Characterization of CDK5RAP2 as a novel regulator in the Hippo signaling pathway

CEP161 is a novel component of the Dictyostelium discoideum centrosome and is the ortholog of mammalian CDK5RAP2. Mutations in CDK5RAP2 are associated with autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder characterized by reduced head circumference, a reduction in the size of the cerebral cortex and a mild to moderate mental retardation. Here we show that the amino acids 1-763 of the 1381 amino acids of CEP161 protein are sufficient for centrosomal targeting and centrosome association. AX2 cells over-expressing truncated and full length CEP161 proteins have defects in growth and development. Furthermore, we identified the kinase SvkA (severinkinase A) as its interaction partner which is the D. discoideum Hippo related kinase designated here as Hrk-svk. Hrk-svk is the direct homolog of human MST1. Both proteins co-localize at the centrosome. We further demonstrate that this interaction is also conserved in mammals. We were able to show that CDK5RAP2 interacts with MST1 and TAZ and it also down-regulates the transcript levels of TAZ in HEK293T cells. Taken together, our data on Dictyostelium CEP161 and human CDK5RAP2 supports the hypothesis that CDK5RAP2 as a novel regulator of Hippo signaling pathway. We propose that CDK5RAP2 mutations may lead to a decrease in the number of neurons and the subsequent reduction of brain size by regulating the hippo signaling pathway.

Phylum-wide transcriptome analysis of oogenesis and early embryogenesis in selected nematode species

Oogenesis is a prerequisite for embryogenesis in Metazoa. During both biological processes important decisions must be made to form the embryo and hence ensure the next generation: (1) Maternal gene products (mRNAs, proteins and nutrients) must be supplied to the embryo. (2) Polarity must be established and axes must be specified. While incorporation of maternal gene products occurs during oogenesis, the time point of polarity establishment and axis specification varies among species, as it is accomplished either prior, during, or after fertilisation. But not only the time point when these events take place varies among species but also the underlying mechanisms by which they are triggered. For the nematode model Caenorhabditis elegans the underlying pathways and gene regulatory networks (GRNs) are well understood. It is known that there the sperm entry point initiates a primary polarity in the 1-celled egg and with it the establishment of the anteroposterior axis. However, studies of other nematodes demonstrated that polarity establishment can be independent of sperm entry (Goldstein et al., 1998; Lahl et al., 2006) and that cleavage patterns, symmetry formation and cell specification also differ from C. elegans. In contrast to the studied Chromadorea (more derived nematodes including C. elegans), embryos of some marine Enoplea (more basal representatives) even show no discernible early polarity and blastomeres can adopt variable cell fates (Voronov and Panchin 1998). The underlying pathways controlling the obviously variant embryonic processes in non-Caenorhabditis nematodes are essentially unknown. In this thesis I addressed this issue by performing a detailed unbiased comparative transcriptome analysis based on microarrays and RNA sequencing of selected developmental stages in a variety of nematodes from different phylogenetic branches with C. elegans as a reference system and a nematomorph as an outgroup representative. In addition, I made use of available genomic data to determine the presence or absence of genes for which no expression had been detected. In particular, I focussed on components of selected pathways or GRNs which are known to play essential roles during C. elegans development and/or other invertebrate or vertebrate model systems. Oogenesis must be regulated differently in non-Caenorhabditis nematodes, as crucial controlling components of Wnt and sex determination signaling are absent in these species. In this respect, I identified female-specific expression of potential polarity associated genes during gonad development and oogenesis in the Enoplean nematode Romanomermis culicivorax. I could show that known downstream components of the polarity complexes PAR-3/-6/PKC-3 and PAR-1/-2 are absent in non-Caenorhabditis species. Even PAR-2 as part of the polarity complex does not exist in these nematodes. Instead, transcriptomes of nematodes (including C. elegans), show expression of other polarity-associated complexes such as the Lgl (Lethal giant larvae) complex. This result could pose an alternative route for nematodes and nematomorphs to initiate polarity during early embryogenesis. I could show that crucial pathways of axis specification, such as Wnt and BMP are very different in C. elegans compared to other nematodes. In the former, Wnt signaling, for instance, is mediated by four paralogous beta-catenins, while other Chromadorea have fewer and Enoplea only one beta-catenin. The transcriptomes of R. culicivorax and the nematomorph show that regulators of BMP (e.g. Chordin), are specifically expressed during early embryogenesis only in Enoplea and the close outgroup of nematomorphs. In conclusion, my results demonstrate that the molecular machinery controlling oogenesis and embryogenesis in nematodes is unexpectedly variable and C. elegans cannot be taken as a general model for nematode development. Under this perspective, Enoplean nematodes show more similarities with outgroups than with C. elegans. It appears that certain pathway components were lost or gained during evolution and others adopted new functions. Based on my findings I can conjecture, which pathway components may be ancestral and which were newly acquired in the course of nematode evolution.