Arf tumor suppressor and miR-205 regulate cell adhesion and formation of extraembryonic endoderm from pluripotent stem cells

Significance

The Arf tumor suppressor gene is not expressed in most normal tissues but when activated by oncogenic stress signals engages a p53-dependent transcriptional program that prevents tumor formation. Surprisingly, expression of the p19^Arf protein in mouse embryoid bodies is required for the timely formation of extraembryonic endoderm (ExEn). Inactivation of Arf down-regulates a single microRNA, miR-205, which can “rescue” ExEn formation in Arf-null embryonic or induced pluripotent stem cells. During ExEn formation, miR-205 regulates a suite of genes that govern cell migration and adhesion, suggesting a conceptual basis for linking the roles of Arf in ExEn differentiation and tumor metastasis.

Abstract

Induction of the Arf tumor suppressor (encoded by the alternate reading frame of the Cdkn2a locus) following oncogene activation engages a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although the p19^Arf protein is not detected in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm (ExEn). Expression of the mouse p19^Arf protein marks late stages of ExEn differentiation in cultured embryoid bodies (EBs) derived from either embryonic stem cells or induced pluripotent stem cells. Arf inactivation delays differentiation of the ExEn lineage within EBs, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of ExEn cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure the development, but not maintenance, of the ExEn lineage. Remarkably, a significant temporal delay in ExEn differentiation detected during the maturation of Arf-null EBs is rescued by enforced expression of mouse microRNA-205 (miR-205), a microRNA up-regulated by p19^Arf and p53 that controls ExEn cell migration and adhesion. The noncanonical and canonical roles of Arf in ExEn development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell attachment and migration.

The Ink4–Arf (Cdkn2a,b) locus, which is only 50 kb in length, encodes three intimately linked tumor suppressor genes. The Ink4a and Ink4b genes encode polypeptides (p16^Ink4a and p15^Ink4b) that inhibit cyclin D-dependent kinases to maintain the retinoblastoma protein (Rb) in its active inhibitory state, thereby limiting cell proliferation. In contrast, the Arf protein (p19^Arf in the mouse, p14^ARF in humans) inhibits the Mdm2 E3 ubiquitin ligase to activate and stabilize p53, a transcription factor that coordinates a complex gene expression program that potently guards against tumor formation (1, 2). The p19^Arf and p16^Ink4a proteins are encoded in part by unique first exons, whose products are spliced to a second shared exon that is translated in alternative reading frames, yielding proteins that bear no shared amino acid sequences and that are functionally distinct. The Ink4a–Arf locus is generally not expressed under normal physiological circumstances but is induced by aberrant mitogenic signals that result from oncogene activation. By engaging Rb- and p53-dependent transcriptional programs, the Ink4–Arf proteins counter tumor cell progression by eliciting cell cycle arrest, apoptosis, or cellular senescence. Deletion of this small gene cluster incapacitates the functional Rb/p53 tumor-suppressive network and is one of the most common events observed in human cancers.

The Ink4a–Arf locus is silenced in stem cells—whether of embryonic, fetal, or adult somatic tissue origin—thereby facilitating their capacity for continuous cellular self-renewal. In contrast, the locus is epigenetically remodeled in more differentiated cell types to allow its engagement in response to oncogenic stress signals. Despite the risk of its deletion in cancer, the evolutionary conservation of the Ink4–Arf locus in mammals may provide a mechanism for limiting the numbers of stem and progenitor cells (2). In agreement with the idea that epigenetic silencing of the locus is necessary to maintain cellular self-renewal, reprogramming of somatic cells to yield induced pluripotent stem (iPS) cells is accompanied by Ink4–Arf repression (see below) and facilitated by Ink4–Arf deletion (3).

Paradoxically, the p19^Arf protein is physiologically expressed in a few disparate tissues during mouse development, including perivascular cells within the hyaloid vasculature of the eye (4–6), mitotically dividing spermatogonia within seminiferous tubules (6, 7), and the fetal yolk sac (8). Inactivation of Arf results in blindness and reduced sperm production, but effects of Arf deletion on yolk sac development have not been investigated. Whether these diverse physiological roles of Arf can be explained through a common mechanism and whether they reflect the canonical role of Arf as a potent tumor suppressor remain a mystery. We demonstrate that a signaling pathway involving Ras/Erk, p19^Arf, p53, and microRNA 205 (miR-205) regulates a cell motility and adhesion program that facilitates formation of extraembryonic endoderm (ExEn) cells from pluripotent embryonic stem (ES) or iPS cell progenitors.

Results

Expression of Arf in ExEn.

Blastocysts harvested from mouse embryos at embryonic day (E) 4.5 exhibit pluripotent Oct4-positive cells in the inner cell mass surrounded by Gata4-marked primitive endoderm (PrE) cells in a generally mutually exclusive pattern (Fig. 1A Upper). By using a particularly sensitive and specific monoclonal antibody (9), p19^Arf expression was not detected in the early PrE lineage at E4.5 (Fig. 1A Lower), whereas embryos recovered at E7.5 revealed p19^Arf expression in ExEn tissues surrounding Oct4-positive cells (Fig. 1B). Inter-crossing Arf^+/− mice yields Arf^−/− offspring at the expected Mendelian ratio, and Arf-null females produce equivalent numbers of blastocysts compared with those of wild-type (WT) mice and generate normal-sized litters (10). Examination of 10 Arf^−/− and 12 Arf^+/+ embryos recovered at E7.5 revealed as much morphological variation within individual cohorts as between them.

Fig. 1.

p19^Arf protein expression in ExEn. (A) WT E4.5 embryos were fixed and stained with the indicated antibodies and visualized for immunofluorescence with a confocal microscope. Pluripotent cells (red) in the inner cell mass and differentiated primitive endoderm cells (green) were visualized with antibodies to Oct4 and Gata4, respectively; p19^Arf protein was not detected. DAPI was used to visualize cell nuclei. (Scale bars, 25 µm.) (B) Section of WT embryo recovered from the decidua of the uterus at E7.5 stained as above. p19^Arf-positive ExEn cells (green, asterisks) surround the Oct4-positive epiblast (red). (Scale bars, 200 µm, Upper; 100 µm, Lower.) (C) Cryosectioned WT EBs were stained and visualized as above. Expression of p19^Arf (green) and Bmi1 (red) colocalize (yellow) in a single ExEn cell layer at the EB periphery. (Scale bar, 50 µm.) (D) Lineage tracing. Knock-in mice with Cre under the regulatory control of the cellular Arf promoter were crossed to an indicator strain that expresses LacZ in response to Cre-mediated excision of a “lox–stop–lox” cassette. ES cells obtained from these blastocysts were induced to differentiate to EBs. β-galactosidase was detected at the periphery of EBs expressing Arf–Cre (Lower; magnification: 50×).

Given difficulties in defining any overt developmental deficiency in Arf-null embryos, we attempted to model the earliest stages of ExEn formation in embryoid bodies (EBs) derived from cultured ES cells. When undifferentiated ES cells are suspended in hanging drops and transferred to ultralow attachment dishes in medium lacking leukemia-inhibitory factor (LIF), the well-organized EBs that develop are rimmed by a single ExEn layer, which surrounds the pluripotent epiblast and other cells within the inner mass that are differentiating to form the three other germ cell lineages. Cryosections of EBs that arose after 4 d of culture clearly revealed p19^Arf expression restricted to the outer ExEn layer, which was marked by expression of the Polycomb group protein Bmi1 (11) (Fig. 1C). Bmi1 is essential for silencing of the entire Ink4–Arf locus in adult hematopoietic and neural stem cells and is required for formation of the early ExEn lineage (12), where, in contrast, it does not interfere with p19^Arf expression (Fig. 1C).

At early times of embryonic development (E4.5) when p19^Arf is not detected (Fig. 1A), some cells within the inner cell mass express the ExEn markers Gata6, Dab2, and Lrp2 and exhibit diminished expression of the pluripotency markers Nanog and Oct4 (13). PrE cells then migrate to the EB periphery to surround pluripotent cells. Because we never detected p19^Arf in the inner cell mass, we assumed that the emergence of p19^Arf-positive cells only occurs after the migration of PrE cells to form the outer EB layer. We crossed females of a knock-in strain that expresses Cre recombinase under the control of the endogenous cellular Arf promoter (6) to reporter male mice that conditionally express Cre-dependent LacZ from the Rosa-26 locus. Although there was no expression of LacZ in pluripotent ES cells due to the silenced Arf promoter, the Arf–Cre allele was activated in EBs as expected, and β-galactosidase expression was largely limited to the EB periphery (Fig. 1D).

Retardation of ExEn Differentiation in Cultured Arf-Null ES Cells.

Unlike WT EBs that spontaneously develop in cultures deprived of LIF for 4 d, Arf-null EBs expressed the pluripotency marker Oct4 in lieu of Dab2 at their periphery (Fig. 2 A and B). Independently derived Arf-null ES cell clones that underwent differentiation after LIF withdrawal exhibited highly robust Nanog protein expression, but reduced levels of the ExEn markers Dab2 and Lrp2, compared with those in WT EBs (Fig. 2C), consistent with the failure to detect ExEn cells on the surface of Arf-null EBs. Given that an inability of Arf-null progenitors to form ExEn cells might be compensated during early embryonic development in the mouse, the duration of EB cell culture was extended to determine whether ExEn cell development was temporally delayed. EB cells derived from Arf-null ES clones recovered Dab2 expression after 10 d of suspension culture and produced Dab2 at levels equivalent to those detected in differentiating WT cells cultured for only 4 d (Fig. 2D). By using Arf-null ES cells in which a cDNA encoding green fluorescent protein (GFP) was substituted for Arf exon-1β sequences under the control of the cellular Arf promoter (14), Arf^Gfp/Gfp ES clones produced GFP- and Gata4-positive ExEn cells after 10 d of prolonged EB culture (Fig. 2E). Hence, the functionally null Arf^Gfp alleles were induced as Gata4-positive ExEn cells eventually emerged.

Fig. 2.

Delayed formation of ExEn cells in Arf-null EBs. (A and B) WT (A) and Arf-null (B) EBs were processed for immunofluorescence. Dab2 expression was greatly reduced when Arf was inactivated. DAPI was used to stain cell nuclei. The intense halo of blue cells in B Upper Left is an artifact dependent upon the confocal plane; these same cells expressed Oct4 (Lower Left). (Scale bars, 50 µm.) (C) Immunoblotting of lysates from WT and three pooled Arf-null ES cell-derived EBs generated after 4 d of suspension culture exhibited quantitative differences in the expression of the indicated proteins. (D) Immunoblotting with the indicated antibodies showed that Arf-null EBs express very low levels of the ExEn marker Dab2 after 4 d of culture, but EBs cultured for 10 d recovered Dab2 expression. (E) EBs derived from functionally Arf-null Arf^Gfp/Gfp ES cells after 10 d of culture express Gata4-positive ExEn cells on their surface. (Magnification: 50×.) Activation of the cellular Arf promoter in “GFP knock-in” cells that do not express the p19^Arf protein was accompanied by GFP signals, which colocalized with Gata4-marked ExEn cells at the periphery.

Arf-null, but Not Ink4a-Deficient, iPS Cells Exhibit Defective ExEn Differentiation.

The creation of iPS cells by introduction of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, into mouse embryo fibroblasts (MEFs) is facilitated by inactivation of p53 (15–18). Because elevated Myc signaling engages Arf to induce p53 (19), Arf-null MEFs might be as susceptible to four-factor iPS reprogramming as their p53-null counterparts. We derived 2 WT, 3 Arf^+/−, and 14 Arf-null iPS cell lines, the latter originating from Arf^−/− or Arf^Gfp/Gfp strains (10, 14). All iPS cell lines selected for subsequent studies maintained normal karyotypes and expressed the pluripotency markers Oct4, Sox2, Nanog, and CD15/SSEA1; all could generate cell types representing three embryonic germ layers; and all formed teratomas in immunodeficient mice (Fig. S1 A–D). Like p53-null MEFs, strains lacking Arf generated iPS cells at ∼50-fold greater efficiency than cells containing a single Arf allele (Fig. S1E).

When allowed to differentiate for 4 d in the absence of LIF, Arf^+/− iPS cells generated EBs that expressed p19^Arf and Gata4 at the periphery (Fig. S1 F and G). However, Arf-null iPS clones mimicked their ES cell counterparts and expressed much lower levels of the ExEn markers Dab2 and Lrp2 than Arf^+/− iPS cells (Fig. S1H) and were defective in producing fully differentiated ExEn cells in EBs (Fig. S1 I and J). Two iPS cell lines derived from p16^Ink4a-null MEFs generated EBs that expressed the ExEn marker proteins Gata4 and Lrp2 at levels equivalent to those of their WT counterparts (Fig. S1K) and exhibited comparatively higher levels of Dab2 and Gata4 mRNAs (Fig. S1L). Thus, although the entire mouse Ink4a–Arf locus is silenced during the reprogramming of iPS cells, and although p19^Arf and p16^Ink4a are both reexpressed as iPS cells differentiate, only Arf loss of function delays ExEn differentiation. Given that Arf-null ES and iPS cells exhibited identical phenotypes, both were exploited subsequently.

Ras/Erk Signaling and p53 Promote ExEn Differentiation.

Generation of ExEn cells in culture depends upon Ras signaling through the Raf–Mek–Erk pathway and can be accelerated by Ras overexpression and blocked by pharmacologic Mek/Erk inhibitors (20). Arf is induced by Ras (14, 21), suggesting that each may be required for ExEn specification. A constitutively active H-Ras effector mutant (G12V/T35S) that specifically targets signaling through the Ras/Erk pathway potentiated ExEn differentiation in the absence of LIF, whereas a dominant-negative form of H-Ras (S17N) abrogated ExEn differentiation in WT iPS cells. WT iPS cells expressing H-Ras–G12V/T35S generated morphologically altered cells typical of the ExEn lineage (Fig. 3A). Equivalent levels of ectopic H-Ras gene expression were induced in cultured WT and Arf-null cells, but only those that retained Arf expressed detectable levels of Dab2 in EBs after 4 d (Fig. 3 B and C). Although complete ExEn differentiation accompanied by Dab2 expression was blocked in an Arf-dependent manner, H-Ras still induced phospho-Erk at the peripheries of EBs (Fig. 3D). Thus, Ras/Erk signaling requires Arf gene function to enforce complete ExEn differentiation.

Fig. 3.

Ras/Erk signaling promotes Arf-dependent ExEn differentiation. (A) WT and Arf-null iPS cells (indicated at left) were transduced with vectors expressing the indicated Ras mutants or with a control (Ctl) vector, selected for puromycin resistance in medium containing LIF, and induced to differentiate for 4 d in the absence of LIF. A constitutively active Ras effector mutant (G12V/T35S) that signals to Erk promoted ExEn cell formation from WT, but not Arf-null, iPS progenitors, an effect blocked by a dominant-negative mutant (HRas–S17N). Phase contrast images are shown. (Scale bars, 200 µM.) (B and C) Quantitative PCR (qPCR) analysis (B) and immunoblotting (C) confirmed Dab2 mRNA and protein up-regulation in WT cells infected with the G12V/T35S Ras mutant (abbreviated as T35S). (D) Activated phospho-Erk was detected at the periphery of both Arf-null and WT day-4 EBs generated from the different iPS progenitors. (Scale bars, 200 µm.)

Ectopic expression of p19^Arf in ES cells is not tolerated, and within 24 h, these cells undergo cell cycle arrest and fail to further differentiate. However, the N-terminal 62 amino acids of p19^Arf encoded by exon-1β retain some ability to induce p53 and to retard cell proliferation, albeit with reduced activity compared with that of the full-length protein (22–24). We fused Arf exon-1β sequences to a tamoxifen-responsive estrogen-receptor moiety (ER^TAM) and separated these cassettes by a 16-amino acid linker peptide (Arf residues 63–78) containing the epitope recognized by the 5C3-1 monoclonal antibody to p19^Arf (9) (SI Materials and Methods). Following its transduction into WT MEFs, the ∼50-kDa fusion protein was detected with antibodies to either p19^Arf or ER (Fig. 4A). Arf-null NIH 3T3 cells, MEFs, and iPS cells expressing the uninduced fusion protein continued to proliferate. After introduction of the minigene into Arf-null iPS cells, whose RNA expression was detected by using PCR primers directed to the ER^TAM cassette (Fig. 4B Upper Left), tamoxifen treatment induced modestly but significantly increased expression of Dab2 and Gata4 mRNAs and decreased expression of Nanog (Fig. 4B Upper Right, Lower Left, and Lower Right). Hence, induction of Arf–exon1β–ER^TAM was capable of driving elements of the ExEn differentiation program.

Fig. 4.

Arf and p53–ER promote expression of ExEn marker proteins. (A) Lysates of WT MEFs (left lane) or from cells transduced with a vector encoding Arf exon-1β sequences (center lane) or with a naked control vector (right lane) were immunoblotted with antibodies directed to p19^Arf (Upper) or to the ER cassette (Lower). The endogenous p19^Arf protein was detected in all three samples, whereas the 50-kDa minigene-coded fusion protein was detected only in the center lane. The asterisk indicates a staining artifact. (B) Arf-null iPS cells transduced with the vector encoding the Arf–exon-1β minigene were subjected to qPCR analysis. Primers that amplified the ER moiety confirmed generally equivalent levels of vector-coded RNA expression in cells treated with different concentrations of tamoxifen (TAM; see legends). Dab2 and Gata4 mRNAs in transduced Arf-null iPS cells were induced, and Nanog expression was decreased in response to minigene expression and tamoxifen treatment. Error bars, mean ± SEM (n = 3 experiments). (C) Phase contrast micrographs reveal that EBs derived from p53-null iPS cells (Right) fail to express a halo of ExEn cells visualized in WT EBs (Left). (D) Like day-4 EBs derived from Arf-null iPS cells, p53-null iPS cells exhibited reduced Dab2 protein levels compared with WT iPS controls. (E) Although p19^Arf protein expression was detected at the periphery of day-4 EBs derived from p53-null iPS cells, neither Gata4 nor Dab2 were detected. Instead, Oct4-positive cells were observed at the periphery. (Scale bars, 200 µm.) (F) Arf-null iPS cells were transduced with a retroviral vector expressing the HRas–G12V/T35 mutant. Cells engineered to coexpress p53–ER^TAM (Center and Right) were treated with tamoxifen (Right) or not (Center), and Dab2 expression was quantified by flow cytometric analysis. The percentages of cells that exhibited increased Dab2 expression are noted.

We determined that EBs derived from p53-null iPS cells failed to exhibit a distinct halo of ExEn cells on their surface (Fig. 4C), and Dab2 levels in pooled day-4 EBs were significantly reduced, similar to that seen in the Arf-null setting (Fig. 4D). Despite the absence of p53, the p19^Arf protein was expressed on the EB periphery, but Dab2 and Gata4 expression could not be detected (Fig. 4E). Oct4-positive cells localized on the surface instead (Fig. 4E). Thus, Arf enforces p53-dependent differentiation of ExEn cells.

EBs derived from Arf-null iPS cells transduced with H-Ras–G12V/T35S expressed increased phospho-Erk activity at their periphery (Fig. 3D), but like p53-null EBs, these cells did not form Dab2-positive ExEn cells in the absence of Arf (Fig. 3 B and C). We expressed p53–ER^TAM in Arf-null iPS cells either alone or in those engineered to stably express H-Ras–G12V/T35S. Conditional induction of Dab2 was achieved following tamoxifen exposure during 4 d of EB development only when Ras was overexpressed (Fig. 4F), indicating that the Ras/Erk pathway complements p19^Arf–p53 signaling to enforce ExEn differentiation.

WT ExEn Cells Form Chimeric EBs When Admixed with Arf-Null ES Cells.

ExEn cell lines, also established from mouse blastocysts (25, 26), exhibit a morphology distinct from cultured ES cells (Fig. 5A) and express a different repertoire of ExEn proteins and mRNAs (Fig. 5 B and C). Because we did not succeed in generating such cultures from Arf-null ES cells, we established ExEn cells from blastocysts containing “floxed” Arf alleles (Arf^FL/FL) (6) and deleted Arf exon-1β with a retrovirus encoding Cre recombinase. These Arf-null ExEn cells continued to proliferate continuously, and expression of ExEn markers was maintained, so Arf is not required for ExEn cell line viability and maintenance.

Fig. 5.

Self-sorting of ExEn cells in chimeric EBs. (A) Morphology of ExEn cells visualized by phase contrast microscopy. (Magnification: 100×.) (B and C) Immunoblotting of proteins (B) and PCR analysis of transcripts (C) in WT ES or ExEn cell lines. (D) WT ExEn cells expressing p19^Arf, Gata4, and Dab2 were transduced with a vector expressing GFP, mixed with an equal number of Arf-null ES cells, and suspended in the absence of LIF to form chimeric EBs. Two days after suspension, EBs were harvested, cryosectioned, and stained with antibodies to the indicated proteins. Marked ExEn cells spontaneously sorted to the periphery of the chimeric EBs and completely segregated from DAPI-stained ES cells in the inner mass. (Scale bars, 100 µm.)

We infected cultured WT ExEn cells with a retrovirus encoding GFP and admixed them in a 1:1 ratio with Arf-null ES cells to form chimeric EBs (26). Two days later, ExEn cells had localized to the EB outer layer, spontaneously segregating from DAPI-stained ES cells that comprised the inner pluripotent compartment (Fig. 5D). Because Arf-null ES cells cannot contribute to ExEn formation under these conditions, the exclusion of GFP-marked ExEn cells from the inner pluripotent cells and their localization and retention at the periphery is an inherent property of differentiated ExEn cells themselves.

miR-205 Can Rescue ExEn Differentiation in Arf-Null Cells.

Arf is expressed in mitotically dividing spermatogonia, but not in primary spermatocyctes derived from them or in other cells within the testis (6, 7). In unrelated studies, we determined that the levels of a single microRNA, miR-205, were selectively decreased by >15-fold in the Arf-null testes of postnatal day-18 mice, a time when p19^Arf levels in spermatogonia are maximal and are synchronously expressed throughout the developing seminiferous tubules. miR-205 is up-regulated by p53 (27), and its promoter is directly bound and induced by other p53-related family members, p63 and p73 (28, 29).

Motivated to test whether Arf–p53 signaling and miR-205 expression are directly correlated in ES cells, we obtained an ES cell line (designated Arf154) in which a miR-30–based shRNA that targets Arf mRNA (but not sequences shared with Ink4a) is induced by doxycycline (Dox) treatment (30). Arf154 ES cells were suspended for EB formation and treated with Dox for 4 d. Expression of miR-205 RNA was decreased following Dox treatment, which was without effect in WT ES cells (Fig. 6A Left). Conversely, introduction of the Arf–exon-1β–ER^TAM fusion protein into Arf-null iPS cells triggered a conditional increase in miR-205 levels following tamoxifen treatment (Fig. 6A Right).

Fig. 6.

miR-205 is regulated by Arf and enhances ExEn formation from WT ES progenitors. (A Left) Arf154 ES cells expressing a Dox-inducible Arf shRNA were cultured to form EBs in the absence of LIF and presence of Dox, resulting in a small decrease (P < 0.01) in miR-205 mRNA expression, quantified by PCR. WT ES cells were unaffected by Dox. Error bars, mean ± SEM (n = 3 experiments). (Right) Arf-null ES cells transduced with Arf–exon-1β–ER^TAM were induced to form EBs and simultaneously treated with tamoxifen (TAM) for 4 d. (B) The V1 vector transcribes miR-205 (hairpin) embedded in a miR-30 backbone, whereas the V2 vector expresses the complete pre-miRNA sequence (gray rectangle). Vectors included a PGK promoter-driven cassette and encoded neomycin resistance (Neo^r); GFP was translated from an internal ribosome entry site (IRES). Long terminal repeat (LTR) sequences required for viral integration and viral promoter-driven mRNA expression and a psi-2 (φ) virion packaging sequence are indicated. (C) WT ES cells infected with a control vector (Ctl GFP; Left) or with miR-205 vectors (Center and Right) were maintained in LIF to retard differentiation. Nonetheless, enforced miR-205 expression generated cells with altered morphology reminiscent of ExEn cells. (Magnification: 100×.) (D) Cells shown in C down-regulated the pluripotency marker Nanog and up-regulated the ExEn marker Dab2. (E) A more comprehensive quantitative RT-PCR analysis of infected WT iPS cells (left two bars) revealed up-regulation of other ExEn markers, Gata4 and Sox7, but insignificant alterations in the expression of markers of other germ-cell layers (mesoderm T, ectoderm Fgf5, and trophectoderm Cdx2). As expected, p19^Arf, p53, and the p53-responsive gene p21^Cip1 were induced as cells assumed the ExEn fate. By contrast, enforced expression of miR-205 in established ExEn cell lines (right two bars) did not affect the mRNA expression of the above genes.

WT ES cells were transduced with miR-205 vectors coexpressing GFP (Fig. 6B) or with a control vector expressing GFP alone, and GFP-positive cells were sorted 2 d later and replated in the presence of LIF to preserve their undifferentiated state. Cells expressing GFP alone maintained their characteristic ES morphology, but those expressing threefold more miR-205 underwent partial differentiation in 2 d, even in the presence of LIF, to yield ExEn-like progeny (Fig. 6C) that exhibited increased Dab2 and decreased Nanog expression (Fig. 6D). Four days after plating these cells in the absence of LIF, expression of Gata4 and Sox7 mRNAs, as well as p19^Arf, p53, and p21^Cip1, were up-regulated as expected, but without effects on the relative expression of markers of ES-derived ectoderm (Fgf5), mesoderm (T/Brachyury), or trophectoderm (Cdx2) (Fig. 6E, left two columns). In contrast, enforced miR-205 expression in established, fully differentiated WT ExEn cell lines did not affect the expression of these RNAs (Fig. 6E, right two columns). Thus, overexpression of miR-205 could up-regulate the Arf–p53–p21 signaling axis and preferentially drive pluripotent WT ES cells to assume an ExEn fate but did not affect the same target genes once ExEn differentiation was established. Given that p19^Arf can induce miR-205 (Fig. 6A) and vice versa (Fig. 6E), a feed-forward mechanism appears to drive ExEn formation, after which miR-205 is without effect.

We used the same strategy to introduce miR-205 into Arf-null ES cells and promoted their spontaneous differentiation by withdrawing LIF. Cells exhibiting a significant (P < 0.0032) 3.2-fold increase in miR-205 expression differentiated within 4 d to form typical ExEn cells (Fig. 7A). Dab2-positive cells were now identified at the EB periphery (Fig. 7B), and other ExEn markers were further up-regulated (Fig. 7C). Strikingly, then, enforced miR-205 expression not only potentiated ExEn differentiation in WT ES cells (Fig. 6) but induced fully differentiated ExEn cells on the EB periphery even when Arf function was completely disabled (Fig. 7).

Fig. 7.

miR-205 induces ExEn formation from Arf-null ES progenitors. (A) Arf-null ES cells infected with a naked control (Ctl) GFP vector or one encoding miR-205 from a miR-30 backbone together with GFP were sorted 2 d after infection, placed in culture, and visualized 2 d later by phase contrast microscopy at magnification of 50× (Left) or 100× (Right). Cells with the characteristic morphology of ExEn cells appeared in response to miR-205. (B) EBs derived from WT or Arf-null ES cells and infected with control or miR-205–encoding vectors (indicated at the top) were stained for Dab2 protein expression. WT cells (Left) and Arf-null EBs transduced with miR-205 (Right) expressed Dab2 at their periphery, whereas Arf-null cells, whether uninfected or transduced with a control vector, did not (second and third from left). (Scale bars, 50 µm.) (C) Quantitative RT-PCR was used to quantify mRNA expression of five ExEn markers (indicated at the top) in vector-transduced Arf-null ES cells grown in the presence of LIF (day 0) and in EBs derived from them (day 4). Cells were transduced with a control virus (Ctl) or a vector encoding miR-205 (205) as indicated in the legend. Error bars, mean ± SEM (n = 3 experiments).

ExEn Gene Expression Program Regulated by miR-205.

Two independently derived Arf-null ES cell lines infected in duplicate with control or miR-205–encoding vectors were sorted 2 d later for GFP expression and cultured for an additional 4 d in the absence of LIF to form EBs. Gene expression was profiled by using Affymetrix Mouse Gene Chips (Version 1.0), which include 33,283 probe sets. We identified 632 genes that were differentially up- or down-regulated at least twofold in response to enforced miR-205 expression vs. their levels in cells infected with the control vector (Fig. 8A). Of 144 probe sets down-regulated in ES cells 2 d after introduction of miR-205 but before LIF withdrawal (Fig. 8A, lane 2 vs. 1), none corresponded to highly predicted miR-205 targets (SI Materials and Methods). ExEn markers (Dab2, Gata4, Gata6, Sox17, and Afp) were further induced in response to miR-205, whereas expression of pluripotency markers (such as Klf4 and Tbx3) was reduced (Fig. 8 B and C). Gene Ontology (Database for Annotation, Visualization, and Integrated Discovery) analysis indicated that the most significantly up-regulated genes encode glycoproteins (2.4E-17; 4.2% of the total genes), affect in utero development (4.29E-05; 0.74% of total genes), and govern cell motility and migration (4.64–8.71E-04; 0.74% of total genes) and cell adhesion (2.37E-04; 0.82% of total genes) (Fig. 8D).

Fig. 8.

Gene expression profiling of Arf-null ES cells and EBs transduced with miR-205. Arf-null ES cells infected with a retroviral vector expressing GFP alone (Ctl) or with a vector coexpressing miR-205 and GFP were sorted for GFP expression, plated in the presence of LIF (designated ES), or induced for 4 d in the absence of LIF to form EBs (designated EB). Isolated RNAs labeled with fluorochromes were used to probe Affymetrix Mouse Gene Chips (Version 1.0). The data were normalized (Z score transformed) across samples so that the heat intensity scales shown in A, C, and D are in units of SD. (A) Indicated in the heat map are 632 probe sets that were significantly up-regulated (red) or down-regulated (green) with twofold or more changes. Control ES cells, ES cells expressing miR-205, control EBs, and EBs expressing miR-205 are indicated and are designated 1–4, respectively, at the top of the heat map. Although these experiments were not designed to identify genes directly targeted by miR-205, we selected the most probable miR-205 mouse target genes in each of three public databases (miRDB.org, TargetScan.org, and microRNA.org) and chose 26 genes for further analysis that were concordantly ranked in the “top 60.” None of the 144 probe sets corresponding to selected candidates exhibited >1.5-fold down-regulation in miR-205–expressing ES cells taken for comparative microarray analysis before LIF withdrawal. Six genes [Cdh11 (see also Fig. S2A), Cldn11, Plcb1, Ralyl, Zfp558, and Zfp758] were not expressed at significant levels in control GFP+ ES cells. The remaining candidates included Acsl1, Akna, BC030336, Cadm1, Ccny, Chn1, Ctps2, Dmxl2, Ezr, Lrch3, Lrp1, Mgrn1, Mllt4, Nacc2, Nfat5, Rfk, Sbf2, Sdha, Sorbs1, and Spata13. (B) qPCR analysis confirmed up-regulation of two ExEn markers in EBs transduced with miR-205. Error bars, mean ± SEM (n = 3 experiments). (C) Heat map of a subset of genes associated with pluripotency and differentiation toward germ layers other than ExEn (terms indicated at the right). Of note, Klf4, Tbx3, Lefty2, Tdgf1, Ncam1, and Fgf5 revealed less than twofold changes. (D) Heat map of genes involved in cell adhesion (i) and cell motility and migration (ii). Designation of lanes in C and D is identical to that in A. The data have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE42210.

The cell-autonomous ability of WT ExEn cells to properly form chimeric EBs when cocultured with Arf-null ES cells (Fig. 5) mimics the classical sorting behavior of cells that express variable levels of cadherins (31, 32). The E-cadherin (Cdh1) repressor Snail (33) was expressed at higher levels in ExEn cells compared with their ES cell progenitors with concomitant Cdh1 down-regulation (Fig. 5). Affymetrix chip gene profiling (Fig. 8D) and PCR analysis (Fig. S2A) confirmed the up-regulation of Snail2 in EBs responding to miR-205, but down-regulation of Cdh1 was not observed when day-4 EBs expressing a control vector and miR-205 were directly compared by microarray analysis. However, introduction of miR-205 into Arf-null ES cells resulted in a very rapid reduction in Cdh1 levels, which occurred even before LIF was withdrawn (Fig. S2A). Although the few reported direct targets of miR-205 include Zeb proteins that also act as Cdh1 corepressors (34), Zeb2 levels were elevated in response to transduction of the miR-205 vs. control vector in Arf-null EBs (Fig. 8D and Fig. S2A). We used imaging techniques to look at the topological distribution of E-cadherin in EBs formed from Arf-null ES cells that had been infected either with the control vector or that encoding miR-205 (Fig. 9). Strikingly, E-cadherin was no longer detected in relative abundance at the periphery of EBs overexpressing miR-205, and this result was accompanied by induction of vimentin (Fig. 8D) and its preferential distribution to the ExEn layer at the EB surface (Fig. 9).

Fig. 9.

miR-205 drives vimentin synthesis and relocalization of E-cadherin within Arf-null EBs. Immunofluorescence staining of Arf-null day-4 EBs derived from cells infected with a control vector (Ctl; Left) and a vector expressing miR-205 (205; Right) is shown. Confocal images of EBs stained with antibodies to E-cadherin or vimentin and with DAPI to visualize nuclei are merged. (Scale bars, 100 µm.)

Significantly increased expression of candidate genes that regulate cell migration and adhesion, including Cdh2 (N-Cadherin), Cdh5, Cdh6, and Cdh11, was observed (Fig. 8D and Fig. S2A). Many other genes reported to be essential for cell migration, invasion, and adhesion also exhibited the same pattern of expression as the latter cadherins (Fig. 8D). Wnt pathway components, including Wnt3, Wnt5a, and Lef1, were enriched in the up-regulated group of genes (Fig. S2B), consistent with previous observations that activation of the β-catenin/Tcf–Lef signaling pathway is an obligatory step required for the retinoic acid-induced differentiation of pluripotent cells into ExEn cells (35). Collectively, these results imply that miR-205–dependent gene regulation can substitute for p19^Arf signaling in altering a program of gene expression required for the proper migration and sorting of differentiating ExEn cells to their final location on the EB periphery.

Discussion

The p53-dependent role of Arf as a tumor suppressor is widely appreciated, but the functions of Arf in other seemingly arcane physiological settings are poorly understood. The p19^Arf protein was not detected in mouse embryos at E4.5 but is expressed in the ExEn lineage by E7.5, consistent with its detection in the yolk sac later during fetal development (8). We modeled the earliest stages of ExEn cellular differentiation and development by studying pluripotent ES and iPS cells of various genotypes that were induced to differentiate in culture into EBs in which cells of the ExEn lineage, born internally, differentiate and migrate to the periphery. The Ink4–Arf locus is silenced in pluripotent ES and iPS cells, and, like p53, Arf inactivation increases the frequency of successful four-factor reprogramming by >50-fold. WT iPS cells mimicked ES cells in reengaging Arf expression during ExEn development.

Unlike other ExEn marker proteins—such as Gata4 and Dab2, which are first detected in select cells within the inner cell mass as they lose expression of pluripotency factors—the induction of p19^Arf occurs only after primitive endoderm cells have migrated to form the single EB outer layer. Notably, the appearance of differentiated ExEn cells in Arf-null EBs (but not in Ink4a-null EBs) was significantly delayed, requiring 10 d of ex vivo culture instead of the 4-d period normally sufficient for Arf^+/+ ES cells to generate mature ExEn derivatives. Given that Arf-null pups are born in appropriate numbers at the expected Mendelian ratio, compensation for Arf loss-of-function might occur during early mouse development.

Development of the ExEn lineage is enforced by Ras and inhibited by Myc. Ras/Erk signaling is central to these effects, and drugs that inhibit either Mek or Erk interfere with this process (20). Although Ras potently elicits phospho-Erk expression at the periphery of Arf-null EBs, other markers of the ExEn lineage, including Dab2, Lrp2, and Gata4, were not detected, implying that p19^Arf acts “downstream” of, or parallel to, Ras/Erk signaling to facilitate later stages of ExEn differentiation. p53-deficient iPS cells also failed to generate EBs that exhibited mature ExEn cells on their surface. However, in this setting, p19^Arf was expressed at the EB periphery, but again without the appearance of Dab2- or Gata4-positive cells, implying that the ability of p19^Arf to induce ExEn differentiation is p53-dependent. In turn, conditional activation of p53–ER^TAM in Arf-null iPS cells engineered to express H-Ras–G12V-T35S bypassed the Arf requirement and restored the appearance of Dab2- and Gata4-positive ExEn cells.

We successfully generated ExEn cell lines from WT and Arf^Fl/Fl embryos but were unable to derive them from Arf-null embryos. Conditional Cre recombinase-mediated deletion of Arf^FL alleles from established ExEn lines had no effect on their viability or ability to be continuously passaged in culture; hence, Arf plays a role in late stages of ExEn lineage differentiation but not in maintaining ExEn cell lines once they have been established. When suspended Arf-null ES and WT ExEn cells were admixed and cocultured to form EBs, GFP-marked ExEn cells segregated from Oct4-positive ES cells and rapidly localized to the periphery of chimeric EBs. Because ExEn cells cannot arise from Arf-null EBs under these conditions, their ability to sort properly is an inherent property.

Adhesion in tissues is predominantly mediated by cadherins, whose quantitative differences in expression are sufficient to govern the spontaneous self-sorting behavior of two cell populations, rendering them immiscible (32). E-cadherin is required for aggregation of ES cells and for the proper formation of EBs (36). In model systems analogous to the formation of chimeric EBs, differences in the levels of cellular surface tension arising from cadherin expression are sufficient to enable two populations to sort out, allowing cells with the lower level of cadherin to envelop the others (37). Consistent with this view, ExEn cells express lower Cdh1 levels than their ES progenitors. We therefore considered the idea that Arf may regulate a p53-dependent program of gene expression that affects ExEn cell migration and adhesion within differentiating EBs.

Arf plays a salutary role in male germ-cell development where its transient expression, restricted to basement membrane-bound spermatogonial progenitors in seminiferous tubules, ensures the survival of detached spermatocytes that have extinguished Arf expression and entered meiotic prophase I (7). Detachment of Arf-null progenitors from the tubular lining triggers DNA damage and p53-dependent apoptosis (anoikis) of primary spermatocytes, resulting in reduced numbers of mature sperm. Sequencing of microRNAs extracted from whole testes, in which relatively synchronous and maximal expression of p19^Arf is achieved at postnatal day 18, revealed that miR-205 was singularly down-regulated by >15-fold when Arf was inactivated. Analysis of EBs in which Arf expression could be conditionally up- or down-regulated provided direct evidence for positively correlated Arf and miR-205 expression. In turn, miR-205 expression is induced by p53, and its promoter is directly regulated by other p53 family members (27–29).

Threefold overexpression of miR-205 in WT ES cells accelerated ExEn formation without affecting differentiation of other germ-cell lineages. Notably, our goal was not to try to identify mRNA targets regulated by miR-205 in ES cells, but, instead, to test whether miR-205, acting as a p19^Arf–p53 signaling response element, might “rescue” Arf deficiency and promote ExEn differentiation. Strikingly, introduction of miR-205 into Arf-null ES cells was sufficient to induce complete ExEn differentiation in EBs after LIF was withdrawn. Given that p19^Arf regulates miR-205 levels and vice versa, a feed-forward mechanism likely drives ExEn formation, after which miR-205 is without observed effects in mature ExEn cells. Unlike the vast majority of microRNAs, inactivation of miR-205 results in early embryonic lethality (38), whereas physiological up-regulation of miR-205 might compensate for Arf loss in the embryo proper.

PCR analysis, immunofluorescence imaging, and gene profiling pointed to widespread miR-205–induced effects on the expression of genes that govern cell migration and adhesion. Introduction of miR-205 into Arf-null ES cells acutely triggered Cdh1 down-regulation even before LIF was withdrawn, and elevated expression of the Cdh1 corepressors Snail and Zeb2 was observed as miR-205-expressing Arf-null EBs emerged and formed ExEn cells. Vimentin was up-regulated in response to enforced miR-205 synthesis, and the protein preferentially localized to the EB outer layer in lieu of E-cadherin. In contrast, another family of Cdh genes (Cdh4, 5, 6, and 11) was induced by miR-205, as were many other genes implicated in regulating cell motility and adherence. Given that qualitative differences in cadherin signaling dictate patterns of mutual adhesive binding and help to define mechanical polarization boundaries (39), the segregation of external ExEn tissue from internal ES cells within EBs likely depends on many such factors, and not simply one. In agreement with observations that activation of β-catenin signaling is required for retinoic acid-induced differentiation of ES cells into ExEn cells (35), Wnt pathway components, including Wnt3, Wnt5a, and Lef1, were also induced. Together, these findings indicate that regulation of miR-205 by p19^Arf–p53 signaling affects the expression of genes required for late stages of ExEn differentiation, including many that govern cell migration and sorting within EBs.

Differential adhesion plays well-documented roles not only in embryogenesis but also in malignancy. Notably, some of the outcomes observed during ExEn formation in EBs were inconsistent with the reported role of miR-205 in maintaining epithelial fates and E-cadherin expression and in forestalling the epithelial to mesenchymal transition (EMT) during tumor progression (29, 33, 34). The few reported targets of miR-205 in tumor cells include Zeb1/2 corepressors, which down-regulate Cdh1. However, when miR-205 expression was enforced in Arf-null ES progenitors that were induced to form EBs, Zeb2 expression was unexpectedly increased, and Cdh1 levels fell. We can only presume that other as-yet-undefined mechanisms intercede in preventing Zeb2 mRNA down-modulation by miR-205 during EB formation.

Despite these differences, our data argue that miR-205 expression is positively regulated by Arf–p53 signaling and that Arf loss compromises miR-205 expression in at least two different physiological settings—ExEn differentiation and spermatogenesis. Down-regulation of miR-205 in tumor cells promotes the EMT and metastasis in several forms of cancer (29, 33, 34, 40, 41). A parsimonious hypothesis is that a role for Arf as a tumor suppressor in somatic cells is mediated, at least in part, by its regulation of EMT in response to oncogene activation. This finding is consistent with a plethora of studies indicating that Arf expression is induced by activated oncogenes and that subsequent Arf inactivation facilitates late stages of tumor progression and metastasis.

Materials and Methods

Animals and Cell Lines.

Work with mice was performed under established guidelines and supervision by the St. Jude Children's Research Hospital's (SJCRH) Institutional Animal Care and Use Committee, as required by the US Animal Welfare Act and National Institutes of Health policy to ensure proper care and use of laboratory animals for research. We previously generated Arf-null (10), Arf–GFP (14), Arf–Flox, and Arf–Cre mice (6). Mouse strains deficient for Ink4a (42) and Ink4a–Arf (43) were derived by others. Genetically engineered mice were back-crossed nine or more times onto a C57BL/6 background to create isogenic strains. C57BL/6 mice deficient for p53 were purchased from Jackson Laboratories (stock no. 2101), and immunodeficient nude mice (stock no. 086) were from Charles River Laboratories.

ES cell lines (44) and ExEn cells (25) from E3.5 embryos were derived as described. E3.5 blastocysts were flushed and cultured in suspension until E4.5 before analysis by immunofluorescence. ES cells (Arf154) expressing an shRNA directed to Arf mRNA (30) were generously provided by Prem Premsrirut (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Mirimus, Cold Spring Harbor, NY). Early passage primary MEF strains were generated as described (10).

A single inducible lentiviral vector containing a Dox-responsive element coregulating Oct4, Klf4, Sox2, and cMyc (STEMCCA-tetO-4F) and another vector expressing the reverse tetracycline activator (rtTA) were used to produce iPS cells (45, 46). Approximately 1 × 10⁵ MEF cells were seeded in 60-mm culture plates and infected with 2.5 mL of medium containing the rtTA vector plus 2.5 mL of medium containing the STEMCCA-tetO-4F vector in the presence of polybrene (5 μg/mL) (Sigma-Aldrich). The medium was replaced after 24 h with ES cell medium and changed every 2–3 d. Dox (Sigma-Aldrich) was added 24 h after infection at a final concentration of 1 μg/mL and then removed at day 14. iPS colonies, picked 20–25 d after infection on the basis of morphology, were expanded on gelatin-coated culture dishes in ES cell medium. Then, 4 × 10⁶ undifferentiated iPS and ES cells were injected into the hind leg muscles of 6- to 8-wk-old nude mice, and teratomas were analyzed 4 wk later.

Plasmid Vectors.

Construction of the Arf–exon1β–ER^TAM plasmid is described in SI Materials and Methods. The p53ER^TAM vector (47) was obtained with permission of Gerard Evan from Douglas Green (SJCRH), and miR-205 vectors were provided by Gregory Hannon (Cold Spring Harbor Laboratory). Vectors encoding H-Ras–G12V/T35S, H-Ras–S17N were provided by Hiroshi Koide (Kanazawa University, Kanazawa, Ishikawa, Japan) (48).

EB Formation.

EBs were generated by trypsinizing undifferentiated mouse ES or iPS cells and placing 300 cells into 50-µL hanging drops. Two days later, aggregates were collected and cultured in ultralow attachment Petri dishes with EB culture medium, consisting of 90% DMEM, 10% FBS (both vol/vol), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine (all from Invitrogen), and 0.1 mM mercaptoethanol (Gibco). The medium was changed every other day. To generate chimeric EBs, ES and ExEn cells were trypsinized, mixed 1:1, and dropped onto ultralow attachment Petri dishes at a density of 300 cells per 50 μL. Two days later, chimeric EBs were collected, embedded in tissue freezing medium (Triangle Biomedical Sciences), and sectioned into (10 µm) slices for immunofluorescence analysis.

RT-PCR.

Total RNA was extracted from cells by using TRIzol (Invitrogen) and transcribed into cDNA by using a high-capacity reverse-transcription kit (ABI), which included oligo(dT)15 and ReverTra Ace reverse transcriptase. For miRNA reverse transcription, universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) was used. For quantitative RT-PCR, raw data were obtained on PRISM 7900 (ABI) by using SYBR Green I double-stranded DNA-binding dye chemistry (Applied Biosystems) as described (44). For detection of miR-205 and endogenous miRNA RNU6B, TaqMan MicroRNA Assay kits (Applied Biosystems) were used. See Table S1 for primers.

Immunofluorescence and Immunoblotting.

Staining was performed as described (49). Primary antibodies listed in Table S2 were diluted in blocking buffer and incubated with sections overnight at 4 °C. Fluorescently coupled secondary antibodies, including Alexa anti-rat 488, anti-mouse 488, anti-rabbit 488, anti-rat 555, anti-mouse 555, and anti-rabbit 555 (all from Invitrogen), were incubated for 1 h at room temperature. Representative images were captured by confocal microscopy (Zeiss LSM 510 NLO Meta). Immunoblotting was performed as described (50).

Affymetrix Microarray Analysis.

RNA from Arf-null iPS cells and EBs infected with a control vector expressing GFP alone or with another vector coexpressing GFP and miR-205 were collected and subjected to hybridization by using Affymetrix Mouse Gene Chips (Version 1.0). Data presentation and analysis are described in SI Materials and Methods. Expression of selected genes was validated by qPCR using the PCR primers listed in Table S1.

RNA sequencing experiments performed on whole mouse testes of WT and Arf-null mice by Kaja Wasik and Gregory Hannon (Cold Spring Harbor Laboratory) first revealed down-regulation of miR-205 in response to Arf inactivation. These insights provided the impetus for studying miR-205 expression during extraembryonic development. Relevant RNA sequencing data were generously provided for our own use.