Identification of factors involved in the maintenance of embryonic stem cell self renewal and pluripotency 3

I was able to show that Lefty2 RNAi induces ES cell differentiation specifically towards the meso/mesendodermal cell fates.. The work presented in this thesis reveals that LEFTY2 is sec

Chapter3

Conclusion

3 Conclusion

The first chapter of this thesis summarizes the current state of knowledge regarding what constitutes the fundamental mechanics governing ES cell self-renewal Much is left to be learnt The primary aim of this project hence serves to identify and delineate the roles of additional factors and pathways that are important for the maintenance of ES cell self-renewal To achieve this aim, a candidate gene approach was adopted and 70 candidates were shortlisted An RNAi screen in mouse ES cells (E14) using Oct4 and Nanog promoter activity as readouts was used to further narrow down the

choice of candidates Lefty2 was one very apparent candidate that emerged from the RNAi screen as Lefty2 RNAi perturbed both Oct4 and Nanog promoter activity Athough Lefty2 has previously been implicated in embryonic development and stem cell-specific expression of Lefty has been observed in mouse and human, its functionalsignificance in embryonic stem cells has never been demonstrated A part of this thesis is hence

dedicated to addressing if Lefty2 plays any functional role(s) in embryonic stem cells To elucidate the biological role(s) of Lefty2, loss of function studies using shRNAs was carried out I was able to show that Lefty2 RNAi induces ES cell differentiation

specifically towards the meso/mesendodermal cell fates The next objective to be met

was the dissection of the molecular mechanism via which Lefty2 operates to maintain ES

cell self renewal The work presented in this thesis reveals that LEFTY2 is secreted by mouse ES cell as a negative feedback mechanism to autoregulate autocrine Nodal signals

How Nodal signals dictate embryonic stem cell fates is also not entirely clear Conflicting roles of Nodal signaling in both the maintenance of ES cell self renewal and induction of ES cell differentiation have both previously been reported A key objective

of this thesis is to reconcile the differences between studies reported by several group Nodal, as a morphogen, is known to elicit effects in embryos in a dose dependent manner

In this thesis, the possibility that Nodal signaling might, as in the in vivo scenario, likewise influence ES cell fate decision in a dose dependent manner in vitro is addressed Interestingly, it was found that Nodal signaling is able to arbitrate at least three distinct fate decisions in ES cells Perturbation of endogenous signaling in ES cells leads to their exit from self renewal towards differentiation Increasing Nodal signals above basal

levels through ALK4* overexpression, Nodal overexpression, Lefty2 RNAi or addition

of Activin A ligand causes differentiation of ES cells into meso/mesendodermal lineages while repression of signaling with the specific chemical inhibitor SB431542 and Lefty protein supplementation induces trophectodermal differentiation This suggests that Nodal/Activin signaling must be tightly regulated to sustain ES cell self renewal, as deviation from this steady state promotes divergent developmental programs

Nodal/Activin signaling is mediated by SMAD2 and/or SMAD3 Part of this thesis serves to dissect the individual contribution of SMAD2 and SMAD3 towards the various cell fate arbitrated by Nodal signaling Here, data showing that inhibition of SMAD3 activity blocks Nodal/Activin signaling induced mesendodermal specification is presented This suggests that mesendodermal specification of ES cells is a SMAD3

specification of ES cells as inhibition of SMAD3 activity does not lead to ES cell differentiation towards trophectoderm

Here, it is also found that graded activation of Nodal signal transduction pathways

is translated into quantitative differences in level of SMAD2 phosphorylation It is plausible that SMAD2 binds differentially to target genes at differential level of phosphorylation, thereby leading to activation of different target gene sets at the various signaling thresholds Such an investigation is currently in progress to verify if the mechanism via which graded Nodal signaling translates into different cell fates is indeed

as such

In conclusion, the data presented in this thesis lead to the establishment of Lefty2

as an important regulator of embryonic stem cell fate Also, this thesis extends the current understanding of ES cell self renewal mechanism beyond the established roles of LIF/STAT3, BMP and WNT pathways and that of the core transcriptional regulatory circuitry governed by OCT4, SOX2 and NANOG Furthermore, this thesis is the first report that puts in perspective how graded Nodal signaling is critical for ES cell fate decisions