ETV4 and ETV5 Orchestrate FGF-Mediated Lineage Specification and Epiblast Maturation during Early Mouse Development

Abstract

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signaling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are critical mediators of FGF signaling in cell lineage specification and maturation in mouse embryos. We show that loss of Etv5 compromises primitive endoderm formation at pre-implantation stages. Furthermore, Etv4/5 deficiency delays naïve pluripotency exit and epiblast maturation, leading to elevated NANOG and reduced OTX2 expression within the blastocyst epiblast. As a consequence of delayed pluripotency progression, Etv4/5 deficient embryos exhibit anterior visceral endoderm migration defects post-implantation, a process essential for coordinated embryonic patterning and gastrulation initiation. Our results demonstrate the successive roles of these FGF signaling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development.

Figure 1:. Etv4 and Etv5 expression during mouse embryonic development

(A) Single-cell RNAseq from (Nowotschin et al 2019) showing Etv4 and Etv5 mRNA expression at E3.5 and E4.5 in inner cell mass (ICM), epiblast (EPI) and primitive endoderm (PrE) lineages. (B) Confocal images of immunofluorescence immunostaining of NANOG, GATA6 and ETV5 in blastocyst stage mouse embryos. Cell numbers (c) indicated. Scale bars 20 μm (C) Quantification of ETV5 levels from (B). Double positive (DP; NANOG+GATA6), epiblast (EPI; NANOG+) and primitive endoderm (PrE; GATA6+)

Figure 2:. Loss of Etv5 compromises the formation of primitive endoderm

(A-B) Confocal images of immunofluorescence staining of CDX2, NANOG and GATA6 in an Etv4;Etv5 allelic series of embryos at mid- (A) and late- (B) blastocyst stages. Scale bars 20 μm (C) Quantification of inner cell mass (ICM) lineage composition in an allelic series of Etv4;Etv5 mutant embryos shown in (A-B). Double positive (DP; NANOG+GATA6+), primitive endoderm (PrE; NANOG−;GATA6+), epiblast (EPI; NANOG+;GATA6− light red, NANOG−;GATA6− dark red). Black dotted line represents wild-type ratio of EPI:PrE at late blastocyst stage. T-test of PrE numbers compared to wild-type, * p < 0.05, ** p < 0.01, otherwise comparison not significant. Etv4^+/+;Etv5^−/− at late-blastocyst stage did not have sufficient n number to performstatistical test. (D) Quantification of NANOG levels in epiblast cells in wild-type embryos and an allelic series of Etv4;Etv5 mutant embryos shown in (A) at mid-blastocyst stage. T-test of mean NANOG levels per embryo compared to wild-type * p < 0.05, otherwise comparison not significant.

Figure 3:. Mechanism of Etv5 action on inner cell mass cell fate decision

(A) ETV5 binding to the Fgf4 locus in mouse embryonic stem cells (mESC) in naïve pluriptency conditions (2i) and upon naïve pluropotency exit (16 hours after 2i withdrawal). ChIP-seq data (Kalkan et al 2019). (B) Expression of Fgf4 in wild-type and Etv5^−/− late-stage blastocyst by qPCR. T-test, * p < 0.05. (C) Confocal images of immunofluorescence staining of CDX2, NANOG and GATA6 in wild-type and Etv5^−/− mutant embryos treated with or without 1μg/ml FGF4 + 1μg/ml Heparin from E2.5 + 48 hours. Scale bars 20 μm. (D) Quantification of inner cell mass (ICM) lineage composition in treated embryos (C). Double positive (DP; NANOG+GATA6+), primitive endoderm (PrE; NANOG−;GATA6+), epiblast (EPI; NANOG+;GATA6− light red, NANOG−;GATA6− dark red).

Figure 4:. Loss of Etv4/5 causes a delay in the progression of pluripotency

(A) Confocal images of immunofluorescence staining of SOX2, OTX2 and KLF4 in an allelic series of Etv4;Etv5 mutant embryos at the late blastocyst stage. (B-C) Quanitifcation of primed marker OTX2 from (A) by individual genotypes (B) or grouped by Etv5 genotype (C). T-test of mean OTX2 levels in epiblast cells per embryo, compared with wild-type, * p < 0.05, otherwise comparison was not significant. (D) ChIP-seq of ETV5 binding to the Otx2 locus in mouse naïve ESC (2i) and after pluripotency exit (2i withdrawal). Data from (Kalkan et al., 2019).

Figure 5:. Compound Etv4/5 mutants have developmental delay and anterior visceral endoderm migration defects

(A) Confocal max intensity projection images of an allelic series of Etv4;Etv5 embryos at mid-streak gastrulation stages, embryonic day (E) 6.5, immunostained for NANOG and OTX2. Arrowheads show abnormal anterior/distal visceral endoderm migration and/or morphology. (B) Confocal images of an allelic series of Etv4;Etv5 embryos at mid-streak gastrulation stages, embryonic day (E) 6.5 immunostained for SOX2, T, and CER1. Arrowheads show abnormal anterior visceral endoderm migration and morphology. MIP = max intensity projection. A = Anterior, P= Posterior. Pr = Proximal. D = Distal. Scale bars 100μm.