The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

The ParaHox cluster is the evolutionary sister of the Hox cluster. Like the Hox cluster, the ParaHox cluster is subject to complex regulatory phenomena such as collinearity. Despite the breakup of the ParaHox cluster within many animals, intact and collinear clusters have now been discovered within the chordate phyla in amphioxus and the vertebrates, and more recently within the hemichordates and echinoderms. The archetypal ParaHox cluster of amphioxus places it in a unique position in which to examine the regulatory mechanisms controlling ParaHox gene expression within the last common ancestor of chordates, and perhaps even the wider Deuterostomia. In this thesis, the genomic and regulatory landscape of the amphioxus ParaHox cluster is characterised in detail. New genomic and transcriptomic resources are used to better characterise the B.floridae ParaHox cluster and surrounding genomic region, and conserved non-coding regions and regulatory motifs are identified across the ParaHox cluster of three species of amphioxus. In conjunction with this, the impact of retrotransposition upon the ParaHox cluster is examined and analyses of transposable elements and the AmphiSCP1 retrogene reveal that the ParaHox cluster may be more insulated from outside influence than previously thought. Finally, the detailed analyses of a regulatory element upstream of AmphiGsx reveals conserved mechanisms regulating Gsx CNS expression within the chordates, and TCF/Lef is likely a direct regulator of AmphiGsx within the CNS. The work in this thesis makes use of new genomic and transcriptomic resources available for amphioxus to better characterise the genomic and regulatory landscape of the amphioxus ParaHox cluster, serving as a basis for the improved identification and characterisation of functional regulatory elements and conserved regulatory mechanisms. This work also highlights the potential of Ciona intestinalis as a ‘living test tube' to allow the detailed characterisation of amphioxus ParaHox regulatory elements.

Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...