Segmentale Musterbildung in der terminalen Abdominalregion des zentralen Nervensystems von Drosophila melanogaster

In dieser Arbeit wurden Mechanismen der Musterbildung in der terminalen Abdominalregion des Zentralnervensystems von Drosophila melanogaster untersucht. Dazu wurden zunächst die Anzahl der angelegten Neuromere und das Muster der dort lokalisierten neuralen Stammzellen (Neuroblasten) analysiert. Dabei zeigte sich, dass sowohl die Größe der Neuromere, als auch die Anzahl an Neuroblasten von anterior nach posterior sukzessiv abnimmt, wobei keine geschlechtsspezifischen Unterschiede in der Anzahl der vorhandenen Neuroblasten festgestellt werden konnten. Durch die Kombination einer Vielzahl von molekularen Markern war es anschließend möglich, die Identität aller Neuroblasten in diesem Bereich aufzuklären und in einer Karte zusammenzutragen. Sie weisen alle eine serielle Homologie zu Neuroblasten in weiter anterior gelegenen Segmenten auf. Des Weiteren wurde die embryonale Identität der geschlechtsspezifischen Neuroblasten untersucht und deren postembryonalen mänchenspezifischen Zellstammbäume charakterisiert. Diese detaillierten Beschreibungen bildeten die Grundlage für die funktionelle Analyse von geschlechts- und segmentspezifischen Faktoren, die zur Musterbildung in dieser Region des Zentralnervensystems beitragen. So konnte gezeigt werden, dass die weibliche Isoform von doublesex den programmierten Zelltod der geschlechtsspezifischen Neuroblasten induziert, während die männliche Isoform diesen verhindert. Das Hox-Gen Abdominal-B zeigt relativ milde Effekte auf das Überleben dieser Neuroblasten, was darauf hindeutet, dass weitere Faktoren benötigt werden, um diesen Prozess in segmentspezifischer Weise zu kontrollieren. Die Funktion von Hox-Genen wurde ferner im Hinblick auf die abgeleitete Morphologie der terminalen Neuromere untersucht. Es konnte herausgefunden werden, dass die regulatorische Isoform von Abdominal-B auf mehreren Ebenen wirkt: Sie beeinflusst die Zusammensetzung bestimmter Zellstammbäume durch Modifikation von Zelldeterminationsprozessen und durch die Kontrolle des programmierten Zelltods. Außerdem unterdrückt sie die Bildung einer spezifischen Subpopulation von Neuroblasten. Allerdings benötigt Abdominal-B.r die Co-Expression des ParaHox-Gens caudal, um sein gesamtes Potenzial bezüglich der Suppression dieser Neuroblasten zu entfalten. Die vorliegende Arbeit hat somit erste Einblicke in die geschlechtsspezifische und segmentspezifische Spezifizierung der terminalen Abdominalregion des Zentralnervensystems von Drosophila auf Ebene des Neuroektoderms, der daraus hervorgehenden Neuroblasten und deren Tochterzellen gewährt. Die vollständige und detailgetreue Beschreibung des Neuroblasten-Musters und der postembryonalen männchenspezifischen Zellstammbäume hat zudem attraktive Modellsysteme für zukünftige Untersuchungen etabliert, an denen sich weitere Mechanismen der Musterbildung im Zentralnervensystem analysieren lassen.

Haematological parameters are normal in dominant white Franches-Montagnes horses carrying a KIT mutation

The KIT receptor protein-tyrosine kinase plays an important role during embryonic development. Activation of KIT is crucial for the development of various cell lineages such as melanoblasts, stem cells of the haematopoietic system, spermatogonia and intestinal cells of Cajal. In mice, many mutations in the Kit gene cause pigmentation disorders accompanied by pleiotropic effects on blood cells and male fertility. Previous work has demonstrated that dominant white Franches-Montagnes horses carry one copy of the KIT gene with the p.Y717X mutation. The targeted breeding of white horses would be ethically questionable if white horses were known to suffer from anaemia or leukopenia. The present study demonstrates that no statistically significant differences in peripheral blood parameters are detectable between dominant white and solid-coloured Franches-Montagnes horses. The data indicate that KIT mutations may have different effects in mice, pigs, and horses. The KIT p.Y717X mutation does not have a major negative effect on the haematopoietic system of dominant white horses.

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

[Lineage tracing of epicardial cells during development and regeneration].

Tracing the history of individual cells during embryonic morphogenesis in a structure as complex as the cardiovascular system is one of the major challenges of developmental biology. It involves determining the relationships between the various lineages of cells forming an organ at different stages, describing the topological rearrangements tissues undergo during morphogenesis, and characterizing the interactions between cells in different structures. However, despite the great expectations raised in the field of regenerative medicine, only limited progress has been made in using regenerative therapy to repair the cardiovascular system. Recent research has highlighted the role of the epicardium during cardiac regeneration, but it is still unclear whether it is important for molecular signaling or acts as a source of progenitor cells during this process. Consequently, increasing knowledge about the origin, diversification and potential of epicardial cells during development and homeostasis and under pathological conditions is of fundamental importance both for basic research and for the development of effective cellular therapies. The aims of this article were to provide a general overview of the classical techniques used for tracing cell lineages, including their potential and limitations, and to describe novel techniques for studying the origin and differentiation of the epicardium and its role in cardiac regeneration.

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^

XIPHOPHORUS ENZYME GENE EXPRESSION: TISSUE SPECIFICITY, GENETIC CONTROL, AND STABILITY IN CULTURED CELLS

Five permanent cell lines were developed from Xiphophorus maculatus, X. helleri, and their hybrids using three tissue sources, including adults and embryos of different stages. To evaluate cell line gene expression for retention of either tissue-of-origin-specific or ontogenetic stage-specific characters, the activity distribution of 44 enzyme loci was determined in 11 X. maculatus tissues, and the developmental genetics of 17 enzyme loci was charted in X. helleri and in helleri x maculatus hybrids using starch gel electrophoresis. In the process, eight new loci were discovered and characterized for Xiphophorus.^ No Xiphophorus cell line showed retention of tissue-of-origin-specific or ontogenetic stage-specific enzyme gene expressional traits. Instead, gene expression was similar among the cell lines. One enzyme, adenosine deaminase (ADA) was an exception. Two adult-origin cell lines expressed ADA, whereas, three cell lines derived independently from embryos did not. ADA('-) expression of Xiphophorus embryo-derived cell lines may represent retention of an embryonic gene expressional trait. In one cell line (T(,3)) derived from 13 pooled interspecific hybrid (F(,2)) embryos, shifts with time were observed at enzyme loci polymorphic between the two species. This suggested shifts in ratios of cells of different genotypes in the population rather than unstable gene expression in one dominant cell type.^ Verification of this hypothesis was attempted by cloning the culture--seeking clones having different genetic signatures. The large number of loci electrophoretically polymorphic between the two species and whose alleles segregated independently into the 13 progeny from which this culture originated almost guaranteed the presence of different genetic signatures (lineages) in T(,3).^ Seven lineages of cells were found within T(,3), each expressing genotypes at some loci not characteristic of the expression of the culture-as-a-whole, supporting the hypothesis tested. Quantitative studies of ADA expression in the whole culture (ADA('-)) and in clones of these seven lineages suggested the predominance in T(,3) of ADA deficient cell lineages, although moderate to high ADA output clones also occurred. Thus, T(,3) has the potential to shift phenotypes from ADA('-) to ADA('+) by simply changing proportions of its constituent cell types, demonstrating that such shifts can occur in any cell culture containing cells of mixed expressional characteristics.^

Cloning and molecular analysis of paraxis: A {\it trans\/}-acting factor required for somite maturation

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic-Helix-Loop-Helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. This dissertation focuses on the cloning, expression and functional analysis of a bHLH protein termed paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in both its amino and carboxyl termini. During the process of mouse embryogenesis, paraxis transcripts are first detected at about day 7.5 post coitum within the primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated within the myotome.^ To determine the function of paraxis during mammalian embryogenesis, mice were generated with a null mutation in the paraxis locus. Paraxis null mice survived until birth, but exhibited severe foreshortening along the anteroposterior axis due to the absence of vertebrae caudal to the midthoracic region. The phenotype also included axial skeletal defects, particularly shortened bifurcated ribs which were detached from the vertebral column, fused vertebrae and extensive truncation and disorganization caudal to the hindlimbs. Mutant neonates also lacked normal levels of trunk muscle and exhibited defects in the dermis as well as the stratification of the epidermis. Analysis of paraxis -/- mutant embryos has revealed a failure of the somites to both properly epithelialize and compartmentalize, resulting in defects in somite-derived cell lineages. These results suggest that paraxis is an essential component of the genetic pathway regulating somitogenesis. ^

Computational methods to create and analyze a digital gene expression atlas of embryo development from microscopy images

Abstract The creation of atlases, or digital models where information from different subjects can be combined, is a field of increasing interest in biomedical imaging. When a single image does not contain enough information to appropriately describe the organism under study, it is then necessary to acquire images of several individuals, each of them containing complementary data with respect to the rest of the components in the cohort. This approach allows creating digital prototypes, ranging from anatomical atlases of human patients and organs, obtained for instance from Magnetic Resonance Imaging, to gene expression cartographies of embryo development, typically achieved from Light Microscopy. Within such context, in this PhD Thesis we propose, develop and validate new dedicated image processing methodologies that, based on image registration techniques, bring information from multiple individuals into alignment within a single digital atlas model. We also elaborate a dedicated software visualization platform to explore the resulting wealth of multi-dimensional data and novel analysis algo-rithms to automatically mine the generated resource in search of bio¬logical insights. In particular, this work focuses on gene expression data from developing zebrafish embryos imaged at the cellular resolution level with Two-Photon Laser Scanning Microscopy. Disposing of quantitative measurements relating multiple gene expressions to cell position and their evolution in time is a fundamental prerequisite to understand embryogenesis multi-scale processes. However, the number of gene expressions that can be simultaneously stained in one acquisition is limited due to optical and labeling constraints. These limitations motivate the implementation of atlasing strategies that can recreate a virtual gene expression multiplex. The developed computational tools have been tested in two different scenarios. The first one is the early zebrafish embryogenesis where the resulting atlas constitutes a link between the phenotype and the genotype at the cellular level. The second one is the late zebrafish brain where the resulting atlas allows studies relating gene expression to brain regionalization and neurogenesis. The proposed computational frameworks have been adapted to the requirements of both scenarios, such as the integration of partial views of the embryo into a whole embryo model with cellular resolution or the registration of anatom¬ical traits with deformable transformation models non-dependent on any specific labeling. The software implementation of the atlas generation tool (Match-IT) and the visualization platform (Atlas-IT) together with the gene expression atlas resources developed in this Thesis are to be made freely available to the scientific community. Lastly, a novel proof-of-concept experiment integrates for the first time 3D gene expression atlas resources with cell lineages extracted from live embryos, opening up the door to correlate genetic and cellular spatio-temporal dynamics. La creación de atlas, o modelos digitales, donde la información de distintos sujetos puede ser combinada, es un campo de creciente interés en imagen biomédica. Cuando una sola imagen no contiene suficientes datos como para describir apropiadamente el organismo objeto de estudio, se hace necesario adquirir imágenes de varios individuos, cada una de las cuales contiene información complementaria respecto al resto de componentes del grupo. De este modo, es posible crear prototipos digitales, que pueden ir desde atlas anatómicos de órganos y pacientes humanos, adquiridos por ejemplo mediante Resonancia Magnética, hasta cartografías de la expresión genética del desarrollo de embrionario, típicamente adquiridas mediante Microscopía Optica. Dentro de este contexto, en esta Tesis Doctoral se introducen, desarrollan y validan nuevos métodos de procesado de imagen que, basándose en técnicas de registro de imagen, son capaces de alinear imágenes y datos provenientes de múltiples individuos en un solo atlas digital. Además, se ha elaborado una plataforma de visualization específicamente diseñada para explorar la gran cantidad de datos, caracterizados por su multi-dimensionalidad, que resulta de estos métodos. Asimismo, se han propuesto novedosos algoritmos de análisis y minería de datos que permiten inspeccionar automáticamente los atlas generados en busca de conclusiones biológicas significativas. En particular, este trabajo se centra en datos de expresión genética del desarrollo embrionario del pez cebra, adquiridos mediante Microscopía dos fotones con resolución celular. Disponer de medidas cuantitativas que relacionen estas expresiones genéticas con las posiciones celulares y su evolución en el tiempo es un prerrequisito fundamental para comprender los procesos multi-escala característicos de la morfogénesis. Sin embargo, el número de expresiones genéticos que pueden ser simultáneamente etiquetados en una sola adquisición es reducido debido a limitaciones tanto ópticas como del etiquetado. Estas limitaciones requieren la implementación de estrategias de creación de atlas que puedan recrear un multiplexado virtual de expresiones genéticas. Las herramientas computacionales desarrolladas han sido validadas en dos escenarios distintos. El primer escenario es el desarrollo embrionario temprano del pez cebra, donde el atlas resultante permite constituir un vínculo, a nivel celular, entre el fenotipo y el genotipo de este organismo modelo. El segundo escenario corresponde a estadios tardíos del desarrollo del cerebro del pez cebra, donde el atlas resultante permite relacionar expresiones genéticas con la regionalización del cerebro y la formación de neuronas. La plataforma computacional desarrollada ha sido adaptada a los requisitos y retos planteados en ambos escenarios, como la integración, a resolución celular, de vistas parciales dentro de un modelo consistente en un embrión completo, o el alineamiento entre estructuras de referencia anatómica equivalentes, logrado mediante el uso de modelos de transformación deformables que no requieren ningún marcador específico. Está previsto poner a disposición de la comunidad científica tanto la herramienta de generación de atlas (Match-IT), como su plataforma de visualización (Atlas-IT), así como las bases de datos de expresión genética creadas a partir de estas herramientas. Por último, dentro de la presente Tesis Doctoral, se ha incluido una prueba conceptual innovadora que permite integrar los mencionados atlas de expresión genética tridimensionales dentro del linaje celular extraído de una adquisición in vivo de un embrión. Esta prueba conceptual abre la puerta a la posibilidad de correlar, por primera vez, las dinámicas espacio-temporales de genes y células.

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Desarrollo de un procedimiento para la observación y caracterización mecánica de células sobre sustratos opacos mediante microscopía de fuerzas atómicas : aplicación al estudio de linfocitos

Las células en los tejidos biológicos están continuamente sometidas a estímulos físicos tales como la presión hidrostática y esfuerzos de tracción, compresión o cortante, entre otros. La importancia de los estímulos mecánicos en el comportamiento de las células se ha reconocido recientemente al comprobarse cómo la naturaleza de estas fuerzas puede cambiar en patologías tales como las enfermedades vasculares o el cáncer. En respuesta a estos cambios, las células reaccionan modificando desde su forma o aspecto hasta su ciclo celular. Consecuentemente, el interés por el comportamiento mecánico de las células ha experimentado un auge creciente que ha requerido el desarrollo de varias técnicas de caracterización. En este contexto, se puede afirmar que una de las técnicas que ha irrumpido con más fuerza en esta nueva área, situada entre el mundo biológico y el físico, es la microscopía de fuerza atómica. En esta Tesis se ha abordado el estudio mediante microscopía de fuerza atómica de linfocitos de ratón que constituyen un linaje celular especialmente difícil de caracterizar mediante esta técnica por su tamaño y naturaleza no adherente. Los linfocitos, como actores fundamentales del sistema inmune, tienen gran importancia en la determinación de la respuesta que un organismo desencadena ante la presencia de un biomaterial. Bajo esta premisa, y como condición previa a la caracterización de los linfocitos, ha sido necesario el desarrollo de una metodología robusta y de amplia aplicabilidad que permita el estudio de células sobre biomateriales. Finalmente y con el objetivo de correlacionar el comportamiento mecánico de los linfocitos con alguna característica fisiológica relevante, se ha analizado la hipótesis de que el comportamiento mecánico pueda ser utilizado como marcador de la edad biológica. Consecuentemente se ha abordado el estudio del comportamiento mecánico de los linfocitos clasificados por grupos de edad, de manera que se han obtenido los primeros resultados que indican cómo puede manifestarse el proceso de inmunosenescencia -depresión del sistema inmune relacionada con el envejecimiento- en el comportamiento mecánico de las células del sistema inmune. Cells within tissues are continuously exposed to physical forces including hydrostatic pressure, shear stress, and compression and tension forces. The relevance of these mechanical stimuli has recently been recognised by different works in which significant changes were observed in these forces when they were measued in individuals affected by cardiovasvular diseases or cancer. Cells may alter their orientation, shape, internal constitution, contract, migrate, adhere, modify the synthesis and degradation of extracellular constituents, or even their life cycle in response to perturbations in their mechanical environment. As a consequence of this, the attention in cell mechanical behavior has undergone a significant thrust and novel techniques have been developed. In this context, atomic force microscopy has become a basic tool for the progress of this field. In this Thesis, the mechanical behavior of living murine T-lymphocytes was assessed by atomic force microscopy. Lymphocytes play a main role in the immune system of the individual and, consequently, in the immune response triggered by the presence of a biomaterial. The observation and characterization of the lymphocytes required the development of a robust experimental procedure that allowed overcoming the difficulties related to the analysis of this cell lineage, in particular their relatively large size and non-adherent character. These procedures could be easily transferred to other non-adherent cell lineages. Finally, to check the viability of developed method, we study the lymphocyte mechanical behavior as a function of the murine ageing. The obtained data represent a first step in the knowledge about how mechanical stimuli can affect the age-dependent decrease in immunological competence, i.e., the immunosenescence.

A lineage-specific protein kinase crucial for myeloid maturation

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-β (PKC-β) and several stable PKC-β transfectants, we found that PRKX gene expression is under control of PKC-β; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/− mice that exhibit defective NK cell development. In the Id2−/− FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/− FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/− fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/− and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

A power law for cells

Darwin observed that multiple, lowly organized, rudimentary, or exaggerated structures show increased relative variability. However, the cellular basis for these laws has never been investigated. Some animals, such as the nematode Caenorhabditis elegans, are famous for having organs that possess the same number of cells in all individuals, a property known as eutely. But for most multicellular creatures, the extent of cell number variability is unknown. Here we estimate variability in organ cell number for a variety of animals, plants, slime moulds, and volvocine algae. We find that the mean and variance in cell number obey a power law with an exponent of 2, comparable to Taylor's law in ecological processes. Relative cell number variability, as measured by the coefficient of variation, differs widely across taxa and tissues, but is generally independent of mean cell number among homologous tissues of closely related species. We show that the power law for cell number variability can be explained by stochastic branching process models based on the properties of cell lineages. We also identify taxa in which the precision of developmental control appears to have evolved. We propose that the scale independence of relative cell number variability is maintained by natural selection.