In order for a human being to process complex thought, cells within the brain must communicate with each other in a very precise manner. The mechanisms which underlie the development of these connections, however, are poorly understood and thus require a thorough investigation. In this dissertation, we attempt to identify components involved in stabilizing synaptic contacts and the mechanisms by which synaptic proteins are trafficked to newly forming contact sites. Interestingly, we also identify a gene involved in the formation of the myotome. To identify proteins involved in stabilizing synaptic contacts, we characterized the function of 4.1B in developing zebrafish embryos. 4.1B is a scaffolding molecule involved in stabilizing protein complexes at sites of cell adhesion. We identified two 4.1B genes in the zebrafish genome, 4.1B-a and 4.1B-b, which are differentially expressed and have evolved divergent functions. 4.1B-a is expressed within the central nervous system, specifically within primary motor neurons. Knockdown studies show a reduction in the number of synapses and altered kinetics of touch evoked-responses, suggesting a role in synaptic stabilization. In contrast, 4.1B-b is primarily expressed in muscle cells. Knockdown of 4.1B-b results in severe muscle fiber disorganization as well as altered locomotor behaviors. Together, these data suggest the basic functions of 4.1B are evolutionarily conserved, with new roles described in the development of synapses and muscle fibers. To determine the mechanisms that underlie protein recruitment to newly forming synapses, we examined the recruitment of three distinct transport packets in the zebrafish spinal cord. During presynaptic assembly, we found synaptic vesicle protein transport vesicles preceded piccolo-containing active zone precursor transport vesicles, which in turn preceded synapsin transport vesicles. We identified the last transport packet as a unique and independent mechanism for the recruitment of synapsin, a protein involved in regulating the reserve pool of synaptic vesicles. Importantly, we found cyclin-dependent kinase 5 regulated the late recruitment of synapsin transport packets to synapses, thus identifying kinases as a key signaling molecule in the formation of synaptic contacts. Together, this work provides new insight into the mechanisms that underlie synaptogenesis. This dissertation includes both previously published and unpublished co-authored material.

Mutations of the gene encoding the inositol polyphosphate 5-phosphatase OCRL1 are responsible for causing two disorders in humans: Lowe syndrome and type 2 Dent's disease (Dent-2). Lowe syndrome (oculocerebrorenal syndrome of Lowe) is an X-linked genetic disorder that causes multisystem defects affecting predominantly the eyes, brain and kidneys. Dent-2 disease is very similar to Lowe syndrome, but it affects primarily the kidneys with little or no symptoms in the brain and eyes. The enzymatic activity, structure and binding partners of the OCRL1 protein have been described and progress on the cellular functions of OCRL1 has been made. However the studies to date have not provided the necessary insight to explain the tissue-specific defects observed in Lowe syndrome and Dent-2 patients. In order to investigate the role of OCRL1 and the consequences of its deficiency in a physiological context an animal model is required. We have chosen the zebrafish for this study due to its suitability for investigating vertebrate early development and the abundance of research techniques available for this model organism. We have studied the expression of OCRL1 in zebrafish and its role in the early embryonic development. We have also investigated its role in the endocytic function of the zebrafish larval pronephric kidney. Finally we have investigated its role in ciliogenesis and function of pronephric cilia. Our studies show that OCRL1 depletion does not cause gross developmental defects, nor affects the development of pronephros, but impairs their endocytic activity. We have also shown, that efficient pronephric uptake requires OCRL1 interactions with clathrin, Rab GTPase family proteins, APPL1 and IPIP27A/B. Our studies link the reduced uptake with lowered levels of megalin receptor, which is responsible for the bulk of protein reabsorption in the kidney. Together our results strongly suggest that defects in this process are responsible for low molecular weight proteinuria present in Lowe syndrome and Dent-2 patients and zebrafish is a suitable model to study the renal aspect of these diseases.

Development and the associated cellular differentiation are some of the most fundamental processes in biology. Since the early conception of the Waddington landscape, with cells portrayed as rolling down a landscape, understanding these processes has been at the forefront of biology. Progress in tissue regeneration, organoid culture, and cellular reprogramming relies on our ability to unfold cellular decision making and its dynamics. In this thesis, we ask to what extent development follows such landscape. Secondly, we address whether cellular branching points are discrete events. Given the recent surge in single-cell genomics data, we can now address these fundamental questions. To this end, we analyzed two large-scale single-cell RNAseq time course datasets from vertebrate embryogenesis in zebrafish. From the Waddington analogy, we expect the cell-to-cell correlation to increase across development as cells specialize. Our analysis does not show a linear trend, but rather, that cell-to-cell variability is lowest during gastrulation. Interestingly, the two different datasets from two different laboratories display a qualitatively similar trend, providing internal consistency of our analysis. To uncover the branchpoint dynamics, we extended our analysis to include computations of gene-to-gene correlations. It has been shown, using PCR data, that the transition index, the ratio between cell-to-cell and gene-to-gene correlations, displays a peak during such branchpoints, suggesting discrete transitions. To this end, we tracked individual developmental trajectories, and characterized both correlations, enabling computation of the transition index. However, the cell-to-cell correlation and gene-to-gene correlation did not follow a generic inverse relationship, as previously suggested. No unique signal corresponding to the branchpoints could, thus, be detected. Therefore, our analysis does not support the view that branchpoints during vertebrate embryogenesis are discrete, well-defined transition events. In conclusion, this first large-scale single-cell based analysis of time-resolved developmental data does not support a downhill rolling ball notion where cells decide their fate at discrete transition points. The temporal organization of an undulating developmental landscape appears to be more complex than initially conceptualized by Waddington. Therefore, it is of paramount interest to extend this type of analysis to other systems and to develop techniques to compute such landscape in a data-driven manner.

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No description available.

Previous research showed mutations in muscle sarcoplasmic reticulum-bound calcium handler proteins cause swimming defects in embryonic zebrafish. CaMK-II is a highly conserved Ca2+/calmodulin-dependent protein kinase expressed in all vertebrates has been defined to activate and inactivate multiple Ca2+ handler proteins involved in excitation- contraction coupling and relaxation of cardiac and skeletal muscle. In this study, evidence is provided through pharmacological and genetic intervention that CaMK-II inhibition and overexpression causes swimming defects, particularly response to stimuli and swimming ability, reinforced by immunolocalization of skeletal muscle. Transient CaMK-II inactivation does not have any long-term defects to swimming behavior. Overexpression of wild-type, constitutively active, and dominant-negative CaMK-II-GFP in embryos tended to co-localize in fast muscle which led to defects in swimming behavior. This study concludes that inhibition or overexpression of CaMK-II in skeletal muscle diminishes normal swimming behavior specifically in response to mechanical stimulation and swimming ability.

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical cell division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Making use of extended time-lapse, multi-photon microscopy and a careful three-dimensional analysis of cell division orientation, we show that SDO is required for neural rod midline formation during neurulation, but dispensable for body axis elongation during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the non-canonical Wnt receptor Frizzled 7 (Fz7), and that interfering with cell division orientation leads to severe defects in neural rod midline formation, but not body axis elongation. These findings suggest a novel function for Fz7 controlled cell division orientation in neural rod midline formation during neurulation. They also shed new light on the field of cell division orientation by uncoupling it from tissue elongation.

As some of the most important and widely utilised intercellular signalling molecules, transforming growth factor βs (TGFβs) play critical roles in normal development and in human disease. Establishing appropriate levels of signalling involves positive and negative feedback, driven by the same signal transduction components, but whether or how the two are distinguished has not previously been understood. Here we show that LIM domain binding proteins (Ldbs) drive the Smad6/7-mediated negative feedback of TGFβ signalling, but they are not required for the ligand-driven positive feedback or other downstream transcriptional activation. In Ldb-deficient zebrafish embryos, the homeostasis of TGFβ signalling is perturbed. As a consequence, signalling of TGFβ family members, Nodal and BMP, is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing Nodal and BMP. Later in development, conditional ldb2a knockdown causes defective vascular, angiogenic and haemogenic development, likely also by elevating TGFβ signalling. Thus, Ldbs control the homeostatic regulation of TGFβ signalling and therefore play critical roles in diverse developmental processes.

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical cell division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Making use of extended time-lapse, multi-photon microscopy and a careful three-dimensional analysis of cell division orientation, we show that SDO is required for neural rod midline formation during neurulation, but dispensable for body axis elongation during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the non-canonical Wnt receptor Frizzled 7 (Fz7), and that interfering with cell division orientation leads to severe defects in neural rod midline formation, but not body axis elongation. These findings suggest a novel function for Fz7 controlled cell division orientation in neural rod midline formation during neurulation. They also shed new light on the field of cell division orientation by uncoupling it from tissue elongation.

No description available.