A framework for TRIM21-mediated protein depletion in early mouse embryos: recapitulation of Tead4 null phenotype over three days

Abstract

Background: While DNA and RNA methods are routine to disrupt the expression of specific genes, complete understanding of developmental processes requires also protein methods, because: oocytes and early embryos accumulate proteins and these are not directly affected by DNA and RNA methods. When proteins in the oocyte encounter a specific antibody and the TRIpartite Motiv-containing 21 (TRIM21) ubiquitin-protein ligase, they can be committed to degradation in the proteasome, producing a transient functional knock-out that reveals the role of the protein. However, there are doubts about whether this targeted proteolysis could be successfully used to study mammalian development, because duration of the transient effect is unknown, and also because amounts of reagents delivered must be adequate in relation to the amount of target protein, which is unknown, too.

Results: We show that the mouse egg contains up to 1E-02 picomoles/protein, as estimated by mass spectrometry using the intensity-based absolute quantification (iBAQ) algorithm. However, the egg can only accommodate ≈1E-04 picomoles of antibody or TRIM21 without incurring toxic effects. Within this framework, we demonstrate that TRIM21-mediated protein depletion efficiently disrupts the embryonic process of trophectoderm formation, which critically depends on the TEA domain family member 4 (Tead4) gene. TEAD4 depletion starting at the 1-cell stage lasts for 3 days prior to a return of gene and protein expression to baseline. This time period is long enough to result in a phenotype entirely consistent with that of the published null mutation and RNA interference studies: significant underexpression of trophectodermal genes Cdx2 and Gata3 and strongly impaired ability of embryos to cavitate and implant in the uterus. Omics data are available via ProteomeXchange (PXD012613) and GEO (GSE124844).

Conclusions: TRIM21-mediated protein depletion can be an effective means to disrupt gene function in mouse development, provided the target gene is chosen carefully and the method is tuned accurately. The knowledge gathered in this study provides the basic know-how (prerequisites, requirements, limitations) to expedite the protein depletion of other genes besides Tead4.

Keywords: Mouse; Oocyte; Preimplantation embryo; Proteome; TEAD4; TRIM21; Trophectoderm.

Fig. 1

a. Venn diagram overview [38] of the number of protein identities detected in oocytes and preimplantation stages of the mouse following LC-MS/MS. b. Distribution of individual protein abundances (riBAQ) in oocytes and blastocysts. c. Survey of the top 50 most abundant proteins detected in oocytes, plus TEAD4. d. Demonstration that the subunits of four known macromolecular complexes are quantified at similar riBAQ values within each complex. e. Tentative picogram amounts of protein p predicted with the formula M_p = riBAQ_p × MW_p are consistent with those found in historical immunoblotting data Abbreviations: MS, mass spectrometry; WB, western blotting; MII, metaphase II oocyte

Fig. 2

a. Picture of a metaphase II mouse oocyte with the picture of a micrometre grid (10-μm intervals) superimposed. b. Zygotes were filmed during injection with a gentle flow of concentrated suspension (0.2 mg/mL), but only up to the filling of the perivitelline space with an injected volume ‘x’; selected frames were extracted from movie at indicated time points. Note that the perivitelline space is completely filled at 25 s, but recovers partly after 2 min and completely after 4 min. c. Serial doubling dilutions of a standard of green-fluorescent dextran beads (Oregon Green dextran beads, OGDB). Zygotes were blown up with a maximum flow of OGDB suspension applied continuously for 30 s, for each of six concentrations (0.2 mg/mL halved by serial dilutions down to 0.00625 mg/mL), causing the zona to be evacuated and the cytoplasm to be completely replaced by OGDB. The green fluorescence intensity was recorded for each dilution with the same excitation and time exposure (d). Dilution factor: A zygote injected as in (B), shown in the small inset photo, had a fluorescence intensity corresponding to 42% (1/2.4) of the fluorescence of the standard alone. These data allow one to solve a simple equation for the injected volume ‘x’: dilution factor = 2.4 = (x + 220 pl) / x = 157 pl. Size bar, 50 μm. AU, arbitrary units of fluorescence intensity

Fig. 3

a. Dose-effect curves in zygotes injected with various concentrations of TRIM21 protein or mCherry-Trim21 mRNA (mg/mL) in a volume of ≈ 100 picoliters. N = 43 zygotes were inspected for each mRNA concentration, N = 22 zygotes for each protein concentration. b Q-PCR for Trim21 sequence conducted at 24-h intervals after microinjection of mCherry-Trim21 mRNA in the zygote. N = 5 embryo equivalents from a lysate of 20 embryos per stage. Height of bars indicates the excess amount of microinjected Trim21 mRNA over the endogenous Trim21 mRNA. c The fluorescent protein product of mCherry-Trim21 mRNA is visible already 3 h after microinjection and accumulates in the blastocysts. d Stability of microinjected antibody in the absence of mCherry-Trim21 mRNA, demonstrated via immunofluorescence against primary antibody in zygotes microinjected with anti-GFP antibody. Left, representative picture of immunofluorescence against microinjected anti-GFP; right, measured fluorescence intensity (n = 7 embryos per time point). Size bar, 50 μm. AU, arbitrary units of fluorescence intensity

Fig. 4

a. Experimental design of the selectivity test. All zygotes were injected with mCherry-Trim21 mRNA and OGDB, cultured to the 2-cell stage and injected in one blastomere either with water (b), with anti-GFP antibody (c), with anti-OCT4 antibody (d) or with anti-PDIA3 antibody (e). f. The same as (c), followed up to the blastocyst stage (day E3.5). In the right-hand column, mCherry fluorescence was quantified using Image-J. Size bar, 50 μm. OGDB, Oregon Green dextran beads. Statistical significance tested with Student’s t test. n.s., not significant. AU, arbitrary units of fluorescence intensity

Fig. 5

a. Experimental design for the investigation of developmental consequences of TRIM21-mediated protein depletion in zygotes. b. Representative images of blastocysts developed in KSOM (aa) after the microinjection of mCherry-Trim21 mRNA and OGDB tracer with or without TEAD4 antibody. Effect of anti-TEAD4 was due to TRIM21-mediated depletion, as shown by the lack-of effect of TEAD4-antibody alone. c. Representative images of embryos with mCherry-Trim21 mRNA and OGDB tracer; embryos with mCherry-Trim21 mRNA, OGDB tracer and anti-TEAD4; non-injected oocytes with no fluorescence. Plot shows Cherry fluorescence intensities of zygotes and subsequent stages preloaded with mCherry-Trim21 mRNA and OGDB tracer, and then injected with water (), anti-GFP antibody () or anti-TEAD4 antibody (), compared to the background fluorescence of non-manipulated cells (). N = 3 zygotes or embryos per stage per treatment. Note the secondary right axis used in plot to better discern the background fluorescence values. d. Representative immunofluorescent signals (largest cross section, nucleus fluorescence) of TEAD4 and CDX2 in TRIM21-only and TEAD4-depleted embryos at day E3.5 (n = 7 TEAD4-depleted and n = 8 TRIM21-only embryos for TEAD4 immunofluorescence; n = 11 TEAD-depleted and n = 8 TRIM21-only embryos for CDX2 immunofluorescence). DNA stained with YO-PRO-1. Arrows point at peripheral nuclei that are TEAD4- or CDX2-positive in controls but negative in TEAD4-depleted embryos. Size bar, 50 μm. OGDB, Oregon Green dextran beads. Error bars = standard deviations. Statistical significance tested with Student’s t test. AU, arbitrary units of fluorescence intensity

Fig. 6

a. Morphologies of blastocysts and outgrowths with and without TEAD4 depletion. b. Developmental rates during preimplantation and outgrowth formation. Zygotes injected with mCherry-Trim21 mRNA, n = 212; zygotes injected with mCherry-Trim21 mRNA and anti-GFP antibody, n = 245; zygotes injected with mCherry-Trim21 mRNA and anti-TEAD4 antibody, n = 501. Blastocysts tested for outgrowth formation: n = 12 after mCherry-Trim21 mRNA, n = 8 after mCherry-Trim21 mRNA and anti-GFP antibody, n = 12 after mCherry-Trim21 mRNA and anti-TEAD4 antibody. c. Developmental rates after blastocyst transfer to uterus and representative pictures of uteri. Postimplantation development of TEAD4-depleted embryos was lower than that embryos injected with mCherry-Trim21 mRNA or with mCherry-Trim21 mRNA + anti-GFP antibody. Day E3.5 blastocysts containing mCherry-Trim21 mRNA, n = 30 in three recipients; day E3.5 blastocysts containing mCherry-Trim21 mRNA and anti-TEAD4 antibody, n = 30 in three recipients; day E3.5 blastocysts containing mCherry-Trim21 mRNA and anti-GFP antibody, n = 48 in five recipients. Implantation rate: (fetuses + empty decidua + miscarriages) / transferred embryos. Fetal rate: fetuses / transferred embryos (see Methods). d. Raw Affymetrix signal intensities of selected mRNAs in blastocysts, in logarithmic scale. Two pools of 10 blastocysts each were analyzed for each group (mCherry-Trim21 mRNA, anti-GFP, anti-TEAD4). Size bar, 50 μm. OGDB, Oregon Green dextran beads. Error bars = standard deviations. Statistical significance tested with Student’s t test. n.s., not significant. AU, arbitrary units of Affymetrix hybridization intensity

Fig. 7

a Representative immunofluorescent signals of TEAD4 and CDX2 in TRIM21-only and TEAD4-depleted embryos at day E4.5 (n = 8 TEAD-depleted and n = 6 TRIM21-only embryos for TEAD4 immunofluorescence; n = 10 TEAD-depleted and n = 6 TRIM21-only embryos for CDX2 immunofluorescence). DNA stained with YO-PRO-1. Size bar, 50 μm. b. Developmental rates during preimplantation. Zygotes injected with mCherry-Trim21 mRNA, n = 212; zygotes injected with mCherry-Trim21 mRNA and anti-TEAD4 antibody, n = 501; zygotes injected with mCherry-Trim21 mRNA and anti-OCT4 antibody, n = 464. c. Developmental rates after blastocyst transfer to uterus. Day E3.5 blastocysts containing mCherry-Trim21 mRNA, n = 30 in 3 recipients; E 3.5 blastocysts containing mCherry-Trim21 mRNA and anti-TEAD4 antibody, n = 30 in 3 recipients; E 3.5 blastocysts containing mCherry-Trim21 mRNA and anti-OCT4 antibody, n = 160 in 20 recipients. Implantation rate: (fetuses + empty decidua + miscarriages) / transferred embryos. Fetal rate: fetuses / transferred embryos (see Methods). Statistical significance tested with Student’s t test. n.s., not significant