FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst

Abstract

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.

Lineage specification in the mouse preimplantation embryo

“…Fgfr2 is expressed in all early ICM cells at E3.25 before its restriction to PE cells by E3.5, suggesting that all early ICM cells are capable of responding to FGF ligands (Ohnishi et al, 2014;Boroviak et al, 2015). Blocking FGF signalling is sufficient for all ICM cells to adopt an EPI fate (Chazaud et al, 2006;Nichols et al, 2009;Yamanaka et al, 2010;Kang et al, 2013;Krawchuk et al, 2013), whereas the addition of excess FGF4 is sufficient to differentiate all ICM cells into PE . Interestingly, GATA6 is expressed until the early blastocyst stage in Fgf4 mutants Krawchuk et al, 2013), meaning that FGF4 is required for the salt-and-pepper patterning but that other factors regulate the initial expression of GATA6.…”

“…Blocking FGF signalling is sufficient for all ICM cells to adopt an EPI fate (Chazaud et al, 2006;Nichols et al, 2009;Yamanaka et al, 2010;Kang et al, 2013;Krawchuk et al, 2013), whereas the addition of excess FGF4 is sufficient to differentiate all ICM cells into PE . Interestingly, GATA6 is expressed until the early blastocyst stage in Fgf4 mutants Krawchuk et al, 2013), meaning that FGF4 is required for the salt-and-pepper patterning but that other factors regulate the initial expression of GATA6. In addition, FGF4 administration can repress NANOG expression without GATA6 (Bessonnard et al, 2014), while blocking FGF signalling can repress GATA6 expression in the absence of NANOG (Frankenberg et al, 2011).…”

The molecular underpinnings of totipotency

“…Exogenous FGF is also required to maintain TS cells in vitro [72]. Additionally, FGF signalling is required for the segregation of PE and Epi in vivo [40,71,73], although the initial expression of the endoderm transcription factor GATA6 is independent of FGF signalling [74,75]. However, this early GATA6 expression is in cells that also express NANOG, and FGF signalling appears essential for the downregulation of NANOG in NANOG/GATA6 coexpressing cells and the subsequent initiation of the downstream endodermal transcription programme.…”

Spécification de l’épiblaste et de l’endoderme primitif lors du développement embryonnaire préimplantatoire chez la souris

“…Afin de restaurer l'organisation en « poivre et sel », des cultures en présence de FGF4 exogène ont été réalisées. Une concentration homogène de FGF4 permet de changer l'identité de toutes les cellules de la MCI en EPr, mais elle ne recrée pas le patron en « poivre et sel » [13,14]. L'hétérogénéité du taux de FGF4 est donc un élément clé pour expliquer la spécification cellulaire en Epi et en EPr au sein de la MCI.…”

“…Nous avons postulé que la baisse hétérogène de la concentration de FGF4 extracellulaire pouvait jouer ce rôle. Cette hypothèse s'est vue renforcée par les résul-tats obtenus lors de l'analyse des mutants Fgf4 [13,14]. Néanmoins, tout autre facteur stimulant l'expression de NANOG, y compris la variabilité stochastique des taux cellulaires initiaux (la valeur aléatoire du taux de NANOG dans chaque cellule), pourrait aussi expliquer le déclenchement du mécanisme de spécification de la MCI [15] (➜).…”