No description available.

A segmented vertebral column is one of the major innovations vertebrates. In mice and chicks – amniotes – a subpopulation of the somites, the sclerotome, is sole source of vertebral tissue. It is unclear, however, how applicable this amniote-based ‘canonical’ mechanism is across the vertebrates. In fact, the vast majority and diversity of vertebrates are not amniotes, but are members of ‘fish’ groups where there has been relatively little investigation into vertebral development. Indeed, there is great diversity in vertebra form throughout ‘fish’ groups and fossil evidence suggests that the components of the vertebra, the neural arches and the vertebral bodies, arose separately and that vertebrates have evolved multiple ways of building vertebral bodies. In teleosts fish, the vertebral bodies initially form as mineralised rings within the notochord sheath (chordacentra) and then secondarily, bone is deposited around this (perichordal centra and arches). Notochord cells (chordoblasts) have been implicated in chordacentrum mineralisation and patterning in zebrafish and Atlantic salmon, though the question of how the overtly unsegmented notochord could direct segmental mineralisation still remains. My project first aims to address this dual mechanism in the zebrafish model, by testing whether the chordoblasts can mineralise and pattern the chordacentra. The second aim is to elucidate the role of the sclerotome in teleost vertebral development. To do this, I explored CRISPR knock-in tools to label the sclerotome and used a Gal4 gene trap line to investigate sclerotome ablation. I characterised the chordacentra and chordoblasts in our model system and verified the specificity of a promoter as a chordoblast marker. With this promoter, I established a method to target the chordoblasts for KillerRed-induced phototoxicity. I demonstrated that intact chordoblasts are necessary for chordacentrum formation, but that vertebral arches are unaffected. Fused perichordal centra are still able to form, but the underlying sheath has a very different structure. This supports the ‘duality’ hypothesis that in teleosts the role of the sclerotome in vertebra formation is limited to the arches and perichordal centra, whereas the chordoblasts are responsible for the chordacentra.

During development of the vertebrate embryo, a highly conserved tissue called the organizer forms during gastrulation, and is required for establishment of the basic body plan. In mouse, the organizer gives rise to the node and notochord, which are both transient signaling centres involved in patterning the body axes. The genetic regulation and morphogenesis of these tissues, particularly in the mouse, is not well understood. To follow the formation of these tissues we used time-lapse live imaging together with conventional cell lineage tracking. This showed that the notochord has distinct morphogenetic origins along the anterior-posterior axis: anterior head process forms by condensation of dispersed midline organizer cells; trunk forms by convergent extension of node cells; tail forms from posteriorly migrating node cells—this challenges the previously accepted model that tail notochord forms by node regression. We have also found there are distinct genetic requirements within these different regions. Previous mouse mutant analysis showed that conserved transcription factors Foxa2 and Noto are required for either all notochord regions or just tail notochord, respectively. We found a novel genetic interaction between the two demonstrated Foxa2 compensates for Noto specifically in the trunk notochord. Furthermore, we found Noto has a conserved role in regulating axial (notochord) versus paraxial (somite) cell fate. Therefore, we proposed there are three distinct regions within the mouse notochord, each with its own unique morphogenetic origins and genetic control. We have also conducted two microarray-based screens to identify novel gene expression patterns in the node and notochord. First, we compared Foxa2 mutant and wild type gastrula embryos. Second, we isolated notochord progenitors from early somite stage embryos. Extensive in situ hybridization screening based on both data sets revealed over 50 node and notochord expression patterns. Lastly, we screened Foxa2-bound chromatin regions near these notochord-specific genes using a transient zebrafish expression assay, and identified two novel notochord cis-regulatory modules. Together, we found a combination of classical genetics, embryology, and novel imaging techniques, has given us a better understanding of the morphogenesis and genetic regulation of pattern formation in the developing mouse embryo.

During development of the vertebrate embryo, a highly conserved tissue called the organizer forms during gastrulation, and is required for establishment of the basic body plan. In mouse, the organizer gives rise to the node and notochord, which are both transient signaling centres involved in patterning the body axes. The genetic regulation and morphogenesis of these tissues, particularly in the mouse, is not well understood. To follow the formation of these tissues we used time-lapse live imaging together with conventional cell lineage tracking. This showed that the notochord has distinct morphogenetic origins along the anterior-posterior axis: anterior head process forms by condensation of dispersed midline organizer cells; trunk forms by convergent extension of node cells; tail forms from posteriorly migrating node cells—this challenges the previously accepted model that tail notochord forms by node regression. We have also found there are distinct genetic requirements within these different regions. Previous mouse mutant analysis showed that conserved transcription factors Foxa2 and Noto are required for either all notochord regions or just tail notochord, respectively. We found a novel genetic interaction between the two demonstrated Foxa2 compensates for Noto specifically in the trunk notochord. Furthermore, we found Noto has a conserved role in regulating axial (notochord) versus paraxial (somite) cell fate. Therefore, we proposed there are three distinct regions within the mouse notochord, each with its own unique morphogenetic origins and genetic control. We have also conducted two microarray-based screens to identify novel gene expression patterns in the node and notochord. First, we compared Foxa2 mutant and wild type gastrula embryos. Second, we isolated notochord progenitors from early somite stage embryos. Extensive in situ hybridization screening based on both data sets revealed over 50 node and notochord expression patterns. Lastly, we screened Foxa2-bound chromatin regions near these notochord-specific genes using a transient zebrafish expression assay, and identified two novel notochord cis-regulatory modules. Together, we found a combination of classical genetics, embryology, and novel imaging techniques, has given us a better understanding of the morphogenesis and genetic regulation of pattern formation in the developing mouse embryo.

The notochord is a conserved structure in the phylum chordate, which includes all vertebrates and some closely related invertebrates. In mouse embryos, the notochord is a midline structure underneath the neural tube and it consists of a rod of cells constrained by a thick extracellular sheath, which is rich in fibronectin and other extracellular matrix molecule. During notochord formation, two key processes are involved: cell migration and convergent extension. Two molecules are essential for these processes: fibronectin and Protein Kinase C iota (prkci). For cell migration, fibronectin regulates this process by modulating cell protrusion via a signaling pathway in which atypical protein kinase C (aPKC) is an essential factor. For convergent extension, fibronectin has been shown to be important in this process by regulating both cell migration and cell adhesion. The role of aPKCin convergent extension is revealed as it is asymmetrically expressed in Ciona notochord during convergent extension, indicating possible function of aPKC in convergent extension process and in the morphogenesis of notochord. These studies raised the possibility that fibronectin and prkci are important in notochord formation, by regulating cell migration and convergent extension. In this thesis, Cre/loxP system was used to study the function of fibronectin and prkci in the development of notochord. I provided evidence that conditional deletion of fibronectin by Foxa2-Crein the notochord resulted in a notochord of smaller volume and fewer notochordal cells. The nucleus pulposus was also smaller in are and less in cell number. Fibronectin in the notochord was not affected at E9.5, but diminished in the core of the notochord at E12.5 and in nucleus pulposus at E15.5. The phenotypes of smaller notochord and the nucleus pulposus might be the results of reduced notochordal cell proliferation and increased cell death. However, more samples are needed to analyze to confirm this and perform statistical analysis. In addition, convergent extension of notochord seemed less effective. These results are consistent with previous study about fibronectin and α5β1 integrin. The results suggest that fibronectin is required for notochordal cell proliferation, survival, migration and efficient convergent extension, but not for notochordal cell fate determination. The results also demonstrated that prkci seemed not to be important for notochord development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

The mouse node is the structural equivalent of Spemann's Organizer. The transcription factor Foxa2 (HNF3beta) is required for node and notochord specification and its enhancer has been characterized to a 500bp element that drives the expression of a reporter gene to the node and notochord in transgenic embryos. / The aim of the study was to identify sequence elements, within this enhancer, required to drive node/notochord specific-expression. Since, in the Xenopus organizer, Siamois activates organizer-specific gene expression, it was tested whether this factor could activate expression from the Foxa2 node-specific enhancer. Using deletion analysis, the response element was mapped to a 33bp region and it was shown that this element was both necessary and sufficient for reporter gene activation by X-Siamois . Furthermore, it was shown that X-Siamois can bind this DNA element and two sequence motifs required for binding and transactivation by X-Siamois were identified. Preliminary results suggest that the 33bp element, within the 500bp enhancer, is required for the maintenance of expression in the notochord of transgenic mice.

No description available.

Dyslexia is a neurodevelopmental disorder that affects between 5% and 12% of school-aged children. Individuals with dyslexia have difficulties in learning to read despite normal IQ levels and adequate socio-economical and educational opportunities. Dyslexia has a strong genetic component, but only a few candidate genes have been characterized to date. The KIAA0319 gene is a strong dyslexia candidate found to be associated with dyslexia in independent studies. The KIAA0319 genetic variants associated with dyslexia reside in a regulatory region. Studies in rat suggested that this gene is required for neuronal migration during early cortex formation. The KIAA0319-like (KIAA0319L) is a KIAA0319 homolog in structure and has recently been shown to play a role in dyslexia. I used zebrafish as a model organism both to study the effects of non-coding variants and to characterise kiaa0319 gene function. I used Gateway Tol2 technology to study the role of regulatory sequences. While these experiments led to inconclusive results, they highlighted some of the challenges but also the feasibility of using zebrafish as model organism to study genetic associations. In parallel, I studied the kiaa0319 function with knockout and knockdown experiments. Additionally, I conducted a detailed gene expression analysis with different in situ hybridisation protocols showing kiaa0319 ubiquitous expression in the whole embryo before 12 hours post fertilisation, with later specification to the eyes, brain, otic vesicle and notochord. Additionally, I have tested for the expression of kiaa0319l and showed similar expression pattern to the kiaa0319, but with significantly lower expression of kiaa0319l in zebrafish notochord. My data show, for the first time, that kiaa0319 has stage-specific expression in the brain and notochord during zebrafish early development, suggesting kiaa0319 specific role in the development of these structures. These results are in line with recent mouse studies. With this project I support the idea of kiaa0319 role being extended beyond the brain function and propose a role for kiaa03019 in the visual system and in the notochord.

Thesis (Ph. D.)--Cornell University, May, 2009. / Vita. Includes bibliographical references (leaves 207-228).

The notochord is the defining structure of all chordates. A semi-­‐flexible elongated tube of cells, it forms along the central axis of the embryo and provides axial support during development. It also acts as a signalling centre during early embryogenesis, controlling the patterning of a number of tissues and establishing the early body axis of the embryo. In vertebrates, the function of the notochord expands beyond early development. It creates morphogenic gradients for the patterned formation of the vertebral bodies and, in adults, the remnants of the notochord form the nucleus pulposus, a gel-­‐like structure with an integral role in the distribution of vertebral pressure in the intervertebral disc. Little is known about how the notochord copes with damage during embryogenesis, but degeneration of the nucleus pulposus can lead to debilitating spinal disorders. In this thesis, I use a zebrafish model system to present new data that describes the cellular behaviours associated with how the notochord copes with external damage and how this damage can influence the future development of the vertebrae. I have uncovered a novel damage response in the notochord of zebrafish larvae and characterised the morphogenetic changes involved in the process using transgenic fluorescent lines. I have explored the damage in the context of the Wilms’ Tumour 1 (Wt1) gene, a vertebrate-­‐conserved transcription factor, which has recently been associated with several regenerative responses, and discovered that one of its zebrafish orthologues, wt1b, becomes upregulated in the notochord damage response. I have used fluorescent confocal imaging and immunohistochemistry to present new evidence that shows that upon injury, the outer notochord sheath cells upregulate the expression of wt1b. Additionally, I have used time-­‐lapse microscopy to show that damage to the notochord induces novel morphological changes in the injured organ, which include the loss of cellularity of the inner vacuolated cells and the movement of the wt1b-­‐positive outer sheath cells into the injured lumen. Long-­‐term imaging experiments have also demonstrated the capacity of the notochord to heal the damage over time, which ultimately leads to the formation of an extra, smaller vertebra in the wounded area. Skeletal staining of these fish has revealed a previously unknown putative cartilage switch at the site of damage, which leads to the formation of the new vertebral body. This finding has been supported by the microarray analysis of the injured area, which shows the unexpected de-­‐novo expression of cartilage markers at the site of damage The work in this thesis identifies for the first time an endogenous repair mechanism in the notochord of zebrafish larvae and describes the cellular, genetic and molecular processes cotrolling this novel wt1b-­‐associated damage response.