Severe phenotypes in Eri1-knockout mice 83


4.1.1 Growth defects, postnatal death and male sterility

The Eri1-knockout mice were systematically analyzed for their phenotypes to get an impression of the function of murine Eri1. It was observed that Eri1-knockout mice show a reduced body size and weight throughout development and till adulthood (Figure 10). Consistent with this finding, it could be shown that primary MEF cell cultures from Eri1-knockout embryos have a prolonged doubling time (Figure 19). The cell screen analysis cannot distinguish between (1) prolonged duration between cell divisions of all cells in the culture or (2) some of the cells in the culture dividing normally while others become impaired to divide. Experiments to test cell apotosis were not performed in the frame of this thesis, so this cannot be excluded.

The growth defects resemble those associated with Minute Drosophila mutants, Belly spot and tail (Bst) mouse mutants and Diamond-Blackfan anemia patients. All three of them have been linked to mutations in ribosomal genes (Draptchinskaia et al., 1999; Lambertsson, 1998; Oliver et al., 2004). In this work it could be shown, that Eri1 binds to and processes 5.8S ribosomal RNA (section 3.1.4). Therefore a connection might be suggested between impaired ribosome biogenesis, a prolonged doubling time of primary Eri1-deficient MEF cells and decreased growth of Eri1- knockout mice.

Mice of an inbred strain are genetically as alike as possible by being homozygous for virtually all alleles. This makes them favorable for knockout studies, because the phenotypic variability between individual animals is very small. On the other hand, inbred strain animals are more vulnerable to mutations, because they are less able to compensate for experimentally generated loss of function of specific gene products. In the case of Eri1 deletion, deficient mice on an inbred C57BL/6 stain background died soon after birth (Figure 11, Table 6). A strategy to combat this neonatal mortality was found by crossing in an outbred strain that had strongly increased allelic variability. Generating Eri1-knockout mice on an NMRI x C57BL/6 background could indeed largely rescue the phenotype (Table 6). These data suggest that Eri1 deficiency in combination with one or many specific other alleles decreases survival, which can be overcome by compensatory mechanisms present in mixed strains.

Tissue Western blotting revealed relatively high levels of Eri1 in the testes of mice (Figure 13). The deficiency of Eri1 led to the phenotype of male sterility, which was also reflected by an underdevelopment of the testes (Figure 12). It may be speculated that this phenotype is a consequence of Eri1’ s impact on cell cycle- dependent histone mRNAs (Figure 23). A unique regulation of histones occurs in mammalian spermatogenesis. During meiosis, the replication-dependent histones are replaced with testis-specific variants and these themselves are then substituted by protamines in the mature sperm (Kimmins and Sassone-Corsi, 2005). Potentially, Eri1 could be involved in the exchange of replication-dependent histones to testis- specific histones. If in the Eri1-deficient testis this crucial step is impaired one could imagine that spermatogenesis is arrested.

4.1.2 Results from the screen in the German Mouse Clinic

For a more detailed analysis of phenotypes in Eri1-deficient mice, Eri1-knockout, Eri1-heterozygous and wildtype littermates were sent to the German Mouse Clinic to test them in a number of standardized screens. Tests were performed in 14 different categories. In five categories no abnormalities were found: allergy, cardiovascular function, eye, lung function and nociception, but in nine categories Eri1-dependent phenotypes were discovered: behavior, clinical chemistry and hematology, dysmorphology, energy metabolism, immunology, molecular phenotyping, neurology, pathology, and steroid metabolism (Table 7). The diversity of categories exhibiting a

phenotype already suggests that Eri1 is a regulator in many different cell types either of the same cellular pathway or even of several different pathways. This work has provided support for the latter by showing that Eri1 is involved in such crucial processes as ribosome biogenesis, canonical histone mRNA regulation and RNAi. For example, in the clinical chemistry and hematology screen Eri1-deficient mice showed a macrocytic anemia phenotype. The cause of Diamond-Blackfan anemia has been described to be abnormal processing of ribosomal RNA due to mutations in the ribosomal protein S19, which leads to abnormalities in ribosomal function (Draptchinskaia et al., 1999). Consistent with this finding, the macrocytic anemia in Eri1-deficient mice might be connected to impaired ribosomal RNA processing as well (Figure 18). The behavior phenotype of increased anxiety in the Eri1-knockout mice could be due to loss of Eri1 function in neurons. Eri1 has been shown to be highly expressed in a subset of neurons in C. elegans (Kennedy et al., 2004). Further, in Eri1-deficient worms an increased RNAi sensitivity was reported. Testing the worms for siRNA-mediated silencing of a neuron-specific reporter gene, the Eri1- knockout worms showed a 70% decrease in reporter gene expression upon feeding with dsRNA, while the dsRNA had no affect in wildtype animals (Kennedy et al., 2004). These findings suggest that Eri1 might have an important function on miRNAs in the neurons of mice, but this has not been demonstrated yet.