It was shown that Bx42 interacts both in vitro and in vivo, as demonstrated by yeast two hybrid protein-protein studies and Ni-NTA pull-down assays, with the TGF-ß/ Dpp components Mad and Medea. Domains of Smads (Mad and Medea) required for Bx42 interaction were examined using deletion constructs of Smads and the importance of the well conserved MH2 domains of Mad and Medea for this interaction was revealed. Moreover, the rescue of the Bx42-RNAi phenotype by the simultaneous overexpression of Medea demonstrated the genetic interaction between Bx42 and Medea. Furthermore, evidences for the interaction of Bx42 with the TGF-ß/Activin pathway component dSmad2 and with the oncogene protein dSno were obtained from interaction assays. The human homologue of Bx42, Skip, also interacts with Smad2/3 or Sno. The meaning of this interaction in Drosophila has yet to be analysed. The influence of Bx42-RNAi induction on the expression of Dpp target genes distal less (dll), optomotor blind (omb) and spalt (sal) was also investigated using enhancer trap lines and RNA in situ hybridisation. In this way it was proven that these genes are suppressed as they are by elimination of Dpp signalling. These results suggest that Bx42 may be able to modulate positively TGF-ß/Dpp signalling through an interaction with the signalling transducer Mad and Medea.

A major goal of evolutionary developmental biology is to explore mechanisms and events underlying evolution of the myriad body plan morphologies expressed both genetically and phenotypically within the animal kingdom. Arthropods exhibit an astounding array of morphological diversity both within and between representative sub-phyla, thus providing an ideal phylum through which to address questions of body plan innovation and diversification. Major arthropod groups are recognised and defined by the distinct form and number of articulated appendages present along the antero-posterior axis of their segmented bodies. A great deal is known about the developmental genetics of limb development in the model insect Drosophila melanogaster, added to which, much comparative gene expression data and a growing body of functional genetic data is emerging for other arthropod species. Arthropod limb primordia are consistently marked by expression of the homeobox gene Distal-less (Dll), and the focus of this thesis is to compare signalling mediated by early Dll regulatory genes activity along antero-posterior and dorso-ventral embryonic axes during limb specification in Drosophila, with the activity of their orthologs in the widely disparate chelicerate, the spider mite Tetranychus urticae - interpreting new data with that available for other arthropods. Having made a detailed study of spider mite embryonic (and post-embryonic) development, to provide a basis for understanding mRNA transcription and protein activity patterns, I confirmed typical expression of Tetranychus Dll in prosomal limb primordia. I obtained limited results for the candidate antero-posterior positioning genes wingless and engrailed, although one of the two engrailed paralogs I identified is reportedly expressed in posterior segmental compartments, consistent with possible conservation of Engrailed-Wingless interactions in metameric patterning and positive regulation of Dll in arthropod limb specification. In Drosophila, wingless-dependent Dll transcription is restricted along the dorso-ventral axis by dorsal Dpp-mediated and ventral EGFR-mediated signalling gradients. Based on data from Tetranychus and other arthropods, neither dorsal nor ventral signalling regimes appear conserved outside the Drosophila system. Dll suppression in fly abdominal segments occurs due to powerful Hox (Ubx/AbdA) repression of the early Dll cis-regulatory element; this is discussed in relation to the independently evolved limbless chelicerate opisthosoma, informed by hypothetical scenarios of cis (regulatory DNA) and trans (coding sequence) evolution. Given practical difficulties and limitations encountered while working with spider mites, I offer a final assessment of the place of Tetranychus urticae as a non-model, and yet still valuable chelicerate species to consider carrying into the exciting future of evolutionary developmental biology.