Tuqan, Duniazad F
(2011)
Expression pattern and function of eIFE4 isoforms in planarian adult stem cells.
MRes thesis, University of Nottingham.
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PDF (A dissertation presented to the University of Nottingham in consideration for the degree of masters by research in Stem Cell Biology)
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Abstract
The eukaryotic translation initiation factor 4E (eIF4E) is central to translation initiation in eukaryotic cells. Recent studies revealed that there are multiple isoforms of eIF4E present in different species, some of which were found to have specific functions other than translation initiation. Furthermore, some were found to be expressed during specific developmental stages and/or in specialized tissue types. Multiple isoforms of elF4E have been found in several model organisms. For example, five eIF4Es were found in Caenorhabditis elegans, three in mammals and sea urchins, two in zebrafish and eight in Drosophila (Henderson et al., 2009). Among these species, stem cell specific isoforms were found in mouse, C. elegans, and Drosophila (Amiri et al., 2001). None of the eIF4E isoforms have yet been investigated in planarians which is an ideal model organism for studying stem cell biology in vivo.
Planarians are flatworms that belong to the phylum Platyhelminthes. They are known to have a large population of totipotent stem cells called neoblasts which provide the animal with an almost infinite ability to regenerate. Neoblasts are also known to have electron-dense structures called chromatoid bodies. These structures are similar in nature to germ granules which are present in the germ line cells of almost every metazoan (Saffman and Lasko, 1999). Given the close relationship between germ line cells and planarian neoblasts, and in light of the fact that other examined animals show eIF4E isoforms that are germ line specific, we speculate that neoblast specific eIF4E isoform(s) exist in planarians.
In 2007, Yoshida-Kashikawa and co-workers published a paper discussing RNA-binding proteins in planarian stem cells and neurons (Yoshida-Kashikawa et al., 2007). One of the genes found to be neoblast specific was an eIF4E isoform. Based upon this finding, we intended to further investigate and confirm the expression and function of this neoblast specific eIF4E isoform. This isoform will be referred to as eIF4E-A for ease of reference in this text.
In the initial research, the neoblast specific expression of eIF4E-A was confirmed via performing whole mount in situ hybridization (WMISH) on intact and irradiated (stem cell eliminated) worms, and in situ hybridization on sexual planarian histological sections. The function of eIF4E-A was then studied by RNAi which in some cases resulted in phenotypes that in turn were then studied by immunohistochemistry using a chromatoid body marker. eIF4E-A RNAi experiments resulted in a few mild phenotypes despite the confirmation of the neoblast expression pattern of eIF4E-A. The knock down of the gene’s expression was not enough to cause regeneration aberration, nor did it affect the number or size of the chromatioed bodies. This finding led to the supposition that there might be additional eIF4E isoforms that are neoblast specific in planarians.
Genome screening and planarian transcriptome analysis revealed four additional eIF4E isoforms. For ease of reading, they are named eIF4E-B, eIF4E-C, eIF4E-D, and eIF4E-E. WMISH of the remaining isoforms and irradiation transcriptome data analysis revealed two additional strong candidates for neoblast specificity, namely eIF4E-B and eIF4E-D. These results suggest that there is more than one isoform specific to neoblasts in planarians working redundantly towards neoblast specific post-transcriptional regulation.
This study contributes to the knowledge as related to the key factors that govern stem cell regulation (eukaryotic translation initiation factors), thus adding value to the understanding of stem cells and regeneration, while contributing to the evolutionary data of gene members in the eIF4E family.
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