During animal development, epithelial tissues undergo morphogenesis in order to build tissues, organs and body structure. A key driving force in epithelial morphogenesis is cell rearrangement, which results from the remodelling of cell-cell contacts. In epithelia, cell-cell contacts are connected via vertices, where 3 or more cells meet. It is unknown whether cell vertices play an active role in regulating cell rearrangement, however recent work from a number of model systems has suggested that cell vertices are important sites for sensing and regulating tissue tension and providing geometric cues for morphogenetic cell behaviours. I have used the Drosophila model system to investigate the behaviour and role of vertices in epithelial morphogenesis, using as an entry point a newly discovered marker of epithelial vertices, the Immunoglobulin-superfamily domain protein Sidekick. I have characterised the localisation of Sidekick tagged with YFP in a number of Drosophila epithelial tissues and found that in most epithelial tissues, Sidekick-YFP localises to vertices at the level of adherens junctions. Using super-resolution microscopy, I investigated the localisation of Sidekick-YFP during germband extension in the early embryo and found that Sidekick-YFP forms string-like structures at cell vertices at the level of adherens junctions, indicating that large multi-protein complexes containing Sidekick define a novel cortical domain in Drosophila epithelia. Through characterisation of a null allele of sidekick in Drosophila early embryos using both manual and automated large-scale quantitative analysis, I have found that tissue geometry, cell adhesion and morphogenetic cell behaviours are abnormal in the absence of sidekick. Together, my work shows that Sidekick at apical vertices is a novel regulator of epithelial morphogenesis.

Orientador: Sergio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T16:18:21Z (GMT). No. of bitstreams: 1 Ribeiro_MarianaMartins_M.pdf: 3243931 bytes, checksum: d6fb7208f8b58921ce7badece04e363b (MD5) Previous issue date: 2010 / Resumo: A evolução dos primatas foi marcada por uma redução em tamanho das mandíbulas e maxilas acompanhada por uma redução geral de tamanho de dentes. Esta redução foi facilitada pela organização da dentição em módulos que são autônomos em função e evolução. Esta redução segue uma regra simples: quanto mais tardio o desenvolvimento do dente maior será sua redução. Modelos foram propostos para explicar as variações no padrão de dentição mamária, porém nenhum destes modelos leva em consideração o tamanho da mandíbula e maxila já que a falta de espaço parece ter gerado esta redução. O objetivo deste trabalho é desenvolver um modelo novo que considera o espaço disponível medindo o palato e área e comprimento de molares. Neste estudo foram medidas 85 maxilas de primatas e os dados submetidos a análises estatísticas. Este estudo além de prover um modelo que pode estimar o tamanho de cada molar e tamanho de palato secundário em primatas também salienta algumas tendências observadas nos primatas estudados como, por exemplo, a presença de um terceiro pré-molar em alguns primatas e a grande variação de tamanho do terceiro molar / Abstract: The primate evolution was marked by a reduction in size of the jaws accompanied by a general reduction of teeth size. This reduction was facilitated by the organization of the dentition into modules that are autonomous in function and evolution. This reduction follows a simple rule: the later the tooth develops the greater will be its reduction. Models have been proposed to explain the variations in the pattern of mammalian dentition however none of these models takes into account the jaws size, since the lack of space seems to have triggered this reduction. The aim of this paper is to develop a new model that considers the space available by measuring the palate and molars area and length. In this study 85 upper jaws of primates were measured and statistical analysis was carried out with the data. This study not only provides a model that can estimate the size of each molar and secondary palate in primates but also points out some trends observed in the primates studied as, for instance, the presence of a third premolar in some primates and the great variation in size of the third molar / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

The complex and sophisticated circuitry of the neocortex is assembled from an extraordinarily diverse repertoire of neuronal subtypes that reside in distinct functional areas. In recent years, a number of key regulators over neocortical projection neuron subtype and area specification have been identified. It is becoming increasingly clear that these regulators function within a highly-interconnected network, acting in parallel, synergistically, and cross-repressively to orchestrate cortical development. Moreover, an emerging understanding of cortical development has revealed that subtype and area identity are intimately interrelated, and that specification occurs based on several sequential molecular decision points. Although great strides have been made in recent years toward understanding molecular controls over neocortical projection neuron development, many important controls remain to be discovered, and mechanisms by which recently-identified regulators act to delineate subtype and area identity are not well understood. In this dissertation, I characterize functions of two zinc finger transcription factors, Ctip2 and Ctip1, in postmitotic projection neuron subtype and area identity acquisition, using in vivo gain- and loss-of-function approaches in the mouse. I find that Ctip2, known for several years as a central functional control over corticospinal motor neuron (CSMN) terminal differentiation and connectivity, is required both cell-autonomously (within CSMN) and non-cell-autonomously (within striatal medium-sized spiny neurons that surround CSMN axons traveling in the internal capsule) for CSMN to achieve proper connectivity with the spinal cord. In addition, I find that Ctip1, a transcription factor not previously functionally investigated in neocortical development, is a novel control over 1) corticothalamic and callosal projection neuron development and projection neuron migration; and 2) postmitotic area identity acquisition and the formation of sensory maps. Taken together, these results reveal previously unknown functions of Ctip1 in neocortical development, and novel sites of action for Ctip2 control over CSMN connectivity. Ctip1 and Ctip2 are transcriptional controls over the postmitotic specification of neocortical projection neuron subtype and area identity, and over projection neuron connectivity with distant targets.

The zebrafish (Danio rerio) has emerged as a model to study vertebrate development due to rapid ontogenetic processes with external embryonic development. It is also an excellent model to study the mechanisms of regeneration and in this respect, the caudal fin is particularly convenient because it is easily accessible for experimental manipulation. Collection of quantitative data and postulation of theoretical models have become an attractive practice to explain complex biological problems. These models are used to test hypothetical mechanisms and predict results, but they also require calibration and validation with analysis of experimental data. This thesis aims at studying the developmental control of fin joint formation, which determines segment patterns of the rays in the caudal fin of zebrafish, before and after an amputation event, through a computational approach and imaging morpho-dynamics. We used a computational approach based on a quantitative framework developed for the analysis of fish fin development and regeneration and more specifically focused our analysis on the pattern of bone segments forming the ray. This allowed us to generate visual maps of the developing and regenerating caudal fins based on average fin data. The results from our experimental set show that bone segments at the amputation plane are longer after regeneration than segments at the same position in non-amputated fins. We also optimized a previously proposed morphogen driven model for fin growth and regeneration to accurately recreate segment numbers based on experimental data. Finally, we collected segment regression data that could be integrated into a new visual map method to analyse fin bony segment patterns.

Experience of stress and/or cortisol, the end-product of activation of the hypothalamic-pituitary-interrenal (HPI) axis, may serve as a cue to trigger developmental plasticity. In fish, most research in this area has focused on effects of maternal stress or maternal cortisol levels on development, particularly with respect to the HPI axis and stress responses, and little attention has been paid to the effects of an endogenous stress response during early life. In the current study, zebrafish (Danio rerio) at four developmental stages (4, 7, 15 or 35 days post fertilization, dpf) were subjected to an air exposure stressor twice a day for two days. Individuals stressed early in life exhibited decreased survival and growth, increased whole-body Na+ and Ca2+ concentrations, and altered HPI axis activity associated with changes in anxiety-related behaviour at 7 to 35 dpf, with most effects diminishing with increasing age. Stress at 7 dpf was particularly effective at eliciting phenotypic changes, suggesting this age represents a critical window for cortisol to influence development. Finally, stress at 35 dpf induced masculinization, suggesting that cortisol influences sexual differentiation in zebrafish. These findings demonstrate that early-life stress in zebrafish triggers developmental plasticity, with effects on physiology and behaviour mediated by the HPI axis in an age-dependent manner.

The primary aim of my PhD thesis was to identify and characterise novel modulators of intracellular signalling during early vertebrate development. The first phase of my thesis was to design and execute a large-scale gain of function screen in order to identify novel modulators of various important signal transduction pathways during early Xenopus development. From this screen I identified twenty novel of growth factor signalling. In the second phase of my PhD study, I concentrated on the characterization and mode of action of one of the genes I identified in the screen; namely fezf2. I showed that Fezf2 regulates neurogenesis in the diencephalon by locally promoting Wnt signalling through repression of lhx2 and lhx9. Notably, this investigation on the function of fezf2 not only revealed the previously undiscovered role of fezf2-mediated Wnt regulatory mechanism during diencephalon development, but also confirmed our in vivo screening approach in identifying potential regulators of signalling pathways. To the end, my PhD project has provided me with a fruitful journey of discovery, which started with the design and execution of a large-scale screen, ending with the detailed characterization of a factor involved in the modulation of signalling and forebrain development. This study is has broadened our understanding of how intracellular and extracellular signals are integrated during embryonic development process, which forms an interactive network ultimately resulting in appropriate cell differentiation, organ formation, and regional patterning.

Gastrulation is a fundamental process that reorganizes the primary germ layers to shape the internal and external features of an early embryo. Morphogenetic movements underlying this process can be classified into a variety of different types of cellular movements. I will focus on investigating in this thesis two types of cell movements in the dorsal mesoderm; mediolateral cell intercalation and convergence and extension. During gastrulation, mesoderm cells send protrusions to gain traction on neighboring cells and the surrounding extracellular matrix; a process called mediolateral cell intercalation. Mesoderm cells use this type of cell movement to converge and extend the dorsal mesoderm tissue during gastrulation; a process called convergence and extension. These morphogenetic movements are essential to form the early embryo and are important for later development. There are a number of different proteins involved in regulating the morphogenetic movements during gastrulation. The Planar Cell Polarity Signaling Pathway helps establish individual cell polarity and is activated in dorsal mesoderm cells undergoing convergence and extension. In addition, dorsal mesoderm cells migrate by using integrin receptors and the surrounding extracellular matrix to correctly position the mesoderm in the embryo. I will focus my efforts on analyzing the function of ADAM proteins during Xenopus laevis gastrulation. The ADAM family of metalloproteases is important for a variety of biological processes. ADAM proteins function as ectodomain sheddases by cleaving membrane bound proteins involved in signal transduction, cell-cell adhesion, and cell-extracellular matrix adhesion. I will focus on investigating the roles of two ADAM family members; ADAM13 and ADAM19 during gastrulation. Both ADAM13 and ADAM19 are expressed in the dorsal mesoderm during gastrulation. Throughout early embryonic development, ADAM13 is expressed in the somitic mesoderm and cranial neural crest cells. ADAM19 is expressed in dorsal, neural and mesodermal derived structures such as the neural tube, notochord, the somitic mesoderm, and cranial neural crest cells. Since ADAM13 and ADAM19 are expressed in similar tissues, I investigated if both proteins functionally interacted. I show that a loss of ADAM13 protein in the embryo reduces the level of ADAM19 protein by 50%. In the opposite experiment, a loss of ADAM19 protein in the embryo reduces the level of ADAM13 protein by 50%. This suggests that both ADAM13 and ADAM19 are required to maintain proper protein levels in the embryo. This might be explained through their physical interaction in a cell. The ADAM19 Proform binds to the ADAM13 Proform in cultured cells. Through domain analysis, I show that ADAM19 binds specifically to the cysteine-rich domain of ADAM13. When co-overexpressed in a cell, the level of Mature ADAM13 (compared to the Proform) is reduced suggesting a complex form of regulation. I propose a few models that discuss how ADAM19 may function as a chaperone to stabilize and regulate the further processing of ADAM13 protein. Some of the unpublished work discussed in this thesis focuses on the roles of ADAM13 and ADAM19 in the dorsal mesoderm during gastrulation. Specific emphasis is made on investigating the axial mesoderm during notochord formation. I show that ADAM19 affects gene expression important for the A-P polarity of the notochord while ADAM13 does not. The changes in gene expression can be partially rescued by the EGF ligand Neuregulin1β, a known substrate for ADAM19 in the mouse. ADAM13 and ADAM19 are important for convergence and extension movements of the axial mesoderm during gastrulation. Specifically, a loss of ADAM13 or ADAM19 causes a delay in mediolateral cell intercalation resulting in a significantly wider notochord compared to control embryos. These defects occur without affecting dishevelled intracellular localization or the activation of the PCP signaling pathway. However, a loss of ADAM13 or ADAM19 reduces dorsal mesoderm cell spreading on a fibronectin substrate through α5β1 integrin. To conclude, the work presented in this thesis focuses on the similarities and differences of ADAM13 and ADAM19 in the early embryo. Although ADAM13 and ADAM19 are required for normal morphogenetic movements during gastrulation, my data suggests they have different functions. ADAM13 appears to function in regulating cell movements while ADAM19 appears to function in regulating cell signaling. I propose a few models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation.

Epidermal appendages are found on every vertebrate this world has to offer. In fish, these are commonly represented by scales. While we have a solid grasp of how scales develop, little is known about the underlying genetic mechanisms behind these phenotypic changes. Using two species of African cichlids (Labeotropheus fuelleborni and Tropheops “red cheek”) with varying scale phenotypes, we sought to examine their F2 hybrid offspring and statistically link the responsible genetic elements to their respective parental phenotypes through Quantitative Loci Trait (QTL) analysis. Scales were removed from six different locations across the midline of each individual. Then, numerous traits on each scale were measured, and these values were used in the QTL analysis. 42 significant QTL were identified, with multiple QTL intervals possessing promising candidate genes. These genes include: fgfr1b, efna5a, TGIF1, eIF6, and col1a1a. Previous studies have implicated these particular genes and gene families to play important roles in scale and placode development. However, they represent the minority of QTL intervals discovered, providing direction for future research towards the other QTL intervals represented by this study.

Modularity plays a pivotal role in evolution, as the compartmentalization of components of a system allows their independent optimization in isolation, minimizing the effect on the system as a whole. As a manifestation of this universal design principle, evidence suggests modularity plays a key role in the function of the brain as well, allowing the compartmentalization of specific structural and functional units before their integration. Despite this, it’s unknown how modularity arises during the development of neural circuits. Accordingly, observing the development of the modularity of the nervous system and correlating this with the emergence of specific behaviors has the potential to highlight features of the functional role of modularity in the mature nervous system. To explore this issue, we work with the small cnidarian Hydra vulgaris, a representative of some of the simplest nervous systems in evolution. Depending on the size of the animal, Hydra’s isometrically scaling nervous system of 300-2,000 neurons is organized in two independent nerve nets in its ectoderm and endoderm and is distributed through the body of the animal without any cephalization or ganglia. Moreover, under the right conditions Hydra can reassemble itself into a normal animal after complete dissociation into individual cells. Using transgenic Hydra which express the calcium sensor GCaMP6s in every neuron (Dupre and Yuste, 2017) we have imaged the neuronal activity of dissociated preparations as they re-aggregate then regenerate over a period of several days. We demonstrate the robust synchronization of Hydra’s neural nets during the process. Of the possible routes toward synchronization, we observe that an initially random structure takes on a hierarchical organization as small groups of neurons synchronize. As these proto-circuits further synchronize, the modularity of the system increases, accompanied by a loss of the hierarchical depth of the network structure as normal behavioral rhythms resume during regeneration.

During development DNA-binding transcription factors are deployed downstream of patterning events to enable specific gene regulatory programs that define diverse cell identities. Within a given eukaryotic cell only a subset of potential binding targets in the genome, called cis-regulatory modules, are available due to the distribution of nucleosomes which restrict access to the underlying DNA. The accessible landscape of cells is highly dynamic over time and across different cell types, although how this process is regulated and influences the function of transcription factors in patterning of complex tissues is not well understood. In this thesis I focused on dissecting the cell type-specific chromatin accessibility landscapes that distinguishes different cell populations within the Drosophila dorsal appendages. The patterning of this system is extremely well characterized allowing for a detailed understanding of how transcription factors at the top of cell fate hierarchies influence, or respond to, the chromatin landscape during development. In Chapter 2 I describe the differences in chromatin accessibility along the proximal-distal axis of the wing imaginal disc which gives rise to distinct populations of the thoracic body wall and appendage in the second thoracic segment (T2). I found that a major driver of chromatin differences in these populations is the repressive input of the conserved insect wing marker Nubbin, whose function in the appendage is associated with decreasing accessibility of select chromatin regions relative to their conformation in body wall cells. In Chapter 3 I characterized the serially homologous body wall and appendage cells in the adjacent third thoracic body segment (T3), which diverge extensively in morphology from the T2 state due to influence of a single gene, Ultrabithorax (Ubx). Ubx is a member of the Hox gene family which functions to provide cells with spatial identity along the anterior-posterior axis. I show this function for Ubx in specifying T3 cells coincides with widespread changes to chromatin accessibility which contribute to a segment and cell type-specific regulatory program.