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 approach yielded a number of different genes of which two are described in this thesis. The gene cdx1 (Chapter 2) is a member of the caudal class of homeobox genes and is expressed in ventrolateral cells of the embryo prior to gastrulation. During gastrulation, transcripts of this gene accumulate in the posterior half of the embryo. The functions of caudal class genes of Drosophila, mouse and Xenopus indicate that genes of this class mediate the specification of posterior positional values in the embryo. Because of their characteristic distribution, cdx1 transcripts are useful markers of (ventro) posterior position in the embryo and have been used as such in the studies of mesoderm formation and anteroposterior patterning in blastoderm explants, performed in our laboratory. A second gene isolated in the search for homeobox genes was the Hoxb-1 gene, described in Chapter 3. This gene belongs to the class of Hox genes, whose members are organized into clusters in the genome of many species. With expression reaching into the hindbrain, the Hoxb-1 gene is one of the most anteriorly expressed Hox genes. Its most prominent expression, especially during segmentation, is found in rhombomere 4. In the late segmentation stage embryo, Hoxb-1 expression is a valuable marker of this rhombomere and the neural crest cells at that level of the hindbrain. Chapter 4 describes the cloning of genes on the basis of their differential gene expression between the oocyte and the early segmentation stage, using a subtractive hybridization strategy. Fifteen genes, identified from the oocyte stage cDNA library, are expressed in early development, when mesoderm induction occurs, and their expression disappears before the beginning of segmentation. From the early segmentation stage cDNA library, 26 genes were selected whose expression was activated during segmentation but not yet in early development, coinciding with the differentiation of the mesoderm and the patterning of the anteroposterior axis. In total 27 genes appeared to code for novel proteins and are therefore candidates for further studies and may provide a better insight into molecular mechanisms underlying developmental processes. Also in light of the large scale mutagenesis screens of zebrafish that have recently been undertaken in a number of laboratories and for which the affected genes yet need to be molecularly identified, it is important that the search for novel genes continues for only few candidate sequences are available so far. The subtractive hybridization strategy described in this thesis appears a worthwhile technique to obtain such candidate sequences. Further investigations of these novel genes were restricted to a detailed characterization of the expression of one gene: cth1. Chapter 5 gives a description of the distribution of the mRNA transcripts of this gene during cleavage, blastula and gastrula stages. Whereas maternal cth1 mRNA is ubiquitously distributed in the blastomeres, the embryonically transcribed cth1 mRNA is expressed after the late blastula stage, in cells at the blastoderm margin which have mesodermal and endodermal fates. The cth1 transcripts disappear around midgastrulation, coinciding with the commitment of cells to the mesodermal (or endodermal) fate. In the cth1 protein, motifs containing three cysteines and one histidine (C3H) are present that are most likely zinc fingers, structures involved in the regulation of expression of target genes. The cth1 mRNA expression pattern and the gene's homology to the TIS11 family, a family of primary response genes whose expression is activated after treatment with for example growth factors, suggest a function for the cth1 gene of maintenance of the cellular potential in cells with mesodermal (and endodermal) fates, and in cells of cleavage stages. By inhibiting the expression of certain target genes, cth1 could prevent or delay the selection of certain differentiation pathways, such as for example the commitment to a mesodermal fate before midgastrulation. Proteins containing C3H motifs are expressed in a number of species.For example in C. elegans, the PIE-1 protein is required to keep germlineblastomeres totipotent during early development, most likely by suppressing the transcription in these blastomeres. In Chapter 6 the literature on the C3H class of proteins is shortly reviewed and the hypothesis is proposed that they may be widely involved in preserving cellular potency in specification events during development. In Chapter 7 the results presented in previous chapters are discussed, with emphasis on the proposed role for cth1 and how its activity could affect the fate of the cells expressing this gene.