Global ablation of the mouse Rab11a gene impairs early embryogenesis and matrix metalloproteinase secretion

Abstract

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a(null) blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a(null) embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.

Keywords: Embryo; Matrix Metalloproteinase (MMP); Mouse Genetics; Rab; Rab11; Rab11a; Trafficking; rab11.

FIGURE 1.

Rab11a global knockout mice die at the peri-implantation stage. A, schematics of Rab11a^flox and Rab11a^null alleles. P1, P2, and P3 represent genomic PCR primers to identify specific alleles. B, Southern blots confirmed correctly targeted ES cell clones. C, PCR using the primer set P1 and P2 confirmed the derivation of Rab11a^fl/fl mice. D, summary of genotyping results of 166 neonatal pups and 275 embryos produced from intercrossing Rab11a^+/− mice. 67 live Rab11a^null embryos were identified from in vitro cultured blastocysts. No live null embryos or pups were detected in utero or after birth. E, representative implanted embryos (Emb), all of which were Rab11a-positive wild types. H&E and Rab11a staining of uterus sections is shown. Scale bars = 10 μm. F, fertilized eggs (E0.5) from Rab11a^+/− mating were cultured for 1 day in vitro. Scale bar = 50 μm. G, after 4 days of development in culture, Rab11a^null blastocysts underwent hatching (arrows). Scale bars = 40 μm. H, genotyping of individual hatched blastocysts identified Rab11a^null embryos (asterisks). I, Rab11a^null embryos demonstrated equivalent hatching activity as wild types. J, Rab11a was inducibly deleted from E6.5 or E8.5 embryos via tamoxifen-gavaging pregnant Rab11a^fl/fl females, which were plugged by Rab11a^fl/fl;Rosa26-CreER males. A summary of the genotyping results showed the positive detection of null alleles from live embryos collected at the designated time points.

FIGURE 2.

Rab11a deletion affects recycling and export of selected cargos. A, Western blots showed the deletion of Rab11a from retroviral Cre-infected stable Rab11a^fl/fl MEFs. Rab11a^null MEFs were established after retroviral Cre infection and antibiotic selection (see “Experimental Procedures”). B, transferrin recycling assays using live cell confocal fluorescent microscopy. Increased intracellular transferring retention following endocytosis was detected in Rab11a^null MEFs, suggesting reduced TfR recycling in these cells compared with wild-type cells. **, p < 0.01. Data represent three independent experiments. Scale bars = 10 μm. C, flow cytometric analyses of wild-type and Rab11a^null MEFs that were pulse-treated with transferrin 568 to determine endocytosis and chased with non-labeled transferrin to access recycling activity. Rab11a^null MEFs showed reduced recycling activity compared with wild types. FSC-A, forward scatter area. D, live cell fluorescent analyses of wild-type and Rab11a^null MEFs cotransfected with VSVG-mCherry and plasma membrane-targeted enhanced GFP (mEGFP) that labels the cell surface. Cells were imaged 12 h after transfection. Arrows point to cell surface VSVG. E, immunofluorescent analysis of Rab11a (red) and E-cadherin (green) in wild-type and Rab11a^null blastocysts developed in vitro. Note that E-cadherin localized to intercellular junctions in Rab11a^null blastocysts. Scale bars = 10 μm. DIC, differential interference contrast. F, chemiluminescent analysis of the secretion of a heat-resistant alkaline phosphatase (SEAP). Alkaline phosphatase activities in MEF culture media (secreted, left panel) and MEF lysates (intracellular, right panel) were normalized to Renilla luciferase activities. *, p < 0.05; n.s., not significant.

FIGURE 3.

Rab11a vesicles intersect MMP2 vesicular export. A and B, confocal immunofluorescent staining of wild-type MEFs for endogenous MMP2 (green) showed that the large majority of MMP2 was in the ER (red, A, KDEL), whereas a small portion of MMP2 (green) was localized in the TGN (red, B, TGN38-mCherry). Scale bars = 10 μm. KDEL-EGFP was pseudocolored as red. KDEL, ER retention motif with amino acid sequence as lysine-aspartic acid-glutamic acid-leucine. C, confocal immunofluorescent staining of wild-type MEFs for endogenous MMP2 (green) and Rab11a (red) showed that a small fraction of MMP2⁺ vesicles were positive for Rab11a. Scale bars = 10 μm. D, confocal immunofluorescent staining of wild-type MEFs for endogenous GRP94 (blue), MMP2 (green), and Rab11a (red) showed Rab11a-positive, ER-negative MMP2 vesicles. Scale bar = 10 μm. E, confocal immunofluorescent staining of Rab11a^null MEFs for endogenous MMP2 (green) and TGN (red, TGN38-mCherry) showed a clear aggregation of MMP2 in the TGN. Note that all Rab11a^null MEF cells displayed an identical MMP2 aggregation pattern regardless of whether or not they received a TGN38-mCherry transfection. Untransfected cells are indicated by arrows. The bar graph shows the percentages of TGN-associated MMP2 in wild-type versus Rab11a^null MEFs. ***, p < 0.001. Scale bars = 10 μm. F, confocal immunofluorescent staining of wild-type and Rab11a^null MEFs for endogenous MMP2 (green) and LAMP2 (red). No colocalization was detected in either cell type. Scale bars = 10 μm.

FIGURE 4.

Rab11a ablation impairs MMP2 secretion in MEFs. A, extracellular matrix degradation assays showed reduced gelatin degradation by Rab11a^null MEFs (bottom row), compared with wild-type MEFs (top row). Degradation areas were quantified from three independent experiments. ***, p < 0.001. B, in-gel zymography assays showed reduced MMP2 and MMP9 secretion by Rab11a^null MEFs. Shown are quantified zymography measurements of MMP2 and MMP9 from three independent experiments. ***, p < 0.001. C, Western blot analyses detected the expression of exogenous mCherry-RAB11A in stably infected Rab11a^null MEFs. D, confocal immunofluorescent staining of mCherry-RAB11A rescued Rab11a^null MEFs. Colocalization of some endogenous MMP2 vesicles (green) with mCherry-RAB11A (red) was detected (arrows). Scale bars = 10 μm. E, zymography assays using the mCherry-RAB11A rescued Rab11a^null MEFs showed partially restored MMP2 activities. F, MMP2-specific ELISA showed a decreased MMP2 secretion by Rab11a^null MEFs compared with wild-type MEFs. Monensin also inhibited MMP2 secretion in wild-type MEFs. mCherry-RAB11A-rescued Rab11a^null MEFs showed a partial restoration of MMP2 secretion, which remained sensitive to monensin blockage. Data represent two independent experiments. *, p < 0.05; **, p < 0.01. DMSO, dimethyl sulfoxide.

FIGURE 5.

Rab11a deficiency impairs MMP7 secretion in blastocysts. A, RT-PCR detected Rab11a and Rab11b in embryos at the four-cell, morula, and blastocyst stages. Mouse gut total RNAs served as a control. B, immunofluorescent staining for Cdx2 (green) and Rab11a (red) in blastocysts. Both trophectoderm cells (Cdx2⁺) and the inner cell mass (Cdx2⁻) express Rab11a. The arrow points to intense Rab11a signals at a cleavage furrow within the inner cell mass. Scale bars = 20 μm. C, immunofluorescent staining for MMP7 (green) and Rab11a (red) in wild-type embryos. Scale bars = 10 μm. Some MMP7⁺ vesicles were positive for Rab11a (arrows). D, MMP7 enzymatic kinetics were measured from the medium of cultured Rab11a^+/+, Rab11a^+/−, and Rab11a^−/− hatching blastocysts. Bovine serum served as positive control. Values obtained from blank M16 medium were subtracted from all experimental values. E, accumulative MMP7 enzyme activities were averaged from the entire measuring duration. **, p < 0.01.

FIGURE 6.

Impaired gelatin degradation by Rab11a^null blastocysts. A, confocal immunofluorescent staining on wild-type blastocysts showed colocalization between MMP2⁺ vesicles (green) and Rab11a (red). Scale bars = 10 μm. B and C, individual blastocysts (hatched) were seeded on chamber slides precoated with gelatin 568 (red). Extracellular matrix degradation activities, indicated by the loss of red fluorescent signals, by individual blastocysts were measured 3 days after embryo seeding. Rab11a staining (green) was used to identify null embryos. The leading edges of the embryo C were outlined by phalloidin 350 staining. Scale bars = 20 μm. DIC, differential interference contrast. D, the percentage of degradation areas were quantified and compared between wild-type and Rab11a^null blastocysts. Tc, trophoblastic cells; ICM, inner cell mass.