The sorting and formation of the PrE layer in the H2BGFP knock-in blastocysts. and post implantation blastocysts. Our investigation of the heterozygote and null GFP reporter embryos led to several interesting findings on the regulation of promoter and the role and mechanism of FGF/Ras/Erk1/2 signaling pathway in primitive endoderm development. 2. Results 2.1. Production of an H2BGFP Gata6 knock-in mouse line Previously we observed that promoter activity in living embryos, we generated a GATA6 reporter mutant mouse line using homologous recombination in ES cells by substitution of a GFP coding sequence for a fragment of the gene starting at the translational start site and covering the following exon 2 (here, indicates the mouse gene, promoter. (B) Correlation of GFP signal and GATA6 protein in (+/?)) ES cells were treated with retinoic acid (1 M) for 4 days to induce primitive endoderm differentiation. The cells were stained with anti-GATA6 and counter stained with DAPI. The fluorescence signals of GFP, GATA6 immunostaining, and DAPI were captured and compared. (C) E3.5 wild type, (+/?)), and (?/?)) embryos from matings of (+/?)) and (?/?)) embryos, but were absent in wild type embryos. Positive GATA6 immunostaining was observed in wild type and (+/?)) embryos, but was absent in (?/?)) embryos. (D) Examples of GFP and GATA6 expression in (+/?)) and (?/?)) blastocysts at E4.5 stage are shown. The GFP signals and GATA6 immunostaining were compared. The GATA6 reporter line NCT-502 was first characterized rigorously HSPA1 in cells and tissues from gene dosage caused a reduced number of PrE cells in (+/?) blastocysts (Schrode et al., 2014), though the heterozygous embryos appeared to develop normally afterward (Cai et al., 2008; Bessonnard et al., 2014; Schrode et al., 2014). Also, discrepancies between reporter expression and Gata6 protein were found in another GATA6 reporter line using H2B-Venus transcriptional reporter allele (Freyer et al., 2015). Our analysis of the promoter activity. 2.2. GFP expression in the H2BGFP knock-in blastocysts Mutant pre-implantation embryos were harvested from timed matings of (?/?)) embryos from top to bottom indicated absence of PrE and diminutive GFP signal in the entire ICM (Supplementary Data Movie 3, 4). Open in a separate window Fig. 2 GATA6 is required for primitive endoderm differentiation. (A) (+/?)) heterozygous embryos were collected at E3.5 and matured in culture to around the E4.5 stage. The embryos were examined for GFP and analyzed by immunostaining for GATA4, GATA6, and Dab2 proteins. PrE cells are positive for GFP, Dab2, GATA4, and GATA6. Trophectoderm cells are positive for GFP and GATA6 but are negative for GATA4. NCT-502 (B) Images of GFP and immunofluorescence of (?/?)) blastocysts at the E4.5 stage are shown as representative examples. The embryos were examined for GFP signal and immunostaining for GATA4, GATA6, and Dab2 proteins. (C) Embryos in uterine tissues from a mating between (+/?)) heterozygous mice were collected at E5.5 stage and analyzed following cryo-sectioning. The embryos on slides were for assessed for GFP fluorescence, and were evaluated for Gata6 positivity by immunostaining as well as PCR genotyped of cells collected from the slides. A representative (+/?)) heterozygous embryo was shown for GFP fluorescence, and counterstained with DAPI. (D) A representative (?/?)) homozygous mutant embryo is shown. Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.ydbio.2018.02.007 By E5.0 stage, the newly implanted embryos exhibited a GATA6-positive PrE layer covering NCT-502 the GFP weak epiblast in (+/?)) blastocysts were collected.