Characterization of genes expressed during mesoderm formation and anteroposterior patterning in carp (Cyprinus carpio)

The formation of germ layers during gastrulation and the specification and patterning of the body axes are important events in the development of the embryo. The investigations described in this thesis aimed to isolate and characterize the distribution of transcripts of genes, in particular novel genes, that are expressed during the formation of mesoderm and the patterning of the anteroposterior axis in the carp (Cyprinus carpio), a cyprinid teleost fish. Such studies may contribute to a better insight in the molecular mechanisms underlying the above processes, in two respects. Firstly, the characterization of a gene's expression pattern is one of the first steps towards the elucidation of its function. Especially the characterization of novel genes may provide a key to new insights in development. Furthermore, in studies of development, it is of importance to have markers that identify specific cell types, for example early mesodermal precursor cells. The isolation of genes that are specifically expressed in certain cell types provides such markers.Two molecular approaches were chosen for gene isolation. Firstly, we specifically searched for homeobox genes, which encode transcription factors with important regulatory functions during development. A particular class of homeobox genes, the Hox genes, provides cells along the anteroposterior axis with positional information and the expression patterns of members of this class are excellent markers of position on this axis. Our second approach was a subtractive hybridization strategy. It was applied to isolate genes that are differentially expressed between the oocyte and the early segmentation stage, a period during which mesoderm is induced.For the identification of homeobox-containing sequences in a carp early segmentation stage cDNA library, we used a probe that was composed of a mixture of homeobox fragments, produced by PCR. The PCR primers were designed against the most conserved regions of the homeobox. This ap