On 2014 Aug 16, A Martinez-Arias commented:

There is much that is interesting and significant in this work but the section on ES cells needs to be looked at closely, particularly as the conclusions drawn here (that YAP/TAZ loss of function favours pluripotency) are at odds with others published (that YAP/TAZ are necessary for pluripotency [1]).

Here Azzolin et al. show that loss of function for YAP/TAZ can substitute for Chiron in 2i conditions. As Chiron leads to the upregulation of ß-catenin, the authors conclude that in keeping with their other findings, the effects of YAP/TAZ in pluripotency, are due to ß-catenin. Something supported by the dependence of this effect on the presence of ß-catenin. The experiments are clear but the interpretation should be given some thought.

There are three facts that need to be considered in the interpretation of these results. (a) 2i conditions are special non-physiological conditions which set cells in a peculiar, though experimentally useful, state; in fact, 2i really means “2 out of 3 (PD03, CHIR and LIF)” as LIF+PD03 and LIF+CHIR will work as well as PD03+CHIR [2] in maintaining pluripotency. In fact, ß-catenin mutant cells can be maintained in PD03+LIF, an observation which has been used to state that ß-catenin is not required for pluripotency [2]. (b) There is evidence that the levels of Oct4 are crucial for the maintenance of pluripotency [3-5] and that there is a very close relationship between ß-catenin and Oct4 [6-8]. This might be important in the interpretation of the results here. (c) The function of ß-catenin is pluripotency is not mediated by its transcriptional activity. It is curious that the authors quote Wray et al [2] in support of ß-catenin transcriptional activity in pluripotency when the manuscript shows and states that the transcriptional activity of ß-catenin is dispensable for pluripotency. Something that is further supported by studies in GSK3 mutants that lead to high levels of ß-catenin transcriptional activity which upon introduction of a dominant negative Tcf4, can be shown to be irrelevant for pluripotency [8 see also 2, 6 and 7].

The results on transcriptional activities of YAP/TAZ in ES cells and their overlap with ß-catenin/Tcf are therefore not particularly relevant to the interpretation of the results but the other two might be, as there is a need to understand the discrepancy with previous experiments. It would have been good if the authors had repeated some of their experiments in Serum and LIF or LIF and BMP to have a proper comparison. As it is, we can only consider possibilities. Lin et al. [1] show that YAP/TAZ leads to upregulation of Oct4 and it is known that lowering the levels of ß-catenin leads to increases in Oct4. As low Oct4 favours pluripotency [3, 4], could it be that lowering YAP/TAZ in 2i lowers the levels of Oct4 which then balance the ratios of the different factors and maintains pluripotency? This would provide a partial explanation for the discrepancy. There are two other possibilities worth considering. Could it be that in the same manner that 2i is ‘two out of three’, could be that it is “two out of four”, the fouth being YAP/TAZ? Finally, a key event in pluripotency is the downregulation of Tcf3 whose mechanism remains open [9]. Could it be that YAP/TAZ helps ß-catenin to downregulate Tcf3?

Removal of YAP/TAZ in 2i conditions has no effect on pluripotency, which is not surprising as in 2i condition cells are, for the most part, at the maximum capacity for pluripotency. However, to date the factors involved in 2i have been shown to also maintain pluripotency in Serum or Serum free conditions (at this it is surprising to observe the inability of PD03 to maintain pluripotency, which contradicts multiple previous results). For this reason it would be good to know if the authors have performed knock downs of YAP/TAZ in conditions other than 2i as only then one can consider the portrayed discrepancy.

References

[1] Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel MA, Goldstein LS, Abujarour R, Ding S, Guan KL. (2010) The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24,1106-18.

[2] Wray, J., Kalkan, T., Gomez-Lopez, S., Eckardt, D., Cook, A., Kemler, R. and Smith, A. (2011) 'Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation', Nat Cell Biol. 13, 838-45.

[3] Radzisheuskaya A, Le Bin Chia G, dos Santos R, Theunissen TW, Castrro LF, Nichols J, Silva J (2013) Defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nature Cell Biology 15, 579-90. [4] Karwacki-Neisius V, Goke J, Osorno R, Halbritter F, Ng JH, Weisse AY, Wong FC, Gagliardi A, Mullin NP, Festuccia N, Colby D, Tomlinson SR, Ng HH, Chambers I (2013) Reduced oct4 expression directs a robust pluripotent state with distinct signaling activity and increased enhancer occupancy by oct4 and nanog. Cell Stem Cell 12: 531-45.

[5] Muñoz Descalzo S, Rué P, Faunes F, Hayward P, Jakt LM, Balayo T, Garcia-Ojalvo J, Martinez Arias A. (2013) A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells. Mol Syst Biol. 9:694.

[6] Faunes F, Hayward P, Muñoz-Descalzo S, Chatterjee SS, Balayo T, Trott J, Christophorou A, Ferrer-Vaquer A, Hadjantonakis AK, DasGupta R, Martinez Arias A (2013) A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells. Development 140: 1171-83.

[7] Livigni A, Peradziryi H, Sharov AA, Chia G, Hammachi F, Migueles RP, Sukparangsi W, Pernagallo S, Bradley M, Nichols J, Ko MS, Brickman JM. (2013) A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance. Curr Biol. 123:2233-44.

[8] Kelly, K. F., Ng, D. Y., Jayakumaran, G., Wood, G. A., Koide, H. and Doble, B. W. (2011) 'beta-catenin enhances Oct-4 activity and reinforces pluripotency through a TCF-independent mechanism', Cell Stem Cell 8, 214-27.

[9] Yi F, Pereira L, Hoffman JA, Shy BR, Yuen CM, Liu DR, Merrill BJ. (2011) Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal. Nat Cell Biol. 13, 762-70.

On 2014 Aug 16, A Martinez-Arias commented:

There is much that is interesting and significant in this work but the section on ES cells needs to be looked at closely, particularly as the conclusions drawn here (that YAP/TAZ loss of function favours pluripotency) are at odds with others published (that YAP/TAZ are necessary for pluripotency [1]).

Here Azzolin et al. show that loss of function for YAP/TAZ can substitute for Chiron in 2i conditions. As Chiron leads to the upregulation of ß-catenin, the authors conclude that in keeping with their other findings, the effects of YAP/TAZ in pluripotency, are due to ß-catenin. Something supported by the dependence of this effect on the presence of ß-catenin. The experiments are clear but the interpretation should be given some thought.

There are three facts that need to be considered in the interpretation of these results. (a) 2i conditions are special non-physiological conditions which set cells in a peculiar, though experimentally useful, state; in fact, 2i really means “2 out of 3 (PD03, CHIR and LIF)” as LIF+PD03 and LIF+CHIR will work as well as PD03+CHIR [2] in maintaining pluripotency. In fact, ß-catenin mutant cells can be maintained in PD03+LIF, an observation which has been used to state that ß-catenin is not required for pluripotency [2]. (b) There is evidence that the levels of Oct4 are crucial for the maintenance of pluripotency [3-5] and that there is a very close relationship between ß-catenin and Oct4 [6-8]. This might be important in the interpretation of the results here. (c) The function of ß-catenin is pluripotency is not mediated by its transcriptional activity. It is curious that the authors quote Wray et al [2] in support of ß-catenin transcriptional activity in pluripotency when the manuscript shows and states that the transcriptional activity of ß-catenin is dispensable for pluripotency. Something that is further supported by studies in GSK3 mutants that lead to high levels of ß-catenin transcriptional activity which upon introduction of a dominant negative Tcf4, can be shown to be irrelevant for pluripotency [8 see also 2, 6 and 7].

The results on transcriptional activities of YAP/TAZ in ES cells and their overlap with ß-catenin/Tcf are therefore not particularly relevant to the interpretation of the results but the other two might be, as there is a need to understand the discrepancy with previous experiments. It would have been good if the authors had repeated some of their experiments in Serum and LIF or LIF and BMP to have a proper comparison. As it is, we can only consider possibilities. Lin et al. [1] show that YAP/TAZ leads to upregulation of Oct4 and it is known that lowering the levels of ß-catenin leads to increases in Oct4. As low Oct4 favours pluripotency [3, 4], could it be that lowering YAP/TAZ in 2i lowers the levels of Oct4 which then balance the ratios of the different factors and maintains pluripotency? This would provide a partial explanation for the discrepancy. There are two other possibilities worth considering. Could it be that in the same manner that 2i is ‘two out of three’, could be that it is “two out of four”, the fouth being YAP/TAZ? Finally, a key event in pluripotency is the downregulation of Tcf3 whose mechanism remains open [9]. Could it be that YAP/TAZ helps ß-catenin to downregulate Tcf3?

Removal of YAP/TAZ in 2i conditions has no effect on pluripotency, which is not surprising as in 2i condition cells are, for the most part, at the maximum capacity for pluripotency. However, to date the factors involved in 2i have been shown to also maintain pluripotency in Serum or Serum free conditions (at this it is surprising to observe the inability of PD03 to maintain pluripotency, which contradicts multiple previous results). For this reason it would be good to know if the authors have performed knock downs of YAP/TAZ in conditions other than 2i as only then one can consider the portrayed discrepancy.

References

[1] Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel MA, Goldstein LS, Abujarour R, Ding S, Guan KL. (2010) The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24,1106-18.

[2] Wray, J., Kalkan, T., Gomez-Lopez, S., Eckardt, D., Cook, A., Kemler, R. and Smith, A. (2011) 'Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation', Nat Cell Biol. 13, 838-45.

[3] Radzisheuskaya A, Le Bin Chia G, dos Santos R, Theunissen TW, Castrro LF, Nichols J, Silva J (2013) Defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nature Cell Biology 15, 579-90. [4] Karwacki-Neisius V, Goke J, Osorno R, Halbritter F, Ng JH, Weisse AY, Wong FC, Gagliardi A, Mullin NP, Festuccia N, Colby D, Tomlinson SR, Ng HH, Chambers I (2013) Reduced oct4 expression directs a robust pluripotent state with distinct signaling activity and increased enhancer occupancy by oct4 and nanog. Cell Stem Cell 12: 531-45.

[5] Muñoz Descalzo S, Rué P, Faunes F, Hayward P, Jakt LM, Balayo T, Garcia-Ojalvo J, Martinez Arias A. (2013) A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells. Mol Syst Biol. 9:694.

[6] Faunes F, Hayward P, Muñoz-Descalzo S, Chatterjee SS, Balayo T, Trott J, Christophorou A, Ferrer-Vaquer A, Hadjantonakis AK, DasGupta R, Martinez Arias A (2013) A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells. Development 140: 1171-83.

[7] Livigni A, Peradziryi H, Sharov AA, Chia G, Hammachi F, Migueles RP, Sukparangsi W, Pernagallo S, Bradley M, Nichols J, Ko MS, Brickman JM. (2013) A conserved Oct4/POUV-dependent network links adhesion and migration to progenitor maintenance. Curr Biol. 123:2233-44.

[8] Kelly, K. F., Ng, D. Y., Jayakumaran, G., Wood, G. A., Koide, H. and Doble, B. W. (2011) 'beta-catenin enhances Oct-4 activity and reinforces pluripotency through a TCF-independent mechanism', Cell Stem Cell 8, 214-27.

[9] Yi F, Pereira L, Hoffman JA, Shy BR, Yuen CM, Liu DR, Merrill BJ. (2011) Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal. Nat Cell Biol. 13, 762-70.