The Development of The Germ Line During Embryogenesis of Caenorhabditis elegans

Die Prozesse, die bei der Entwicklung der embryonalen Keimbahn entscheidend sind, sind bisher weitgehend unbekannt. In embryonal letalen Mutanten von C. elegans in den Genen cib-1, cib-2 und cib-3 (cib steht für changed identity of blastomeres) geht die Keimbahnidentität in den Keimbahn-Zellen P1, P2 oder P3 verloren. Diese Blastomeren verzögern die Zellteilung und teilen sich dann symmetrisch statt asymmetrisch. Im Rahmen dieser Arbeit wurden Mutanten in allen drei Genen analysiert. cib-1 codiert für die einzige Thymidylat Synthase in C. elegans. Nach einer Inaktivierung der Zellzyklus-Kontrolle teilen sich in cib-1-Embryonen die P-Zellen nicht mehr verzögert. Die dTTP-Synthese ist über die DNA-Integrität und die Zellzyklus-Kontrolle mit der Entwicklung der Keimbahn verbunden. Die Sequenz von cib-3 ist nematodenspezifisch ohne bekannte Funktion. Als Interaktionspartner wurden in einem Yeast-two-Hybrid die konservierten Proteine RACK-1 und eine Cathepsin L Cystein Proteinase identifiziert, die in RNAi-Experimenten den typischen cib-Phänotyp zeigen. CIB-3 beeinflusst die Segregation von keimbahnspezifischen Proteinen wie PIE-1 und somatischen Proteinen wie MEX-5. In cib-3-Embryonen werden beide Proteine nicht verteilt und gelangen bei der Zellteilung in die gleichen Tochterzellen. Als Folge exprimieren die Tochterzellen gleichzeitig somatische und keimbahnspezifische Proteine. cib-2 ist ein bis zum Menschen sehr konserviertes Gen, für das bisher keine Funktion bekannt ist. Die drei cib-Gene sind an zwei verschiedenen, neuen Prozessen beteiligt. Der eine Prozess steuert die Transmission der Zellpolarität in eine cytoplasmatische Polarität für die Segregation keimbahnspezifischer Proteine. Der andere Prozess reguliert die Keimbahnentwicklung mit einer Qualitätskontrolle für die DNA-Integrität. Möglicherweise steuern die beiden Prozesse mit sehr konservierten Proteinen auch in anderen Organismen die Entwicklung einer gesunden Keimbahn.The essential processes for embryonic germ line development are so far largely unknown. In embryonic lethal C. elegans mutants of the genes cib-1, cib-2 and cib-3 (cib means changed identity of blastomeres) is the germ line identity of the germ line cells P1, P2 or P3 lost. These blastomeres show delayed cell divisions and cleave symmetric instead of asymmetric later on. Mutants of all three genes were analysed during this work. cib-1 codes for the only Thymidylate Synthase of C. elegans. After inactivation of the cell cycle control in cib-1-embryos the P cells are not dividing delayed anymore. The synthesis of dTTP is connected with germ line development via DNA integrity and cell cycle control. The sequence of cib-3 is nematode specific without assigned function. In a Yeast-two-Hybrid were the conserved proteins RACK-1 and a Cathepsin L Cystein Proteinase identified as interaction partners, which show the typical cib-phenotype in RNAi-experiments. CIB-3 has an influence on the segregation of germ line specific proteins like PIE-1 and somatic proteins like MEX-5. In cib-3-embryos both proteins are not segregated and locate after the next cell division in the same daughter cells. So these daughter cells express somatic and germ line specific proteins at the same time. cib-2 is highly conserved to human without assigned function. The three genes participate in two different and new processes. The first process controls the transmission of cell polarity to a cytoplasmic polarity, which is essential for the segregation of germ line specific proteins. The second process regulates the germ line development with a quality check for DNA integrity. It is possible that these two processes control with highly conserved proteins the development of a healthy germ line also in other organisms

Functional dissection of Caenorhabditis elegans CLK-2/TEL2 cell cycle defects during embryogenesis and germline development

CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development