Cilia are cellular organelles involved in processing components of the hedgehog (hh) signalling pathway and determining left-right (L-R) axis formation in the embryo. An embryonic lethal mouse mutant, called gasping 6 (gpg6), was identified that demonstrated morphological and molecular defects associated with L-R development and hh signalling. gpg6 mutant embryos also demonstrate abnormally short cilia, which was hypothesised to be the primary morphological defect in gpg6 mutants. The underlying genetic lesion in gpg6 is a mutation in the DNA repair gene Atmin. The base pair change results in an amino acid substitution in a critical residue in the third zinc finger of Atmin. The consequence of this change is the failure to activate transcriptional targets of Atmin. This raised the possibility that previously unidentified Atmin target genes are important for ciliogenesis. Consistent with this hypothesis, Dynein light chain-LC8 (Dynll1) is downregulated in gpg6 mutants. LC8 (a homolog of mouse Dynll1) is required, in the single cell eukaryotic organism Chlamydomonas, for retrograde intraflagellar transport (IFT), a process crucial for ciliogenesis. These data led to the following hypothesis: Atmin activates expression of Dynll1, which functions in retrograde IFT to enable normal ciliogenesis. Knockdown of Atmin in a ciliated kidney cell line resulted in abnormally short cilia. Thus, Atmin functions in ciliogenesis. Investigation of gpg6 has therefore identified a novel role for Atmin in ciliogenesis and has added to the growing knowledge of genes that control cilia formation and embryonic development.

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.