Positional cloning and characterization of a mutation in the condensin zCAP-G gene of the zebrafish, Danio rerio

In dieser Arbeit habe ich eine Mutation in der CAP-G-Untereinheit des Condensin-Komplexes beim Zebrafisch kloniert. Die Zebrafisch-Mutante creature from the black Lagoon (cbl) zeichnet disch durch kleine Augen aus, die ab Tag 3 nach der Fertilisation deutlich von der wt-Form zu unterscheiden sind. Meine Untersuchungen zeigen, dass dieser Phänotyp auf einer Reduktion der Zellzahl der neuronalen Retina beruht, die durch eine hohe Apoptose-Rate in der mutanten Retina zustande kommt. Daneben findet sich starke Apoptose auch in weiteren Teilen des zentralen Nervensystems. Durch positionales Klonieren wurde als Ursache für den Phänotyp der cbl-Mutante eine Punktmutation in der CAP-G-Untereinheit des Condensin-Komplexes gefunden. Diese führt zu einem Stop-Codon in der Mitte des Gens und zum Verlust der C-terminalen Hälfte des Proteins mit der UVR-Domäne und der PH-Domäne. Das Muster der Expression von zcap-g korreliert mit dem Muster der Zellproliferation in der Retina. In der cbl-Mutante weisen die Zellen der neuronalen Reina Zellteilungsdefekte in Form von Chromosomenbrücken und deformierten Zellkernen auf. Die Zellen der neuronalen Reinta weisen keine Zellzyklusverzögerungen auf und leiten die Apoptose während der Mitose ein. Mit der cbl-Mutante ist nun ein genetisches Modell verfügbar, um die Funktion des Condensin-Komplexes in der Embryonalentwicklung von Vertebraten zu untersuchen.In this study, I cloned a mutation in the CAP-G subunit of the condensin-complex in the zebrafish (Danio rerio). The zebrafish mutant creature from the black lagoon (cbl) is characterized by its small eyes, which are prominent at 3 dpf. I showed that this phenotype results from a reduction of cell number in the neural retina which is caused by a high rate of apoptosis in the mutant retina. A high rate of apoptosis is also found in other parts of the central nervous system. By positional cloning, a point mutation in the zcap-g gene was identified as the cause for the phenotype of the cbl-mutant. This point mutation leads to a stop codon in the middle of the gene and causes a truncation and loss of the c-terminal UvR- and PH-domains in the protein. The expression pattern of zcap-g correlates with the pattern of cell proliferation in the retina. In the cbl-mutant the cells of the neural retina display defects in cell division which lead to chromatin bridges and deformed nuclei. The cells of the neural retina do not show cell cycle delay and induce apoptosis during mitosis. With the cbl-mutant we now have a genetic model to examine condensin function in the embryonic development of vertebrates

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina.

BACKGROUND: The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described. RESULTS: Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the cap-g locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in cap-g mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes. CONCLUSION: Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Non-SMC condensin I complex proteins control chromosome segregation and survival of proliferating cells in the zebrafish neural retina

<p>Abstract</p> <p>Background</p> <p>The condensation of chromosomes and correct sister chromatid segregation during cell division is an essential feature of all proliferative cells. Structural maintenance of chromosomes (SMC) and non-SMC proteins form the condensin I complex and regulate chromosome condensation and segregation during mitosis. However, due to the lack of appropriate mutants, the function of the condensin I complex during vertebrate development has not been described.</p> <p>Results</p> <p>Here, we report the positional cloning and detailed characterization of retinal phenotypes of a zebrafish mutation at the <it>cap-g </it>locus. High resolution live imaging reveals that the progression of mitosis between prometa- to telophase is delayed and that sister chromatid segregation is impaired upon loss of CAP-G. CAP-G associates with chromosomes between prometa- and telophase of the cell cycle. Loss of the interaction partners CAP-H and CAP-D2 causes cytoplasmic mislocalization of CAP-G throughout mitosis. DNA content analysis reveals increased genomic imbalances upon loss of non-SMC condensin I subunits. Within the retina, loss of condensin I function causes increased rates of apoptosis among cells within the proliferative ciliary marginal zone (CMZ) whereas postmitotic retinal cells are viable. Inhibition of p53-mediated apoptosis partially rescues cell numbers in <it>cap-g </it>mutant retinae and allows normal layering of retinal cell types without alleviating their aberrant nuclear sizes.</p> <p>Conclusion</p> <p>Our findings indicate that the condensin I complex is particularly important within rapidly amplifying progenitor cell populations to ensure faithful chromosome segregation. In contrast, differentiation of postmitotic retinal cells is not impaired upon polyploidization.</p