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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 hypothetical 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 hypothetical models that discuss how each ADAM metalloprotease may function in the dorsal mesoderm and contribute to convergence and extension movements during gastrulation.>

No description available.

<p>Early Xenopus laevis development involves highly complex morphogenic movements. Two key movements are gastrulation, which establishes germ layer spatial arrangement, and neurulation, which results in the folding and closure of the neural tube. Multiple signaling pathways are involved in regulating cell adhesion, migration, shape and polarity during these processes to ensure normal development. Two of the most characterized pathways are the canonical and non-canonical Wnt pathways. However, the roles of all the individual molecules involved are not fully understood. In this thesis I provide initial characterization of the POZ-ZF transcription factor xZnf131. Znf131 is a transcriptional activator and its binding partner Kaiso negatively regulates this function. Since Znf131 and Kaiso display antagonistic roles and Kaiso mediates Wnt signaling and morphogenesis during Xenopus gastrulation and neurulation I hypothesize that xZnf131 is also required to regulate morphogenesis during these key developmental events.</p> <p>Like other POZ-ZF proteins, xZnf131 contains an amino-terminal POZ domain and a carboxy-terminal ZF domain comprised of five zinc fingers. xZnf131 is continuously expressed through early Xenopus development but was spatially localized to the dorsal and anterior structures of the embryo, notably the neural plate. Morpholino oligonucleotide (MO) knockdown of xZnf131 resulted in severe defects in notochord and neural plate formation, with abnormal cell morphology, typical of non-canonical Wnt misregulation. Interestingly, xZnf131 overexpression produced phenotypes very similar to xZnf131 knockdown suggesting that xZnf131 protein levels need to be tightly maintained to regulate the correct/normal morphogenic movements during Xenopus gastrulation and neurulation.</p> <p>Our findings indicate that xZnf131 plays a role in the morphogenic movements during Xenopus gastrulation and neurulation. Our data provides a useful foundation for future experiments to elucidate the biological mechanism of xZnf131 action during these key developmental processes.</p> / Master of Science (MSc)

The Wnts, a family of secreted glycoprotein ligands, act through the frizzled (Fz) receptor, a family of seven-transmembrane (7TM) receptor proteins, to mediate intracellular signaling pathways that regulate cell fate determination, cell migration, or both. Whereas many molecular components of the Wnt signal transduction cascade have been identified, it remains unclear how the signal is transduced from the Fz receptors to the cytoplasm. To address this important question, a membrane-based yeast two-hybrid (MbY2H) screen was performed to identify potential Fz-interacting proteins. For this screen, the Frizzled7 (Fz7) protein was used as the bait and a mouse brain library was used the prey. This screen identified resistance to inhibitors of cholinesterase 8 homolog A (Ric8A), a 542–amino acid cytoplasmic protein, along with other proteins as putative Fz7-binding proteins. Ric8A had been studied previously in C. elegans and D. melanogaster for its function in regulating asymmetric cell division as a receptor-independent guanine nucleotide exchange factor (GEF) for Gα proteins. Additional studies in M. musculus and X. laevis further uncovered a role for this protein during gastrulation and neurulation; however, the mechanisms by which Ric8A regulated these processes remained unclear. In this thesis, I show Ric8A to be a bona fide binding partner for both Fz7; that Ric8A can also bind to the phosphoprotein Dishevelled (Dvl); and that both its interaction with Fz7 and Dvl is Wnt-regulated. The spatial and temporal mRNA expression pattern of the Xenopus homologue of Ric8A suggests a potential role in regulating Wnt signaling. The Xenopus homologue of Ric8A was cloned and gain-of-function and loss-of-function approaches in Xenopus uncovered a role for Ric8A in gastrulation and neural tube closure. Additionally, we found inhibition of Ric8A function mechanistically prevents activation of Rac1 which is required downstream of Wnt/Fz signaling during gastrulation. Overall, this study uncovers a novel regulator of Wnt signaling during early development / Biology

Gastrulation involves a complex series of morphogenetic processes that unfold in precise coordination alongside tissue genetic patterning. During the gastrulation of Drosophila melanogaster, a monolayer of cells known as the blastoderm undergoes a sequence of tissue rearrangements that reveal axis determination and cell fates. The Drosophila model provides a comprehensive toolkit for studying embryogenesis, offering insights into its streamlined developmental program, which efficiently produces the final organism while maintaining robustness. This prompts an exploration of the mechanisms that coordinate individual morphogenetic events. Active forces, generated by cytoskeletal-driven cell shape changes, exert themselves on the viscoelastic blastoderm tissue, resulting in passive transmission. Traditional analysis involved studying these events at molecular, cellular, and tissue scales. However, the emergence of single-plane illumination microscopy, enabling whole volume (in toto) imaging of developing embryos with high temporal and spatial resolution, has reshaped this approach. This advancement in microscopy allows us to investigate how morphogenetic events synergise or hinder one another and how the embryo integrates this information to coordinate gastrulation. A recent development in gastrulation research involves identifying regions of heightened friction between the cells and the eggshell within the embryo. Integrins expressed near the midgut primordium have been found to mediate this interaction, challenging the notion that cell movement is the sole driving force of morphogenesis. Instead, static regions emerge as vital contributors to mechanical stability and the preservation of left-right symmetry during germ band extension. This role of integrins is not confined to Drosophila alone, as similar findings have been observed in the beetle Tribolium castaneum, emphasizing their role in orchestrating tissue flows and raising questions about their conservation. Consequently, my focus centered in understanding the physical forces underlying gastrulation, encompassing their interplay, balance, and conflicts in the developing embryo. I considered not only the active forces generated by cell shape changes, but also the passive forces transmitted in the surrounding tissue and the friction generated by the interaction with the physical constraint of the eggshell. In the initial chapter of this thesis, I delved into a lesser-explored morphogenetic event: the formation of the cephalic furrow. This transient tissue fold delineates the head from the trunk but differs from other invaginations in that it does not give rise to further differentiated structures. Instead, it unfolds later in development without leaving a trace of its origin. By employing in toto imaging alongside genetic and photo-manipulation techniques, I investigated the influence of the active forces driving the furrow formation on the head-trunk boundary tissue, and the consequences upon their absence. I found that the invagination is driven by active forces that propagate in the surrounding tissue. By genetic inhibition of the furrow, the presence of ectopic buckling was confirmed in the head-trunk boundary, appearing in between mitotic domains in a rather stochastic fashion. Next, I assessed the impact of further morphogenetic events in the head-trunk boundary tissue using whole embryo imaging and quantitative strain analysis. From my observations, I was able to conclude that the cephalic furrow primes tissue folding to dissipate forces coming from two sources, the local mitotic domains' expansion and the remote germ band compression. These discoveries suggest that the cephalic furrow and its genetic patterning may have evolved in response to the coalition of compressive forces in the head-trunk boundary tissue. The second chapter focused on understanding the ensemble of tissue flows while considering the newfound importance of static regions, mediated by the integrin subunit scab. The possibility to visualise the whole embryo upon mechanical photo-manipulation and integrin mutation, allowed me to uncover how enhanced friction's localised forces contribute to the directionality of tissue movements during the posterior midgut invagination. I was able to verify that cell-to-shell attachment helps maintaining the speed and direction of germ band extension, a highly mechanically unstable process. At the same time, I explored the effects of two milder expression sites of scab not addressed in the past on the mid-dorsal and ventral-anterior region of the embryo. I determined that the first contributes to the shift of the cephalic furrow posteriorly, which is required to allow mitotic domains divisions in the dorsal side of the head. Upon integrin depletion, ectopic buckling in between mitotic domains was observed, resembling our results in the first chapter. Lastly, thanks to whole volumetric imaging, the newly found expression site in the ventral-anterior region was determined to stabilise the head in face of torque generation of the deviating germ band. These results indicate a potential early safety mechanisms against symmetry breaking in the unstable stages of germ band extension, before posterior midgut invagination. In the concluding chapter, I explored the origins of left-right organismal symmetry instability during germ band extension. Analysing cartographic projections of the whole blastoderm surface, I discovered an inherent chirality identified in symmetric embryos at both the cellular and local tissue levels. By quantification of geometric features, along with tissue strain and curl rates, I found significant and consistent differences in all specimens in a specific region of the embryo in the lateral posterior side. These finding raises questions about the potential role of early chiral determinants in embryogenesis, that could translate local asymmetries into organismal twisting. In summary, this thesis underscores the significance of a multi-scale and interdisciplinary approach to embryonic development. The view of genetic patterning setting up the canvas for morphogenesis is taken to the next level, considering this canvas as an active material that needs fine coordination of single cellular events and the forces generated by them. These forces, in turn, shape molecular components and gene expression, culminating in a dynamic picture of a somewhat unstable, though robust, equilibrium of embryonic development.:I Introduction 1 Tissue morphogenesis 2 Embryonic development of Drosophila melanogaster 3 Modern methods for volumetric imaging 4 Project motivation and questions II Results 5 The role of the cephalic furrow. 6 Integration of tissue flows and static regions 7 Left-Right (a)symmetry 8 Discussion 9 Appendix

Die vorliegende Arbeit befasst sich mit Aspekten der frühen Entwicklung der Phoronida („Hufeisenwürmer“). An drei Arten wird der Furchungsprozess untersucht (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). Dies erfolgt sowohl mithilfe der 4D-Mikroskopie als auch anhand von immunocytochemischen Markierungen der Mitosespindeln und konfokaler Laser-Scanning-Mikroskopie. Verschiedene morphologische Merkmale des Furchungsprozesses werden quantitativ erfasst und innerhalb sowie zwischen den Arten verglichen. Die Ergebnisse zeigen eine weitgehend übereinstimmende Furchung bei P. pallida und P. muelleri Embryonen: Ab dem dritten Zellzyklus teilen sich die Blastomeren meist schräg – und alternierend dextral und sinistral – zur animal-vegetativ Achse. Dieses Muster zeigt überraschende Übereinstimmungen mit dem Muster der Spiralfurchung. Dies kann als morphologische Unterstützung molekular-phylogenetischer Befunde einer Stellung der Phoronida innerhalb der Spiralia/Lophotrochozoa interpretiert werden. Die Furchung bei P. vancouverensis unterscheidet sich von der Furchung der anderen beiden Arten; sie weist jedoch auch Unterschiede zu einer Radiärfurchung auf. Generell zeigt die Furchung aller drei Arten einen gewissen Grad an Variabilität. Anhand von in-vivo Einzelzellmarkierungen untersucht die Studie darüber hinaus das Schicksal der Blastomeren früher P. pallida Embryonen bis zu späten Gastrulationsstadien. Diese Analysen zeigen, dass die ersten beiden Furchungsteilungen durch die spätere Achse Blastoporus-Apikalplatte, jedoch in keinem konstanten Orientierungsverhältnis zur Ebene der Bilateralsymmetrie der Gastrula verlaufen. Dies unterscheidet sich von der Situation, wie sie von spiralfurchenden Tieren bekannt ist. Die Unterschiede und die beobachtete Variabilität des Furchungsprozesses werden im Licht unterschiedlicher Mechanismen der Spezifizierung von Zellschicksalen und Körperachsen bei verschiedenen Taxa der Spiralia und den Phoronida diskutiert. / This study addresses aspects of the early development of Phoronida (“horseshoe worms”). The cleavage process is analyzed for three species (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). These investigations are performed using 4D-microscopy as well as immunocytochemical stainings of the mitotic spindle apparatuses in combination with confocal laser-scanning microscopy. Different morphological features of the cleavage process are quantified and compared within as well as between the species. The results reveal a highly consistent cleavage of P. pallida and P. muelleri embryos: from the third cell cycle onward, the blastomeres divide mostly obliquely – and alternatingly dextral and sinistral – with respect to the animal-vegetal axis. This cleavage pattern shows surprising correspondences to the pattern of spiral cleavage. The finding can be interpreted as morphological support for recent molecule-based phylogenies, which indicate a position of Phoronida within the Spiralia/Lophotrochozoa clade. The cleavage of P. vancouverensis differs from the cleavage in the other two species; however, it also shows differences to a radial cleavage pattern. In all three species, the cleavage process also involves some degree of variability. Furthermore, the study traces the cell fates of early P. pallida embryos up to the state of late gastrulation, by the use of fluorescent in-vivo single cell markings. These analyses reveal that the first two cleavage divisions both pass through the later axis blastopore-apical plate of the gastrula, yet they do not pass in a constant relationship with respect to the later plane of bilateral symmetry. This differs from the situation known from spiral cleaving animals. The differences and the encountered variability of the cleavage process are discussed with respect to different mechanisms of the specification of cell fates and body axes in different taxa of the Spiralia and the Phoronida.

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

No description available.

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