An investigation of cellular and molecular mechanisms of stem cell regulation in Schmidtea mediterranea

The planarian Schmidtea mediterranea discovered by Benazzi, Baguñà, Ballester, Puccinelli & Del Papa, 1975 is a classic model organism for the investigation of regenerative processes. The source of new tissues is a population of proliferative cells called 'neoblasts'. The level of heterogeneity among this population of cells is unknown. However at least a proportion of neoblasts are pluripotent stem cells, and these are sometimes referred to as clonogenic neoblasts (cNeoblasts). Although the Schmidtea mediterranea genome has been sequenced, and RNAi techniques are well established, our knowledge of the molecular regulators of neoblast behaviours such as migration in-vivo remain limited.
This thesis presents an assay for tracking spatio-temporal processes such as stem cell migration and division. Data presented herein supports the assays potential as an adjunct method for functional testing of molecular regulators of stem cell biology. Moreover this thesis demonstrates the effect of various gene knockdowns on cell migration in vivo. The matrix metalloprotease MTMMPA has been shown herein to inhibit stem cell and progeny migration. Conversely the serine threonine kinase and tumor suppressor SMG-1 has been shown to positively effect cell migration and regeneration time frames.
This 'over activity' in SMG RNAi has also been demonstrated to ultimately result in the formation of ectopic growths, analogous with tumor masses seen in cancer.
The characterisation of the Methyl-CpG-binding domain protein MBD 2/3 has been expanded upon to include a migration effect. MBD 2/3 RNAi animals exposed to shielded irradiation fail to regenerate as previously published by Jaber 20141 and through the use of Fluorescent Insitu Hybridisation (FISH) visualisation we can confirm that this phenotype is in part attributable to cell migration failure.
This body of work also demonstrates the ability of the developed assay to uncover otherwise undetectable phenotypes. Knockdown of the well-known cancer implicated zinc finger protein SNAIL has previously failed to give rise to regeneration defects in planarians. However in the shielded irradiation paradigm SNAIL RNAi does result in a lethal regenerate defect. SNAIL RNAi animals are able to maintain their stem cell and progeny populations, suggesting SNAIL does not have a role in cell maintenance and differentiation. However, investigations using a Fluorescent In situ Hybridisation technique (FISH) show that the cells of SNAIL RNAi animals fail to migrate, supporting the broadly proposed role for SNAIL in the promotion of cell migration.
Four additional genes selected using the Oncomine web-based microarray database have been identified as having a role in planarian biological processes. Knockdown of gene UDP-Nacteylglucosamine pyrophosphorylase 1 (UAP1) caused homeostatic animals to regress their heads. Further investigations using FISH to visualise underlying cell behaviours is required, however head regression is associated with stem cell defect in the planarian model2.
Knockdown of ribosomal biogenesis protein (WDR12), thyroid hormone receptor interactor 13 (TRIP13) and Bystin (BYSL) resulted in regeneration defects. To the best of our knowledge these genes have never before been investigated in planarians. The phenotypes, all characterised by a failure of animals to regrow a head, were only observable in shielded irradiation experiments. Detailed characterisation of the underlying cellular and molecular mechanisms of these defects is required. However the observations presented herein are adequate to propose that the assay developed has significant potential as a novel technique for the planarian community to investigate important cell behaviours, particularly cell migration, which has a key role in disease, specifically cancer metastasis.

Advisor: Aboobaker, A