Wnt, Hedgehog and Notch signalling in relation to tapeworm anteroposterior polarity and segmentation

Tapeworms are a group of parasitic flatworms whose uniquely segmented body plan has raised questions regarding their anteroposterior polarity and homology to other animals for more than a century. The Wnt, Hedgehog and Notch pathways are conserved developmental signalling pathways involved in embryonic patterning and segmentation in the Metazoa. In this thesis, these three pathways were explored in a tapeworm for the first time. Spatial expression patterns for components belonging to each of the pathways were investigated in the mouse bile duct tapeworm, Hymenolepis microstoma. Wnt pathway factors showed polarised, conserved patterns of expression of ‘posterior’ ligands (Wnts) and ‘anterior’ inhibitors (Sfrps). The expression of Wnt inhibitors, in particular, identified the true developmental anterior of H. microstoma larvae, answering a previously unresolved question of tapeworm development. In adult worms, expression patterns of these Wnt inhibitors confirmed the scolex (a region of the worm presumed to be anterior) as truly anterior. Expression of inhibitors and ‘posterior’ Wnts during strobilation shows polarised patterns along the anteroposterior boundaries of individual segments. These expression patterns indicate that the role of the Wnt pathway in specifying anteroposterior axes is conserved amongst tapeworms. Furthermore, a tapeworm-­specific Wnt11 paralog is hypothesised to have taken on a novel function during adult development, likely acting as the effector of strobilation. The expression of Hmic-­Wnt11a is highly restricted between the neck and strobila and defines a newly recognised region of the adult body, dubbed the ‘transition zone’. Hedgehog signalling in adult H. microstoma is linked with the nervous system (as it is in free-­living flatworms). During larval development Hedgehog shows canonical specification of the midline. Notch signalling was found to be involved in a number of developmental processes. The expression pattern of Hmic-­Notch1 was of particular interest as it showed polarised posterior expression within all segments. Finally, this study developed single and double fluorescent in situ hybridisation techniques using tyramide signal amplification and lead the first steps towards an in vivo approach to RNA interference method in H. microstoma

Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms

Background Early developmental patterns of flatworms are extremely diverse and difficult to compare between distant groups. In parasitic flatworms, such as tapeworms, this is confounded by highly derived life cycles involving indirect development, and even the true orientation of the tapeworm antero-posterior (AP) axis has been a matter of controversy. In planarians, and metazoans generally, the AP axis is specified by the canonical Wnt pathway, and we hypothesized that it could also underpin axial formation during larval metamorphosis in tapeworms. Results By comparative gene expression analysis of Wnt components and conserved AP markers in the tapeworms Echinococcus multilocularis and Hymenolepis microstoma, we found remarkable similarities between the early stages of larval metamorphosis in tapeworms and late embryonic and adult development in planarians. We demonstrate posterior expression of specific Wnt factors during larval metamorphosis and show that scolex formation is preceded by localized expression of Wnt inhibitors. In the highly derived larval form of E. multilocularis, which proliferates asexually within the mammalian host, we found ubiquitous expression of posterior Wnt factors combined with localized expression of Wnt inhibitors that correlates with the asexual budding of scoleces. As in planarians, muscle cells are shown to be a source of secreted Wnt ligands, providing an explanation for the retention of a muscle layer in the immotile E. multilocularis larva. Conclusions The strong conservation of gene expression between larval metamorphosis in tapeworms and late embryonic development in planarians suggests, for the first time, a homologous developmental period across this diverse phylum. We postulate these to represent the phylotypic stages of these flatworm groups. Our results support the classical notion that the scolex is the true anterior end of tapeworms. Furthermore, the up-regulation of Wnt inhibitors during the specification of multiple anterior poles suggests a mechanism for the unique asexual reproduction of E. multilocularis larvae

Genome-wide transcriptome profiling and spatial expression analyses identify signals and switches of development in tapeworms.

BACKGROUND: Tapeworms are agents of neglected tropical diseases responsible for significant health problems and economic loss. They also exhibit adaptations to a parasitic lifestyle that confound comparisons of their development with other animals. Identifying the genetic factors regulating their complex ontogeny is essential to understanding unique aspects of their biology and for advancing novel therapeutics. Here we use RNA sequencing to identify up-regulated signalling components, transcription factors and post-transcriptional/translational regulators (genes of interest, GOI) in the transcriptomes of Larvae and different regions of segmented worms in the tapeworm Hymenolepis microstoma and combine this with spatial gene expression analyses of a selection of genes. RESULTS: RNA-seq reads collectively mapped to 90% of the > 12,000 gene models in the H. microstoma v.2 genome assembly, demonstrating that the transcriptome profiles captured a high percentage of predicted genes. Contrasts made between the transcriptomes of Larvae and whole, adult worms, and between the Scolex-Neck, mature strobila and gravid strobila, resulted in 4.5-30% of the genes determined to be differentially expressed. Among these, we identified 190 unique GOI up-regulated in one or more contrasts, including a large range of zinc finger, homeobox and other transcription factors, components of Wnt, Notch, Hedgehog and TGF-β/BMP signalling, and post-transcriptional regulators (e.g. Boule, Pumilio). Heatmap clusterings based on overall expression and on select groups of genes representing 'signals' and 'switches' showed that expression in the Scolex-Neck region is more similar to that of Larvae than to the mature or gravid regions of the adult worm, which was further reflected in large overlap of up-regulated GOI. CONCLUSIONS: Spatial expression analyses in Larvae and adult worms corroborated inferences made from quantitative RNA-seq data and in most cases indicated consistency with canonical roles of the genes in other animals, including free-living flatworms. Recapitulation of developmental factors up-regulated during larval metamorphosis suggests that strobilar growth involves many of the same underlying gene regulatory networks despite the significant disparity in developmental outcomes. The majority of genes identified were investigated in tapeworms for the first time, setting the stage for advancing our understanding of developmental genetics in an important group of flatworm parasites

Managerial lead of an independent school refectory - allowance organization

Phylogeny of Forkhead (FOX) transcription factors. The FKH domain of forkhead proteins of H. sapiens, D. melanogaster, E. multilocularis, H. microstoma, and published FoxQ2 homologs from other animals were aligned, and a phylogeny was estimated by Maximum Likelihood analysis (with a JTT model) using MEGA 5.2 [85]. Bootstrap support values from 1,000 replicates are indicated next to the nodes. Nodes with lower than 50 % support were collapsed. GeneDB accession codes are given for E. multilocularis and H. microstoma and Genbank accession codes are given for all other sequences. The FoxQ2 group is outlined, and the foxQ2 genes of E. multilocularis and H. microstoma are indicated by arrows. (PDF 33 kb

Additional file 5: of Comparative analysis of Wnt expression identifies a highly conserved developmental transition in flatworms

Alkaline phosphatase-based development of whole-mount in situ hybridization in H. microstoma : Wnt2 and Wnt5. Bars: 50 Οm. (PDF 1653 kb