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.

Repeated evolution of heat responsiveness among Brassicaceae COPIA transposable elements

Eukaryotic genomes contain repetitive DNA sequences. This includes simple repeats and more complex transposable elements (TEs). Many TEs reach high copy numbers in the host genome, owing to their amplification abilities by specific mechanisms. There is growing evidence that TEs contribute to gene transcriptional regulation. However, excess of TE activity may lead to reduced genome stability. Therefore, TEs are suppressed by the transcriptional gene silencing machinery via specific chromatin modifications. In contrary, effectiveness of the epigenetic silencing mechanisms imposes risk for TE survival in the host genome. Therefore, TEs may have evolved specific strategies for bypassing epigenetic control and allowing the emergence of new TE copies. Recent studies suggested that the epigenetic silencing can be, at least transiently, attenuated by heat stress in A. thaliana. Heat stress induced strong transcriptional activation of COPIA78 family LTR-retrotransposons named ONSEN, and even their transposition in mutants deficient in siRNA-biogenesis. ONSEN transcriptional activation was facilitated by the presence of heat responsive elements (HREs) within the long terminal repeats, which serve as a binding platform for the HEAT SHOCK FACTORs (HSFs). This thesis focused on the evolution of ONSEN heat responsiveness in Brassicaceae. By using whole-transcriptome sequencing approach, multiple Arabidopsis lyrata ONSENs with conserved heat response were found and together with ONSENs from other Brassicaceae were used to reconstruct the evolution of ONSEN HREs. This indicated ancestral situation with two, in palindrome organized, HSF binding motifs. In the genera Arabidopsis and Ballantinia, a local duplication of this locus increased number of HSF binding motifs to four, forming a high-efficiency HRE. In addition, whole transcriptome analysis revealed novel heat-responsive TE families COPIA20, COPIA37 and HATE. Notably, HATE represents so far unknown COPIA family which occurs in several Brassicaceae species but is absent in A. thaliana. Putative HREs were identified within the LTRs of COPIA20, COPIA37 and HATE of A. lyrata, and could be preliminarily validated by transcriptional analysis upon heat induction in subsequent survey of Brassicaeae species. Subsequent phylogenetic analysis indicated a repeated evolution of heat responsiveness within Brassicaceae COPIA LTR-retrotransposons. This indicates that acquisition of heat responsiveness may represent a successful strategy for survival of TEs within the host genome.