Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Feb 1;446(1):94-101.
doi: 10.1016/j.ydbio.2018.12.008. Epub 2018 Dec 12.

FGFR1 regulates trophectoderm development and facilitates blastocyst implantation

Agata Kurowski  1 , Andrei Molotkov  1 , Philippe Soriano  2
Affiliations

FGFR1 regulates trophectoderm development and facilitates blastocyst implantation

Agata Kurowski et al. Dev Biol. .

Abstract

FGF signaling plays important roles in many aspects of mammalian development. Fgfr1-/- and Fgfr1-/-Fgfr2-/- mouse embryos on a 129S4 co-isogenic background fail to survive past the peri-implantation stage, whereas Fgfr2-/- embryos die at midgestation and show defects in limb and placental development. To investigate the basis for the Fgfr1-/- and Fgfr1-/-Fgfr2-/- peri-implantation lethality, we examined the role of FGFR1 and FGFR2 in trophectoderm (TE) development. In vivo, Fgfr1-/- TE cells failed to downregulate CDX2 in the mural compartment and exhibited abnormal apicobasal E-Cadherin polarity. In vitro, we were able to derive mutant trophoblast stem cells (TSCs) from Fgfr1-/- or Fgfr2-/- single mutant, but not from Fgfr1-/-Fgfr2-/- double mutant blastocysts. Fgfr1-/- TSCs however failed to efficiently upregulate TE differentiation markers upon differentiation. These results suggest that while the TE is specified in Fgfr1-/- mutants, its differentiation abilities are compromised leading to defects at implantation.

Keywords: FGF; TS cells; Trophectoderm.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. FGFR1 and FGFR2 are expressed in the TE and the extraembryonic ectoderm.
A, Maximal projection (MP) of confocal immunofluorescence images of FGFR reporter embryos showing strong FGFR1GFP and FGFR2mCHERRY expression in the TE of E4.5 blastocysts. OCT4 expression marks the EPI. The single plane (SP) image through the EPI clearly detects FGFR1GFP expression in the ICM, whereas FGFR2mCHERRY is weakly expressed. The dashed line was drawn around cells which expressed OCT4. B, at E5.5 FGFR1GFP is expressed throughout embryonic- and extraembryonic tissues. FGFR2mCHERRY is restricted to the extraembryonic ectoderm. OCT4 marks the EPI. C, both receptors are expressed in CDX2 positive TSCs. DAPI shows nuclei.
Figure 2.
Figure 2.. E4.5 FGFR1−/− TE is specified but the expression of CDX2 and E-Cadherin is abnormal.
A and B, maximal projection confocal immunofluorescence images of wild type and Fgfr1−/− E4.5 blastocysts showing NANOG, CDX2 and E-Cadherin expression. A, wild type E4.5 blastocysts show downregulated CDX2 expression in the mural TE and basolateral E-Cadherin expression in TE cells. NANOG marks the EPI cells. Heat maps show the intensity of CDX2 and E-Cadherin expression. B, Fgfr1−/− blastocysts maintain high CDX2 expression throughout the TE and show strong basolateral and abnormal apical E-Cadherin expression (arrows).
Figure 3.
Figure 3.. Fgfr1−/− blastocysts at E4.5 show abnormal TE cell polarity and an increased apicobasal distance.
A, Single plane confocal immunofluorescence image of wild type and Fgfr1−/− E4.5 blastocysts showing basolateral E-Cadherin expression in TE cells and basolateral and apical expression in Fgfr1−/−. Insets show enlarged single TE cells and depict the apical (a) and basal (b) cell membranes. B and C, confocal plane section images showing that the apicobasal distance of TE cells of Fgfr1−/− embryos is increased in comparison to TE cells of control embryos. C, quantification of TE cell apicobasal distance of two Fgfr1+/+ and two Fgfr1−/− embryos. Cells measured in A and B are quantified in C.
Figure 4.
Figure 4.. Fgfr1−/− TSCs can be derived and maintained in culture.
A, brightfield images showing similar morphology of control and Fgfr1−/− TSCs. B, confocal immunofluorescence images, showing the expression of the TSC marker CDX2 and absence of PrE or EPI blastocyst markers (GATA6 and NANOG) in derived TSC lines. C, RT-qPCR confirming the loss of Fgfr1 mRNA in Fgfr1−/− TSCs and showing unchanged Fgfr2 levels. D, Immunofluorescence and RT-qPCR, showing unchanged E-Cadherin protein expression and mRNA levels in control and mutant TSCs. E, RT-qPCR for TSCs markers showing no significant changes in Fgfr1−/− compared to wild type TSCs. F, RT-qPCR showing significantly lower expression of TE lineage markers in differentiated Fgfr1−/− compared to wild type TSCs. C-F: graphs represent three individually derived wild type TSC lines and three individually derived Fgfr1−/− TSC lines. * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 5.
Figure 5.. Fgfr1−/− TSCs differentiation is impaired.
A, brightfield images, showing that wild type and Fgfr1−/− TSCs become larger and less dense upon FGF removal for two days. No morphological differences can be detected between wild type and mutant cells. B, mRNA levels of TSC markers are downregulated equally in wild type and Fgfr1−/− TSCs. C, TE lineage markers are not upregulated to the same extent in Fgfr1−/− TSCs compared to wild type. B and C: Graphs represent technical triplicates of the same TSC line. * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 6.
Figure 6.. Fgfr1−/− Fgfr2−/− embryos show reduced TE, and TSCs cannot be obtained from CDX2 overexpressing Fgfr1−/−Fgfr2−/− ESCs or deleted from Fgfr1flox/flox Fgfr2flox/flox TSCs.
A and B, Optical section images of Fgfr1−/− Fgfr2−/− embryos isolated at E3.5 and cultured for 48h show reduced numbers of CDX2 positive TE cells. Seven Fgfr1+/+Fgfr2+/+ and six Fgfr1−/−Fgfr2−/− embryos were quantified, *** P < 0.001. C, Maximal projection images showing Fgfr1+/+-Cdx2ERT2 puromycin resistant clones expressing CDX2 and downregulating NANOG upon 24 hours of 4-OHT treatment. GATA6 is not expressed. D, brightfield images, showing three representative images for each genotype. Seven out of eight Fgfr1+/+Fgfr2+/+-Cdx2ERT2 ESC clones gave rise to TSC colonies, but none out of six Fgfr1−/−Fgfr2−/−-Cdx2ERT2 ESC clones gave rise to TSC colonies after two weeks in TSC medium with 1μg/ml 4-OHT. E, PCR results of genomic DNA showing that two out of three Fgfr1flox/+Fgfr2flox/+-CreERT2 TSC clones exhibit CRE recombination after induction with 4-OHT. Fgfr1flox/flox Fgfr2flox/flox-CreERT2 TSC colonies that escaped cell death did not undergo CRE recombination. F, Brightfield images showing 4-OHT treated Fgfr1flox/flox Fgfr2flox/flox-CreERT2 cultures exhibiting cell death.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith A, 2013. Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell 153, 335–347. - PMC - PubMed
    1. Blij S, Frum T, Akyol A, Fearon E, Ralston A, 2012. Maternal Cdx2 is dispensable for mouse development. Development 139, 3969–3972. - PMC - PubMed
    1. Brewer JR, Mazot P, Soriano P, 2016. Genetic insights into the mechanisms of Fgf signaling. Genes Dev 30, 751–771. - PMC - PubMed
    1. Brewer JR, Molotkov A, Mazot P, Hoch RV, Soriano P, 2015. Fgfr1 regulates development through the combinatorial use of signaling proteins. Genes Dev 29, 18631874. - PMC - PubMed
    1. Chazaud C, Yamanaka Y, Pawson T, Rossant J, 2006. Early lineage segregation between epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev Cell 10, 615–624. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources