Essential requirement of mammalian Pumilio family in embryonic development

Abstract

Mouse PUMILIO1 (PUM1) and PUMILIO2 (PUM2) belong to the PUF (Pumilio/FBF) family, a highly conserved RNA binding protein family whose homologues play critical roles in embryonic development and germ line stem cell maintenance in invertebrates. However, their roles in mammalian embryonic development and stem cell maintenance remained largely uncharacterized. Here we report an essential requirement of the Pum gene family in early embryonic development. A loss of both Pum1 and Pum2 genes led to gastrulation failure, resulting in embryo lethality at E8.5. Pum-deficient blastocysts, however, appeared morphologically normal, from which embryonic stem cells (ESCs) could be established. Both mutant ESCs and embryos exhibited reduced growth and increased expression of endoderm markers Gata6 and Lama1, making defects in growth and differentiation the likely causes of gastrulation failure. Furthermore, ESC Gata6 transcripts could be pulled down via PUM1 immunoprecipitation and mutation of conserved PUM-binding element on 3'UTR (untranslated region) of Gata6 enhanced the expression of luciferase reporter, implicating PUM-mediated posttranscriptional regulation of Gata6 expression in stem cell development and cell lineage determination. Hence, like its invertebrate homologues, mouse PUM proteins are conserved posttranscriptional regulators essential for embryonic and stem cell development.

FIGURE 1:

Pum1 and Pum2 double-knockout embryos died at E8.5. (A) Genotypic results of progeny and embryos produced from male Pum1^F/F; Pum2^−/− and female Ddx4-cre; Pum1^+/−; Pum2^+/− intercross. (B) Representative images of dissected E10.5 embryos from four genotypes, except that yolk sac and placenta were not removed from Pum1^−/−Pum2^−/− embryos. Red arrow, placenta; yellow arrow, yolk sac; scale bar represents 500 μm. (C) Representative images of dissected E8.5 embryos of all four genotypes except that Pum1^−/−Pum2^−/− embryos were within the yolk sac. Arrowhead, embryo; red arrow, placenta; yellow arrow, yolk sac; Scale bar represents 500 μm. (D) E3.5 blastocysts from Pum1^+/−Pum2^+/−, Pum1^−/−Pum2^+/−, Pum1^+/−Pum2^−/−, Pum1^−/−Pum2^−/− were recovered and appeared similar in morphology. Scale bar represents 20 μm. (E, F) Genotypes of embryos for Pum1 and Pum2 were determined by PCR (E) and further confirmed by Western blot (F). Asterisk represents nonspecific band. Both PUM1 and PUM2 proteins are completely absent in the double knockout, validating the loss of function nature for both mutations.

FIGURE 2:

Developmental defects in postimplantation embryos lacking both Pum1 and Pum2 genes at E6.5 and E7.5. (A, B) Representative images of intact embryos and HE sections from E6.5 embryos of various genotypes. Scale bars represent 100 μm. (C, D) Representative images of intact embryos and HE-stained sections from E7.5 embryos of various genotypes. E7.5 double-knockout embryos were very small (C), and HE staining shows that embryonic development in tiny embryos was retarded or disrupted with clear signs of degeneration (D). Scale bars represent 200 μm. (E) HE staining of E8.5 Pum1^+/−; Pum2^+/− and Pum1^−/−; Pum2^−/− embryo section. The double-knockout embryo was degenerated and retained few embryonic cells and little extraembryonic structure. Scale bars represent 200 μm. Al, allantois; Am, amnion; PYS, parietal yolk sac; VYS, visceral yolk sac; EPC, ectoplacental cone; H, head folds; Ch, chorion; PE, parietal endoderm; PS, primitive streak; Ex, extraembryonic ectoderm; Epi, epiblast; Pa, proamniotic cavity; VE, visceral endoderm.

FIGURE 3:

Effects of cell proliferation and apoptosis after depletion of PUMs in E6.5 embryos and ESCs. (A, B) Cell proliferation in wild-type (WT) and Pum1^−/−; Pum2^–/− embryo by E6.5 was detected by BrdU labeling (A) and p-H3 staining (B). Scale bars represent 100 μm in C and D. (C) Apoptotic cells were detected by TUNEL staining in E6.5 embryo. The data represented percentages of positive cells in each embryo from at least three comparable sections of representative embryos structure (n = 3). Scale bars represent 200 μm. (D) Cell growth was examined by CCK8 assay in wild-type and Pum1^−/−; Pum2^−/− ESCs. (E) Cell apoptosis were assessed by Annexin V/PI staining. (F) Cell cycle of mutant cells was analyzed using flow cytometry after propidium iodide staining. (G, H) Colony formation assay of wild-type and Pum1^−/−; Pum2^−/− ESCs when cultured in feeder-coated plates following alkaline phosphatase staining. Scale bars represent 50 μm. (I) Potential Pum targets in cell-cycle regulators were examined using Western blot on single- or double-knockout ESCs. Results are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4:

Developmental defects in postimplantation embryos lacking both Pum1 and Pum2 genes at E6.5 and E7.5. (A) Representative images of wild-type and Pum1^−/−; Pum2^−/− ESC colonies and AP staining. Spontaneous differentiation occurred in double-knockout ESCs when cultured under feeder-free condition. Scale bar represents 20 μm. (B) Relative expression of pluripotency and three germ layers marker genes was compared by qRT-PCR between wild-type and double-knockout ESCs, and results were normalized to wild type and are shown as mean ± SD. Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. (C) Western blot result shows that GATA6 protein level was increased in double-knockout (DKO) ESCs compared with wild type (WT). (D) Relative expression of ectoderm and endoderm markers were detected by qRT-PCR in double-heterozygote and DKO whole E6.5 embryos, and results were normalized to double heterozygote and are shown as mean ± SD. (E, F) OCT4 (E) and GATA6 (F) levels in E6.5 embryos were detected by immunohistochemistry staining. Scale bars represent 100 μm in E and 50 μm in F. (G) GATA6 protein expression was detected by immunohistochemistry staining in E7.5 embryos. Scale bars represent 50 μm. Yellow arrowhead represents parietal endoderm, red arrow represents visceral endoderm, blue arrow represents precardiac mesoderm, black arrow represents amnion, * and # represent amniotic cavity and exocoelomic cavity, respectively. (H) qRT-PCR results demonstrate the association of Gata6 and Lama1 mRNA with PUM1 and PUM2 proteins for ESCs in comparison to IgG precipitates. Results are representing as mean ± SD. Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. (I) Bar graph results from dual-luciferase assay on HEK-293T cells expressing the reporter constructs containing either wild-type or PBE mutated Gata6 3′UTR. The cells were also cotransfected with mouse Pum1, Pum2 and empty vector. Results from A to I are presented as mean ± SD. Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001.