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. 2025 Mar 15;152(6):dev204278.
doi: 10.1242/dev.204278. Epub 2025 Mar 24.

ETV4 and ETV5 orchestrate FGF-mediated lineage specification and epiblast maturation during early mouse development

Claire S Simon  1   2   3 , Woonyung Hur  1   4 , Vidur Garg  1 , Ying-Yi Kuo  1 , Kathy K Niakan  2   3   5   6 , Anna-Katerina Hadjantonakis  1
Affiliations

ETV4 and ETV5 orchestrate FGF-mediated lineage specification and epiblast maturation during early mouse development

Claire S Simon et al. Development. .

Abstract

Cell fate decisions in early mammalian embryos are tightly regulated processes crucial for proper development. While FGF signalling plays key roles in early embryo patterning, its downstream effectors remain poorly understood. Our study demonstrates that the transcription factors Etv4 and Etv5 are crucial mediators of FGF signalling 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 and Etv5 (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/Etv5-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 signalling effectors in early lineage specification and embryonic body plan establishment, providing new insights into the molecular control of mammalian development.

Keywords: ETV; Epiblast; FGF; Pluripotency; Primitive endoderm.

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Conflict of interest statement

Competing interests A.-K.H. is an editor at Development. The authors declare that they have no other competing interests.

Figures

Fig. 1.
Fig. 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) are 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+). Sample size: number (n) of embryos is indicated. (D) Confocal images of immunofluorescence immunostaining of SOX2, GATA6 and ETV5 in post-implantation stage mouse embryos. Dashed lines indicate the Epi-ExE boundary (E5.5) and the extension of the PS (E6.5). Scale bars: 50 μm. A, anterior; P, posterior; Pr, proximal; D, distal; ExE, extraembryonic-ectoderm; Epi, epiblast; PS, primitive streak.
Fig. 2.
Fig. 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 and 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. Unpaired t-test of PrE numbers compared to wild type: *P<0.05, **P<0.01 (comparisons that are not significant are not indicated). Etv4+/+;Etv5−/− at late-blastocyst stage did not have sufficient n values to perform a statistical 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. Unpaired t-test of mean NANOG levels per embryo compared to wild type: *P<0.05 (comparisons that are not significant are not indicated). Boxplots represent the interquartile range (IQR), with the median shown as a central line; whiskers extend to lowest or highest value within 1.5×IQR; individual data points show mean NANOG levels per embryo.
Fig. 3.
Fig. 3.
wMechanism 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 pluripotency conditions (2i) and upon naïve pluripotency exit (16 h after 2i withdrawal). ChIP-seq data are reproduced from Kalkan et al. (2019), where it was published under a CC-BY license (https://creativecommons.org/licenses/by/4.0/). (B) Expression of Fgf4 in wild-type and Etv5−/− late-stage blastocyst by qPCR. Unpaired t-test: *P<0.05. Boxplots represent the interquartile range (IQR), with the median shown as a central line; whiskers extend to lowest or highest value within 1.5×IQR; individual data points show normalised gene expression levels in each embryo. (C) Schematic of embryo culture treatments. (D) Confocal images of immunofluorescence staining of CDX2, NANOG and GATA6 in wild-type and Etv5−/− mutant embryos treated with or without 1 μg/ml FGF4+1 μg/ml heparin from E2.5 for 48 h. Scale bars: 20 μm. (E) Quantification of inner cell mass (ICM) lineage composition in treated embryos (C). DP, double positive (NANOG+GATA6+); PrE, primitive endoderm (NANOG−GATA6+); EPI, epiblast (NANOG+GATA6−, red). Unpaired t-test: *P<0.05, ***P<0.001.
Fig. 4.
Fig. 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) Quantification of primed marker OTX2 from A by individual genotypes (B) or grouped by Etv5 genotype (C). Boxplots represent the interquartile range (IQR), with the median shown as a central line; whiskers extend to lowest or highest value within 1.5×IQR; individual data points show normalised gene expression levels in each embryo. Unpaired t-test of mean OTX2 levels in epiblast cells per embryo, compared with wild type: *P<0.05 (comparisons that are not significant are not indicated). (D) ChIP-seq of ETV5 binding to the Otx2 locus in mouse naïve ESC (2i) and after pluripotency exit (2i withdrawal). Data reproduced from Kalkan et al. (2019), where it was published under a CC-BY license (https://creativecommons.org/licenses/by/4.0/).
Fig. 5.
Fig. 5.
Compound Etv4/5 mutants have developmental delay and anterior visceral endoderm migration defects. (A) Confocal maximum 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 indicate 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 indicate abnormal anterior visceral endoderm migration and morphology. MIP, maximum intensity projection; A, anterior; P, posterior; Pr, proximal; D, distal. Scale bars: 100 μm.
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