Abstract How cell and tissue interactions lead to complex structures and differentiated cell types during organogenesis is still one of the most fundermental questions in modern molecular biology. Laminin appears to have a role in branching morphogenesis during organ development. Laminin5 (α3β3γ2) is an epithelium-specific isoform of laminin and previous report has shown that two alternative transcripts for the γ2 chain, the longer γ2 and the shorter γ2*, result from alternative use of the last exon in the human LAMC2 gene. But the transcription of murine laminin γ2 and γ2* and their biological significance have remained unclear. Type XVIII collagen is a newly identified member of the collagen family. It may be involved in the Wnt signaling pathway, since its longest N-terminal variant contains a frizzled domain, which is part of the Wnt receptor and could antagonize Wnt signaling when secreted. Wnt2b is a new member of the Wnt family. Also its function in organogenesis is unknown. In this study, we have investigated the expressions of laminin γ2 and γ2*, type XVIII collagen and Wnt2b during mouse organogenesis. The function of type XVIII collagen in developing lung, kidney and a recombination of ureteric bud and lung mesenchyme tissue and the function of the Wnt-2b gene during kidney organogenesis were studied by using the combined methods of traditional experimental embryology and modern molecular biology. Two alternative laminin γ2 transcripts were demonstrated in mouse. In the developing kidney, the shorter γ2* form was localized in the mesenchyme, whereas the longer γ2 form was only present in the epithelium of the Wolffian duct and in the ureteric bud, indicating different functions for the γ2 variants. Type XVIII collagen was expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomed localized to the epithelial tips in the early-stage lung, while it was confined to the epithelial stalk region and was absent from the nearly formed ureteric tips in the kidney. In recombinants of ureteric bud and lung mesenchyme, the type XVIII collagen expression pattern in the ureteric bud shifted from the kidney to the lung type, accompanied by a shift in epithelial Sonic Hedgehog expression. The lung mesenchyme was also sufficient to induce ectopic lung Surfactant Protein C expression in the ureteric bud. A blocking antibody for the type XVIII collagen reduced the number of epithelial tips in the lung and completely blocked ureteric development with lung mesenchyme, which was associated with a notable reduction in the expression of Wnt2. The shift in type XVIII collagen expression in ureteric bud and lung mesenchyme tissue recombinant was also accompanied by the significant morphological changes in the branching pattern in ureteric bud development. Wnt2b was expressed in numerous developing organs in the mouse embryo, but it was typically localized in the perinephric mesenchymal cells in the region that partly overlaps the presumptive renal stroma at E11.5. Functional studies of the kidney demonstrated that 3T3 cells expressing Wnt2b were not capable of inducing tubule formation but rather stimulated ureteric development. Recombination of ureteric bud treated with cells expressing Wnt2b and isolated kidney mesenchyme resulted in recovery of the expression of epithelial marker genes and better reconstituted organogenesis. Lithium, a known activator of Wnt signaling, was also sufficient to promote ureteric branching in reconstituted kidney in a manner comparable to Wnt2b signaling. Our data suggest that different organ morphogenesis is regulated by an intraorgan patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen. In the mouse kidney, Wnt2b may act as an early mesenchymal signal controlling morphogenesis of epithelial tissue, and the Wnt pathway may regulate ureteric branching directly.

Abstract The polyamines putrescine, spermidine and spermine are highly regulated polycations present in virtually all cells of higher eukaryotes. They are essential for proper cell growth and differentiation by participating in various physiological processes including DNA, RNA and protein synthesis, apoptosis and interactions with ion-channels. The complexity of polyamine metabolism and the multitude of compensatory mechanisms that are invoked to maintain polyamine homeostasis argue that these molecules are critical for cell survival. The primary aim of this study was to gain a better understanding of the mode of action of polyamines and the regulatory mechanisms in which they are involved. Transgenic mice overexpressing the polyamine biosynthetic enzymes S-AdoMetDC and ODC were found to maintain their polyamine pools by acetylation of spermidine and spermine and an increased export of these acetylated compounds. The expression of various genes was studied as a response to polyamine deprivation in cell- and kidney organ culture. Among these genes acetyl-CoA synthetase and ornithine decarboxylase were demonstrated to be developmentally regulated. Changes in gene expression patterns, with most of the transcripts upregulated in the polyamine-depleted samples, indicated selective stabilization of mRNAs. Polyamines were shown to play an important role in kidney organogenesis as their depletion results in a reduction of ureteric branching and retardation of tubule formation. The selective changes of various genes in the ureteric bud and mesenchyme indicate that polyamines might have a role in the regulation of epithelial-mesenchymal interactions during mouse kidney development.

Many vertebrate organs form via the sequential, reciprocal exchange of signaling molecules between juxtaposed epithelial (E) and mesenchymal (M) tissues. For example, the instructive signaling potential for tooth development (odontogenesis) resides in the dental epithelium at the initiation-stage, and subsequently shifts to the dental mesenchyme one day later at the bud-stage. However, the properties of the gene regulatory networks (GRNs) that control the signaling dynamics during epithelial-mesenchymal (E-M) interactions in organogenesis are largely unknown. This dissertation describes an interdisciplinary effort between developmental and systems biology to elucidate the E-M GRN that controls early odontogenesis. The results provide a molecular mechanism for the longstanding paradigm of sequential, reciprocal E-M tissue interactions in development. We generated large-scale spatiotemporal gene expression data for the developing mouse tooth. Surprisingly, the shift in signaling molecule expression from E to M is accompanied by a striking concordance in genome-wide expression changes in both E-M compartments as development proceeds. We hypothesized that since diffusible signaling molecules can act on either E or M independent of their tissue site of synthesis, signaling molecules are uniquely able to simultaneously synchronize and couple the transcriptional dynamics and hence the developmental progression of E and M. To identify the unifying mechanism behind concordant E and M genome-wide expression changes in the face of the discordant expression changes in signaling molecule expression, we developed a novel probabilistic technique that integrates regulatory evidence from microarray gene expression data and the literature to determine the E-M GRN for early tooth development. This GRN contains a uniquely configured E-M Wnt/Bmp feedback circuit in which the Wnt and Bmp signaling pathways in E cross-regulate the expression of Wnt and Bmp4 signaling molecules, whereas both pathways jointly regulate Bmp4 expression in M. We validated the Wnt/Bmp feedback circuit in vivo using compound genetic mutations in mice that either short-circuit or break the circuit, and used mathematical modeling to show how the structure of the Wnt/Bmp feedback circuit can account for reciprocal signaling dynamics. Collectively, these results provide a simple mechanistic framework for how simultaneous signal transduction in E-M compartments can account for the signaling dynamics in organogenesis.

Some epithelial cancers can be induced to revert to quiescent differentiated tissues when combined with embryonic mesenchyme; however, the mechanism of this induction is unknown. This dissertation is based on the hypothesis that because extracellular matrix (ECM) plays a critical role during organ development in the embryo, it also may mediate the differentiation-inducing effects of embryonic mesenchyme on cancer cells. To test this hypothesis, I first optimized methods to isolate ECMs from whole tissues or cultured cells, and to repopulate them with cultured cells, using embryonic tooth as a model system. In Chapter 2, I describe these studies and use them to demonstrate that embryonic ECM is sufficient to regulate odontogenic signaling, cell fate decisions and histodifferentiation during normal tooth development. In Chapter 3, I adapt these methods to show that culture of breast cancer cells with ECM derived from embryonic mammary mesenchyme decreases tumor cell proliferation, and stimulates differentiation, including formation of hollow acini and ducts as well as enhanced expression of estrogen receptor-alpha and decreased migration. Further, when the inductive ECMs were injected into fast-growing breast tumors in mice, they significantly inhibited cancer expansion. Critically, the differentiation observed with ECM was the same as that observed in co-culture with mammary mesenchyme cells, showing that ECM is playing a dominant role in tumor cell normalization. In Chapter 4, I then set out to determine the mechanism by which embryonic ECM normalizes tumor cells, I analyzed the contributions of bound cytokines, ECM composition and mechanics. Western blot analysis revealed several bound growth factors, which remained following decellularization; however, removal of these growth factors using high salt washes had no effect on ECM-mediated normalization of tumors. Further, using proteomics analysis I identified eleven ECM proteins present only within inductive ECMs and by testing these proteins in 3D culture, I found three proteins -- collagen III, biglycan and SPARC -- that increased lumen formation to a similar extent as embryonic ECM. These data confirm that mesenchyme-induced tumor cell normalization is mediated by the insoluble ECM, and reveal the identity of some of the inductive molecules responsible for these effects.

This thesis is focused on the epithelial mesenchymal interactions in tumors derived from squamous epithelium including tumors arising from minor cell population (melanocytes). This study is also reflecting aspects of epithelial glycobiology resp. the study of endogenous lectins, the galectins, in head and neck squamous carcinomas. Galectins represent, in the current concepts of cell and tumor biology molecules with a remarkable potential. Galectins participate, besides in regulation of pre- and postnatal homeostasis in normal tissues, also in many pathological processes such as autoimmune reactions or malignancies. In this thesis, we demonstrated the presence of galectin-1 and -2 and their glycoligands in interphasic and mitotic nuclei, which may contribute to regulation of the cell cycle. Furthermore, we demonstrated galectin-9 as a sensitive marker of transformation normal to the dysplastic squamous epithelium in head and neck. The epithelial mesenchymal interactions represent mechanisms, which are responsible for dynamic maintenance of the homeostasis of the organism during prenatal development, postnatal growth and during cyclic renewal of certain tissues. These interactions also participate in wound healing. On the other hand they play a crucial role in the process of tumor transformation,...

This thesis is focused on the epithelial mesenchymal interactions in tumors derived from squamous epithelium including tumors arising from minor cell population (melanocytes). This study is also reflecting aspects of epithelial glycobiology resp. the study of endogenous lectins, the galectins, in head and neck squamous carcinomas. Galectins represent, in the current concepts of cell and tumor biology molecules with a remarkable potential. Galectins participate, besides in regulation of pre- and postnatal homeostasis in normal tissues, also in many pathological processes such as autoimmune reactions or malignancies. In this thesis, we demonstrated the presence of galectin-1 and -2 and their glycoligands in interphasic and mitotic nuclei, which may contribute to regulation of the cell cycle. Furthermore, we demonstrated galectin-9 as a sensitive marker of transformation normal to the dysplastic squamous epithelium in head and neck. The epithelial mesenchymal interactions represent mechanisms, which are responsible for dynamic maintenance of the homeostasis of the organism during prenatal development, postnatal growth and during cyclic renewal of certain tissues. These interactions also participate in wound healing. On the other hand they play a crucial role in the process of tumor transformation,...

Melanomas are one of the most aggressive types of tumours, with increasing incidence, high mortality and high potential to metastasize to a variety of diverse locations. The aim of this thesis was to study the tumour as a complex structure consisting not only of tumour cells but also of tumour stroma. Stromal cells play a major role in cancer biology. This is well documented for example in squamous cell epithelium tumours of the head and neck. Similar mechanisms can be expected to occur in melanomas. In the first experiment, we simulated the conditions in vivo during the metastatic process and studied the influence of non-adhesive environment both with and without the influence of stromal fibroblasts. The presented data demonstrates a change of tumour cells' phenotype leading to increased plasticity of the melanoma cells in these conditions. It also indicates the crucial role of stromal fibroblasts in interactions with melanoma cells. Cancer cell lines show variability in their behaviour, which is in accordance with well-known melanoma heterogeneity in clinical practice. The previous experiments in our laboratory indicate that cancer associated fibroblasts are able to influence the phenotype of a tumour cell line and this effect is based on a tumour type-unspecific mechanism. In the second part of...

CHAPER I: Through a review of the literature, this article discusses the genetic mechanisms that control tooth morphogenesis. Emphasis is placed upon the structure and function of some key molecules that participate in interactions between its epithelial-mesenchimal components. In this paper we will can understand the mechanisms that control tooth morphogenesis and the dentistry should pay special attention to possible consequences of tooth number anomalies. CHAPER II: The analysis of DNA is widely employed in the genetic studies. Human DNA in most cases is performed with samples obtained from peripheral blood. The use of buccal epithelial cells as a source of DNA for PCR amplifications has several advantages over blood sampling. In the present study our objective was to standardize DNA extraction from an oral swab, using a simple method. To test DNA quality, we amplified the exons 2 of MSX1 gene and the promoter region of LEF1 gene to patients with hypodontia. In conclusion, we standardized a simple DNA. extraction of oral cells, which presented lower costs and faster results, indicating to that DNA from oral brushes/swabs are a reliable source for genetic studies. The quantity and quality of extracted DNA was shown to be adequate for PCR and polymorphism analyses. CHAPER III: Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. In human the participation of MSX1 gene in craniofacial development have been evidenced by the studes that showed mutations in this gene. Hypodontia were shown to be caused by mutations in the MSX1 gene in human however, the mutation in the MSX1 gene cannot explain all types of tooth agenesis. Our data suggest that polymorphisms in MSX1 gene are associated with hypodontia. / CAPITULO I: Este artigo apresenta uma revisão bibliográfica sobre as evidências mais atuais sobre os aspectos genéticos da formação dos dentes. São abordadas nesse artigo as principais moléculas envolvida na interação epitélio-mesênquima, responsável pela formação da estrutura dental. O objetivo é contribuir para um melhor entendimento da expressão genética envolvida na formação do dente, bem como auxiliar na prática odontológica, procurando despertar a atenção do profissional para o conhecimento científico e facilitar assim a identificação de possíveis problemas relacionados à formação dos elementos dentais. CAPITULO II: A análise do DNA é largamente usada em estudos genéticos. O DNA humano, em muitos casos, é obtido através de amostras de sangue periférico. O uso de células descamadas da mucosa oral, como fonte de DNA para amplificação por PCR, tem apresentado muitas vantagens. Nesse estudo, nosso objetivo foi padronizar extração de DNA de células obtidas da mucosa oral, usando um novo método. Para testar a qualidade do DNA, nós amplificamos o segundo éxon do gene MSX1 em pacientes com hipodontia. Criamos um novo método de extração de DNA através de células da mucosa oral, que apresenta baixo custo e rápidos resultados, indicando que o DNA dessas células,quando extraídos por essa técnica, é suficiente para estudos genéticos. O DNA extraído mostrou-se adequado em quantidade e qualidade, para estudos de PCR e análises de polimorfismos. CAPITULO III: Hipodontia, a ausência congênita de dentes, é uma das alterações mais comuns na dentição humana. Vários dentes podem estar ausentes porém, os mais comuns são os terceiros molares, segundo pré-molares e incisivos laterais superiores. Embora essa alteração de número não represente um problema de saúde pública, ela pode causar disfunções mastigatórias e problemas estéticos graves. Nos humanos, o papel do gene MSX1 no desenvolvimento crânio facial tem sido esclarecido em estudos que identificaram mutações nesses genes, associadas a alterações da normalidade. Mutações-polimorfismos no gene MSX1 têm sido relatadas como responsáveis pela agenesia dental, no entanto, mutações neste gene não explicam todas as formas dessa alteração. Nossos resultados sugerem que polimorfismos no gene MSX1 estão associados com a hipodontia. / Doutor em Genética e Bioquímica