A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo

Abstract

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

Keywords: Eomes; dTAG; degron; embryo; mouse.

Fig. 1.

Generation of an Eomes^deg allele using CRISPR-Cas9 and functional analysis in vitro during HE differentiation. (A) Cas9-based targeting strategy for generation of the Eomes^deg allele. A crRNA targeting the stop codon (in grey) of the endogenous Eomes locus was used along with a ssDNA repair template containing the sequence for the degron (FKBP12^F36V) tag, a 2A self-cleaving peptide, and an mCherry reporter. HA, homology arm; XTEN, unstructured peptide linker; P2A, self-cleaving peptide; DEG, degron tag; AB_38/41, primers used for screening of clones in panel (B). (B) PCR genotyping of wildtype and homozygously tagged ESC clones with AB_38/41 primers denoted in A. (C) Schematic of the HE EB differentiation protocol described in Harland et al. (9). Also indicated are the experimental procedures for the results presented in D–F. (D) Western blot analysis of day 3.0 EBs demonstrates the size shift due to the presence of the degron tag compared to Eomes^+/+ EBs and the rapid loss of Eomes protein within 1 h of dTAG-13 treatment. Dimethylsulfoxide (DMSO) is a vehicle control and MEFs are used as a Eomes negative control. (E) Intracellular flow cytometry in day 4.0 Eomes^deg/deg EBs incubated in dTAG-13-containing medium from day 2.0 onwards demonstrates loss of Eomes protein. (F) Eomes^deg/deg EBs and parental E14-RV Eomes^+/+ ESCs form similar cell populations during HE differentiation, as assessed by live cell flow cytometry for mesodermal/HE markers Flk1 and PdgfRa. Eomes^deg/deg EB cultures treated with dTAG-13 from day 2.0 onwards phenocopy Eomes^–/– EBs, as addition of dTAG-13 to Eomes^deg/deg EBs on day 2.0 results in failure to upregulate Flk1 and disrupts the formation of Flk1⁺/PdgfRa⁺ HE progenitors on day 4.0. mCherry+ cells identify the Eomes expressing cell population (red).

Fig. 2.

Ex vivo treatment of Eomes^deg/deg preimplantation embryos. (A) Schematic representation of the experimental protocol to test Eomes-degron degradation in blastocyst stage embryos. (B) Eomes and Nanog staining of DMSO, dTAG-13, or dTAG^V-1 treated E3.5 Eomes^deg/deg blastocyst stage embryos. The TE of dTAG-treated Eomes^deg/deg embryos lacks Eomes expression. (Scale bars, 50 µm.) (C) Quantification of Eomes expression in Eomes^deg/deg blastocysts treated with DMSO (n = 5), dTAG-13 (n = 6), or dTAG^V-1 (n = 5). Corrected total cell fluorescence is calculated as the Integrated Density – (Area of selected cell × Mean fluorescence of background readings). (D) Cultured Eomes^deg/deg homozygous blastocysts treated with DMSO, dTAG-13, or dTAG^V-1 for 72 h. Eomes^deg/deg blastocysts treated with DMSO attach to the plastic substrate and form trophoblast outgrowths with typical giant cells (arrows). In contrast, Eomes^deg/deg blastocysts treated with dTAG-13 or dTAG^V-1 fail to attach and outgrow phenocopying Eomes null embryos, as described previously (2, 4, 5). DMSO, n = 6; dTAG-13, n = 7; dTAG^V-1, n = 8.

Fig. 3.

dTAG treatment of in utero embryos at 6.5dpc. (A) Schematic representation of the experimental protocol designed to test Eomes-degron degradation in utero at 6.5 dpc. (B) Eomes^deg/deg homozygous embryos treated in utero with DMSO, dTAG-13, or dTAG^V-1 for 4 h were dissected, fixed, and stained for Eomes (green) and Foxa2 (red). Nuclei are counterstained with DAPI (blue). Foxa2 staining identifies the VE and the definitive endoderm progenitors in the PS. Eomes staining shows a heterogeneous pattern of degradation of the tagged Eomes-degron protein. Selected images illustrate the range of Eomes degradation observed. Eomes positive cells are detectable in the PS on the proximal posterior side of the dTAG-13 treated embryo. The dTAG^V -1 treated embryo shown retains Eomes expression in the ExE, EmVE, and PS. (Scale bars, 50 µm.)

Fig. 4.

Eomes expression is differentially reduced in a spatial pattern in Eomes^deg/deg embryos after dTAG-13 injection at 6.5 dpc and 7.5 dpc. (A) Schematic representation of the experimental protocol designed to test Eomes -degron degradation in utero at 6.5 dpc and 7.5 dpc. (B and C) IF staining of E6.5 (B) and E7.5 (C) Eomes^deg/+ and Eomes^deg/deg embryos for Eomes (green) and Foxa2 (red) recovered 2 h after pregnant females were injected IP with dTAG-13, as indicated in schematic diagram. White boxes indicate the magnified areas of Eomes expression displayed in the Right panels. Images representative of 12 embryos (E6.5) and 7 embryos (E7.5). Foxa2 (red) staining highlights VE and definitive endoderm cells. Nuclei were stained with DAPI (blue). (Scale bars, 50 μm.)

Fig. 5.

Ex vivo treatment of Eomes^deg/deg embryos and EBs to assess the time-course of Eomes recovery after dTAG-13 or dTAG^V-1 washout and validation of the mCherry reporter. (A) Schematic representation of experimental protocol for dTAG small-molecule treatment and washout. (B) Eomes (green) and Foxa2 (red) staining of DMSO, dTAG-13, or dTAG^V-1 treated E6.5 ex vivo cultured embryos fixed after 2 h or subsequently washed out and cultured for a further 2 h. Eomes expression recovers in the proximal posterior epiblast/PS region 2 h post washout. Nuclei are stained with DAPI (blue). (Scale bars, 50 μm.) (C) Eomes (green) and Nanog (red) staining of day 3.0 HE EBs cultured under the indicated conditions to assess the kinetics of Eomes recovery during in vitro differentiation conditions. Eomes staining is undetectable in EBs treated for 1 h and 2 h with dTAG-13. Eomes nuclear staining becomes evident 1 h post-washout onwards. Nanog staining identifies pluripotent cells. Nuclei are stained with DAPI (blue). (Scale bars, 50 μm.) (D) RFP (red) and Eomes (green) staining of E7.5 Eomes^deg/+ embryos. RFP staining detects the mCherry reporter expression. Nuclei are stained with DAPI (blue). (Scale bars, 50 μm.) (E) Flow cytometric analysis of mCherry reporter expression in dissociated E7.5 Eomes^deg/deg embryos. Roughly 70% of these cells express the mCherry reporter.