Arf Tumor Suppressor and Mir-205 Regulate Cell Adhesion And

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

Chunliang Lia,b, David Finkelsteinc, and Charles J. Sherra,b,1

aHoward Hughes Medical Institute, and Departments of bTumor Cell Biology and cComputational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105

Contributed by Charles J. Sherr, February 4, 2013 (sent for review December 22, 2012) Induction of the Arf tumor suppressor (encoded by the alternate read- progenitor cells (2). In agreement with the idea that epigenetic ing frame of the Cdkn2a locus) following oncogene activation engages silencing of the locus is necessary to maintain cellular self-renewal, a p53-dependent transcriptional program that limits the expansion of reprogramming of somatic cells to yield induced pluripotent incipient cancer cells. Although the p19Arf protein is not detected in stem (iPS) cells is accompanied by Ink4–Arf repression (see be- most tissues of fetal or young adult mice, it is physiologically ex- low) and facilitated by Ink4–Arf deletion (3). pressed in the fetal yolk sac, a tissue derived from the extraembryonic Paradoxically, the p19Arf protein is physiologically expressed in a endoderm (ExEn). Expression of the mouse p19Arf protein marks late few disparate tissues during mouse development, including peri- stages of ExEn differentiation in cultured embryoid bodies (EBs) de- vascular cells within the hyaloid vasculature of the eye (4–6), mi- rived from either embryonic stem cells or induced pluripotent stem totically dividing spermatogonia within seminiferous tubules (6, 7), cells. Arf inactivation delays differentiation of the ExEn lineage within and the fetal yolk sac (8). Inactivation of Arf results in blindness and EBs, but not the formation of other germ cell lineages from pluripotent reduced sperm production, but effects of Arf deletion on yolk sac progenitors. Arf is required for the timely induction of ExEn cells in development have not been investigated. Whether these diverse response to Ras/Erk signaling and, in turn, acts through p53 to ensure physiological roles of Arf can be explained through a common BIOLOGY the development, but not maintenance, of the ExEn lineage. Remark- mechanism and whether they reflect the canonical role of Arf as fi DEVELOPMENTAL ably, a signi cant temporal delay in ExEn differentiation detected dur- a potent tumor suppressor remain a mystery. We demonstrate that ing the maturation of Arf-null EBs is rescued by enforced expression of Arf Arf a signaling pathway involving Ras/Erk, p19 , p53, and microRNA mouse microRNA-205 (miR-205), a microRNA up-regulated by p19 205 (miR-205) regulates a cell motility and adhesion program that and p53 that controls ExEn cell migration and adhesion. The nonca- facilitates formation of extraembryonic endoderm (ExEn) cells Arf nonical and canonical roles of in ExEn development and tumor from pluripotent embryonic stem (ES) or iPS cell progenitors. suppression, respectively, may be conceptually linked through mechanisms that govern cell attachment and migration. Results Expression of Arf in ExEn. Blastocysts harvested from mouse em- p53 tumor suppressor | epithelial to mesenchymal transition bryos at embryonic day (E) 4.5 exhibit pluripotent Oct4-positive cells in the inner cell mass surrounded by Gata4-marked primitive he Ink4–Arf (Cdkn2a,b) locus, which is only 50 kb in length, endoderm (PrE) cells in a generally mutually exclusive pattern Tencodes three intimately linked tumor suppressor genes. The (Fig. 1A Upper). By using a particularly sensitive and specific Ink4a Ink4a and Ink4b genes encode polypeptides (p16 and monoclonal antibody (9), p19Arf expression was not detected in Ink4b p15 ) that inhibit cyclin D-dependent kinases to maintain the the early PrE lineage at E4.5 (Fig. 1A Lower), whereas embryos retinoblastoma protein (Rb) in its active inhibitory state, thereby recovered at E7.5 revealed p19Arf expression in ExEn tissues limiting cell proliferation. In contrast, the Arf protein (p19Arf in ARF the mouse, p14 in humans) inhibits the Mdm2 E3 ubiquitin Significance ligase to activate and stabilize p53, a transcription factor that coordinates a complex gene expression program that potently Arf guards against tumor formation (1, 2). The p19Arf and p16Ink4a The tumor suppressor gene is not expressed in most normal proteins are encoded in part by unique first exons, whose products tissues but when activated by oncogenic stress signals engages a p53-dependent transcriptional program that prevents tumor are spliced to a second shared exon that is translated in alternative Arf reading frames, yielding proteins that bear no shared amino acid formation. Surprisingly, expression of the p19 protein in mouse embryoid bodies is required for the timely formation of extra- sequences and that are functionally distinct. The Ink4a–Arf locus is embryonic endoderm (ExEn). Inactivation of Arf down-regulates generally not expressed under normal physiological circumstances a single microRNA, miR-205, which can “rescue” ExEn formation in but is induced by aberrant mitogenic signals that result from on- Arf-null embryonic or induced pluripotent stem cells. During ExEn cogene activation. By engaging Rb- and p53-dependent tran- formation, miR-205 regulates a suite of genes that govern cell scriptional programs, the Ink4–Arf proteins counter tumor cell migration and adhesion, suggesting a conceptual basis for linking progression by eliciting cell cycle arrest, apoptosis, or cellular se- the roles of Arf in ExEn differentiation and tumor metastasis. nescence. Deletion of this small gene cluster incapacitates the

functional Rb/p53 tumor-suppressive network and is one of the Author contributions: C.L. and C.J.S. designed research; C.L. and D.F. performed research; most common events observed in human cancers. C.L. and C.J.S. contributed new reagents/analytic tools; C.L., D.F., and C.J.S. analyzed data; The Ink4a–Arf locus is silenced in stem cells—whether of and C.L. and C.J.S. wrote the paper. embryonic, fetal, or adult somatic tissue origin—thereby facili- The authors declare no conﬂict of interest. tating their capacity for continuous cellular self-renewal. In Freely available online through the PNAS open access option. contrast, the locus is epigenetically remodeled in more differ- Data deposition: The data reported in this paper have been deposited in the Gene Ex- entiated cell types to allow its engagement in response to on- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE42210). cogenic stress signals. Despite the risk of its deletion in cancer, 1To whom correspondence should be addressed. E-mail: [email protected]. – the evolutionary conservation of the Ink4 Arf locus in mammals This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. may provide a mechanism for limiting the numbers of stem and 1073/pnas.1302184110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302184110 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 culture clearly revealed p19Arf 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 p19Arf expression (Fig. 1C). At early times of embryonic development (E4.5) when p19Arf 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 p19Arf in the inner cell mass, we assumed that the emergence of p19Arf-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 de- prived 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 progen- Arf fi Fig. 1. p19 protein expression in ExEn. (A) WT E4.5 embryos were xed itors to form ExEn cells might be compensated during early and stained with the indicated antibodies and visualized for immunofluorescence with a confocal microscope. Pluripotent cells (red) in the inner cell embryonic development in the mouse, the duration of EB cell mass and differentiated primitive endoderm cells (green) were visualized culture was extended to determine whether ExEn cell devel- with antibodies to Oct4 and Gata4, respectively; p19Arf protein was not opment was temporally delayed. EB cells derived from Arf-null detected. DAPI was used to visualize cell nuclei. (Scale bars, 25 μm.) (B) ES clones recovered Dab2 expression after 10 d of suspension Section of WT embryo recovered from the decidua of the uterus at E7.5 culture and produced Dab2 at levels equivalent to those detected stained as above. p19Arf-positive ExEn cells (green, asterisks) surround the in differentiating WT cells cultured for only 4 d (Fig. 2D). By Oct4-positive epiblast (red). (Scale bars, 200 μm, Upper; 100 μm, Lower.) (C) using Arf-null ES cells in which a cDNA encoding green fluo- Cryosectioned WT EBs were stained and visualized as above. Expression of β Arf rescent protein (GFP) was substituted for Arf exon-1 sequences p19 (green) and Bmi1 (red) colocalize (yellow) in a single ExEn cell layer at under the control of the cellular Arf promoter (14), ArfGfp/Gfp 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 ES clones produced GFP- and Gata4-positive ExEn cells after to an indicator strain that expresses LacZ in response to Cre-mediated exci- 10 d of prolonged EB culture (Fig. 2E). Hence, the functionally Gfp sion of a “lox–stop–lox” cassette. ES cells obtained from these blastocysts null Arf alleles were induced as Gata4-positive ExEn cells were induced to differentiate to EBs. β-galactosidase was detected at the eventually emerged. periphery of EBs expressing Arf–Cre (Lower; magnification: 50×). Arf-null, but Not Ink4a-Deficient, iPS Cells Exhibit Defective ExEn

− Differentiation. The creation of iPS cells by introduction of four surrounding Oct4-positive cells (Fig. 1B). Inter-crossing Arf+/ − − transcription factors, Oct4, Sox2, Klf4, and c-Myc, into mouse mice yields Arf / offspring at the expected Mendelian ratio, and embryo fibroblasts (MEFs) is facilitated by inactivation of p53 Arf-null females produce equivalent numbers of blastocysts com- (15–18). Because elevated Myc signaling engages Arf to induce pared with those of wild-type (WT) mice and generate normal- − − p53 (19), Arf-null MEFs might be as susceptible to four-factor sized litters (10). Examination of 10 Arf / and 12 Arf+/+ embryos iPS reprogramming as their p53-null counterparts. We derived − recovered at E7.5 revealed as much morphological variation within 2 WT, 3 Arf+/ , and 14 Arf-null iPS cell lines, the latter origi- − − individual cohorts as between them. nating from Arf / or ArfGfp/Gfp strains (10, 14). All iPS cell lines Given difficulties in defining any overt developmental de- selected for subsequent studies maintained normal karyotypes ficiency in Arf-null embryos, we attempted to model the earliest and expressed the pluripotency markers Oct4, Sox2, Nanog, and stages of ExEn formation in embryoid bodies (EBs) derived from CD15/SSEA1; all could generate cell types representing three cultured ES cells. When undifferentiated ES cells are suspended embryonic germ layers; and all formed teratomas in immuno- in hanging drops and transferred to ultralow attachment dishes deficient mice (Fig. S1 A–D). Like p53-null MEFs, strains lacking in medium lacking leukemia-inhibitory factor (LIF), the well- Arf generated iPS cells at ∼50-fold greater efficiency than cells organized EBs that develop are rimmed by a single ExEn layer, containing a single Arf allele (Fig. S1E). which surrounds the pluripotent epiblast and other cells within When allowed to differentiate for 4 d in the absence of LIF, − the inner mass that are differentiating to form the three other Arf+/ iPS cells generated EBs that expressed p19Arf and Gata4 germ cell lineages. Cryosections of EBs that arose after 4 d of at the periphery (Fig. S1 F and G). However, Arf-null iPS clones

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1302184110 Li et al. Downloaded by guest on September 29, 2021 through the Ras/Erk pathway potentiated ExEn differentiation PNAS PLUS 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 mor- phologically 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. Ectopic expression of p19Arf 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 p19Arf 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 BIOLOGY DEVELOPMENTAL

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)Im- munoblotting 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 ArfGfp/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 p19Arf protein was accompanied by GFP signals, which colocalized with Gata4-marked ExEn cells at the periphery.

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 p16Ink4a-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 p19Arf and p16Ink4a are Fig. 3. Ras/Erk signaling promotes Arf-dependent ExEn differentiation. (A) both reexpressed as iPS cells differentiate, only Arf loss of function WT and Arf-null iPS cells (indicated at left) were transduced with vectors delays ExEn differentiation. Given that Arf-null ES and iPS cells expressing the indicated Ras mutants or with a control (Ctl) vector, selected exhibited identical phenotypes, both were exploited subsequently. for puromycin resistance in medium containing LIF, and induced to differentiate for 4 d in the absence of LIF. A constitutively active Ras effector Ras/Erk Signaling and p53 Promote ExEn Differentiation. Generation mutant (G12V/T35S) that signals to Erk promoted ExEn cell formation from of ExEn cells in culture depends upon Ras signaling through the WT, but not Arf-null, iPS progenitors, an effect blocked by a dominant- Raf–Mek–Erk pathway and can be accelerated by Ras over- negative mutant (HRas–S17N). Phase contrast images are shown. (Scale bars, μ expression and blocked by pharmacologic Mek/Erk inhibitors 200 M.) (B and C) Quantitative PCR (qPCR) analysis (B) and immunoblotting (C)confirmed Dab2 mRNA and protein up-regulation in WT cells infected (20). Arf is induced by Ras (14, 21), suggesting that each may be with the G12V/T35S Ras mutant (abbreviated as T35S). (D) Activated phospho- required for ExEn specification. A constitutively active H-Ras Erk was detected at the periphery of both Arf-null and WT day-4 EBs gener- effector mutant (G12V/T35S) that specifically targets signaling ated from the different iPS progenitors. (Scale bars, 200 μm.)

Li et al. PNAS Early Edition | 3of10 Downloaded by guest on September 29, 2021 (ERTAM) 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 p19Arf (9) (SI Materials and Methods). Following its transduction into WT MEFs, the ∼50- kDa fusion protein was detected with antibodies to either p19Arf 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 ERTAM 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–ERTAM was capable of driving elements of the ExEn differentiation program. 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 p19Arf 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–ERTAM 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 over- expressed (Fig. 4F), indicating that the Ras/Erk pathway comple- ments p19Arf–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 gener- Fig. 4. Arf and p53–ER promote expression of ExEn marker proteins. (A) ating such cultures from Arf-null ES cells, we established ExEn Lysates of WT MEFs (left lane) or from cells transduced with a vector FL/FL cells from blastocysts containing “floxed” Arf alleles (Arf ) (6) encoding Arf exon-1β sequences (center lane) or with a naked control vector and deleted Arf exon-1β with a retrovirus encoding Cre recom- (right lane) were immunoblotted with antibodies directed to p19Arf (Upper) binase. These Arf-null ExEn cells continued to proliferate con- or to the ER cassette (Lower). The endogenous p19Arf protein was detected tinuously, and expression of ExEn markers was maintained, so Arf in all three samples, whereas the 50-kDa minigene-coded fusion protein was is not required for ExEn cell line viability and maintenance. 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 We infected cultured WT ExEn cells with a retrovirus minigene were subjected to qPCR analysis. Primers that amplified the ER encoding GFP and admixed them in a 1:1 ratio with Arf-null ES moiety confirmed generally equivalent levels of vector-coded RNA expres- cells to form chimeric EBs (26). Two days later, ExEn cells had sion in cells treated with different concentrations of tamoxifen (TAM; localized to the EB outer layer, spontaneously segregating from see legends). Dab2 and Gata4 mRNAs in transduced Arf-null iPS cells were DAPI-stained ES cells that comprised the inner pluripotent induced, and Nanog expression was decreased in response to minigene ex- compartment (Fig. 5D). Because Arf-null ES cells cannot con- pression and tamoxifen treatment. Error bars, mean ± SEM (n = 3 experi- tribute to ExEn formation under these conditions, the exclusion ments). (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). of GFP-marked ExEn cells from the inner pluripotent cells and (D) Like day-4 EBs derived from Arf-null iPS cells, p53-null iPS cells exhibited their localization and retention at the periphery is an inherent reduced Dab2 protein levels compared with WT iPS controls. (E)Although property of differentiated ExEn cells themselves. p19Arf 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- miR-205 Can Rescue ExEn Differentiation in Arf-Null Cells. Arf is positive cells were observed at the periphery. (Scale bars, 200 μm.) (F) Arf-null expressed in mitotically dividing spermatogonia, but not in pri- iPS cells were transduced with a retroviral vector expressing the HRas– mary spermatocyctes derived from them or in other cells within G12V/T35 mutant. Cells engineered to coexpress p53–ERTAM (Center and the testis (6, 7). In unrelated studies, we determined that the Right) were treated with tamoxifen (Right) or not (Center), and Dab2 ex- fi fl levels of a single microRNA, miR-205, were selectively de- pression was quanti ed by ow cytometric analysis. The percentages of cells that exhibited increased Dab2 expression are noted. creased by >15-fold in the Arf-null testes of postnatal day-18 mice, a time when p19Arf levels in spermatogonia are maximal and are synchronously expressed throughout the developing Motivated to test whether Arf–p53 signaling and miR-205 seminiferous tubules. miR-205 is up-regulated by p53 (27), and expression are directly correlated in ES cells, we obtained an ES its promoter is directly bound and induced by other p53-related cell line (designated Arf154) in which a miR-30–based shRNA family members, p63 and p73 (28, 29). that targets Arf mRNA (but not sequences shared with Ink4a)is

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1302184110 Li et al. Downloaded by guest on September 29, 2021 mechanism appears to drive ExEn formation, after which miR- PNAS PLUS 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).

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 dif- ferentially 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 BIOLOGY markers (Dab2, Gata4, Gata6, Sox17, and Afp) were further in- DEVELOPMENTAL Fig. 5. Self-sorting of ExEn cells in chimeric EBs. (A) Morphology of ExEn duced in response to miR-205, whereas expression of pluri- cells visualized by phase contrast microscopy. (Magnification: 100×.) (B and potency markers (such as Klf4 and Tbx3) was reduced (Fig. 8 B C) Immunoblotting of proteins (B) and PCR analysis of transcripts (C)inWT Arf and C). Gene Ontology (Database for Annotation, Visualization, ES or ExEn cell lines. (D) WT ExEn cells expressing p19 , Gata4, and Dab2 and Integrated Discovery) analysis indicated that the most sig- were transduced with a vector expressing GFP, mixed with an equal number nificantly up-regulated genes encode glycoproteins (2.4E-17; 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 4.2% of the total genes), affect in utero development (4.29E-05; with antibodies to the indicated proteins. Marked ExEn cells spontaneously 0.74% of total genes), and govern cell motility and migration sorted to the periphery of the chimeric EBs and completely segregated from (4.64–8.71E-04; 0.74% of total genes) and cell adhesion (2.37E- DAPI-stained ES cells in the inner mass. (Scale bars, 100 μm.) 04; 0.82% of total genes) (Fig. 8D). The cell-autonomous ability of WT ExEn cells to properly form chimeric EBs when cocultured with Arf-null ES cells (Fig. induced by doxycycline (Dox) treatment (30). Arf154 ES cells 5) mimics the classical sorting behavior of cells that express were suspended for EB formation and treated with Dox for 4 d. variable levels of cadherins (31, 32). The E-cadherin (Cdh1) re- Expression of miR-205 RNA was decreased following Dox pressor Snail (33) was expressed at higher levels in ExEn cells treatment, which was without effect in WT ES cells (Fig. 6A compared with their ES cell progenitors with concomitant Cdh1 TAM Left). Conversely, introduction of the Arf–exon-1β–ER fusion down-regulation (Fig. 5). Affymetrix chip gene profiling (Fig. protein into Arf-null iPS cells triggered a conditional increase in 8D) and PCR analysis (Fig. S2A) confirmed the up-regulation miR-205 levels following tamoxifen treatment (Fig. 6A Right). of Snail2 in EBs responding to miR-205, but down-regulation WT ES cells were transduced with miR-205 vectors coex- of Cdh1 was not observed when day-4 EBs expressing a control pressing GFP (Fig. 6B) or with a control vector expressing GFP vector and miR-205 were directly compared by microarray alone, and GFP-positive cells were sorted 2 d later and replated analysis. However, introduction of miR-205 into Arf-null ES cells in the presence of LIF to preserve their undifferentiated state. resulted in a very rapid reduction in Cdh1 levels, which occurred Cells expressing GFP alone maintained their characteristic ES even before LIF was withdrawn (Fig. S2A). Although the few morphology, but those expressing threefold more miR-205 un- reported direct targets of miR-205 include Zeb proteins that also derwent partial differentiation in 2 d, even in the presence of act as Cdh1 corepressors (34), Zeb2 levels were elevated in re- LIF, to yield ExEn-like progeny (Fig. 6C) that exhibited in- sponse to transduction of the miR-205 vs. control vector in Arf- creased Dab2 and decreased Nanog expression (Fig. 6D). Four null EBs (Fig. 8D and Fig. S2A). We used imaging techniques to days after plating these cells in the absence of LIF, expression of look at the topological distribution of E-cadherin in EBs formed Gata4 and Sox7 mRNAs, as well as p19Arf, p53, and p21Cip1, from Arf-null ES cells that had been infected either with the were up-regulated as expected, but without effects on the relative control vector or that encoding miR-205 (Fig. 9). Strikingly, expression of markers of ES-derived ectoderm (Fgf5), mesoderm E-cadherin was no longer detected in relative abundance at the (T/Brachyury), or trophectoderm (Cdx2) (Fig. 6E, left two col- periphery of EBs overexpressing miR-205, and this result was umns). In contrast, enforced miR-205 expression in established, accompanied by induction of vimentin (Fig. 8D) and its prefer- fully differentiated WT ExEn cell lines did not affect the ex- ential distribution to the ExEn layer at the EB surface (Fig. 9). pression of these RNAs (Fig. 6E, right two columns). Thus, Significantly increased expression of candidate genes that reg- overexpression of miR-205 could up-regulate the Arf–p53–p21 ulate cell migration and adhesion, including Cdh2 (N-Cadherin), signaling axis and preferentially drive pluripotent WT ES cells to Cdh5, Cdh6,andCdh11,wasobserved(Fig.8D and Fig. S2A). assume an ExEn fate but did not affect the same target genes Many other genes reported to be essential for cell migration, once ExEn differentiation was established. Given that p19Arf can invasion, and adhesion also exhibited the same pattern of expres- induce miR-205 (Fig. 6A) and vice versa (Fig. 6E), a feed-forward sion as the latter cadherins (Fig. 8D). Wnt pathway components,

Li et al. PNAS Early Edition | 5of10 Downloaded by guest on September 29, 2021 Fig. 6. miR-205 is regulated by Arf and enhances ExEn formation from WT ES progenitors. (ALeft) 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–ERTAM 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 (Neor); 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, p19Arf, p53, and the p53-responsive gene p21Cip1 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.

including Wnt3, Wnt5a, and Lef1, were enriched in the up- Discussion regulated group of genes (Fig. S2B), consistent with previous The p53-dependent role of Arf as a tumor suppressor is widely observations that activation of the β-catenin/Tcf–Lef signaling appreciated, but the functions of Arf in other seemingly arcane pathway is an obligatory step required for the retinoic acid- physiological settings are poorly understood. The p19Arf protein induced differentiation of pluripotent cells into ExEn cells (35). was not detected in mouse embryos at E4.5 but is expressed in Collectively, these results imply that miR-205–dependent gene the ExEn lineage by E7.5, consistent with its detection in the yolk regulation can substitute for p19Arf signaling in altering a pro- sac later during fetal development (8). We modeled the earliest gram of gene expression required for the proper migration and stages of ExEn cellular differentiation and development by sorting of differentiating ExEn cells to their ﬁnal location on the studying pluripotent ES and iPS cells of various genotypes that EB periphery. were induced to differentiate in culture into EBs in which cells of

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1302184110 Li et al. Downloaded by guest on September 29, 2021 mass as they lose expression of pluripotency factors—the in- PNAS PLUS duction of p19Arf 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 p19Arf 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, p19Arf was expressed at the EB periphery, but again without the appearance of Dab2- or Gata4-positive cells, implying that the ability of p19Arf to induce ExEn differentiation is p53-dependent. In turn, conditional activation of p53–ERTAM 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. Fl/Fl

We successfully generated ExEn cell lines from WT and Arf BIOLOGY

embryos but were unable to derive them from Arf-null embryos. DEVELOPMENTAL Conditional Cre recombinase-mediated deletion of ArfFL 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, Fig. 7. miR-205 induces ExEn formation from Arf-null ES progenitors. (A) rendering them immiscible (32). E-cadherin is required for ag- Arf-null ES cells infected with a naked control (Ctl) GFP vector or one gregation of ES cells and for the proper formation of EBs (36). encoding miR-205 from a miR-30 backbone together with GFP were sorted In model systems analogous to the formation of chimeric EBs, 2 d after infection, placed in culture, and visualized 2 d later by phase differences in the levels of cellular surface tension arising from contrast microscopy at magnification of 50× (Left) or 100× (Right). Cells with cadherin expression are sufficient to enable two populations to the characteristic morphology of ExEn cells appeared in response to miR-205. sort out, allowing cells with the lower level of cadherin to en- (B) EBs derived from WT or Arf-null ES cells and infected with control or miR- velop the others (37). Consistent with this view, ExEn cells ex- – 205 encoding vectors (indicated at the top) were stained for Dab2 protein press lower Cdh1 levels than their ES progenitors. We therefore expression. WT cells (Left) and Arf-null EBs transduced with miR-205 (Right) considered the idea that Arf may regulate a p53-dependent expressed Dab2 at their periphery, whereas Arf-null cells, whether un- infected or transduced with a control vector, did not (second and third from program of gene expression that affects ExEn cell migration and left). (Scale bars, 50 μm.) (C) Quantitative RT-PCR was used to quantify mRNA adhesion within differentiating EBs. expression of five ExEn markers (indicated at the top) in vector-transduced Arf plays a salutary role in male germ-cell development where Arf-null ES cells grown in the presence of LIF (day 0) and in EBs derived from its transient expression, restricted to basement membrane-bound them (day 4). Cells were transduced with a control virus (Ctl) or a vector spermatogonial progenitors in seminiferous tubules, ensures the encoding miR-205 (205) as indicated in the legend. Error bars, mean ± SEM survival of detached spermatocytes that have extinguished Arf (n = 3 experiments). 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, the ExEn lineage, born internally, differentiate and migrate to – resulting in reduced numbers of mature sperm. Sequencing the periphery. The Ink4 Arf locus is silenced in pluripotent ES of microRNAs extracted from whole testes, in which relatively and iPS cells, and, like p53, Arf inactivation increases the fre- synchronous and maximal expression of p19Arf is achieved at quency of successful four-factor reprogramming by >50-fold. WT postnatal day 18, revealed that miR-205 was singularly down- iPS cells mimicked ES cells in reengaging Arf expression during regulated by >15-fold when Arf was inactivated. Analysis of EBs in ExEn development. which Arf expression could be conditionally up- or down-regulated Unlike other ExEn marker proteins—such as Gata4 and provided direct evidence for positively correlated Arf and miR- Dab2, which are first detected in select cells within the inner cell 205 expression. In turn, miR-205 expression is induced by p53,

Li et al. PNAS Early Edition | 7of10 Downloaded by guest on September 29, 2021 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.

and its promoter is directly regulated by other p53 family mem- cells. Vimentin was up-regulated in response to enforced miR-205 bers (27–29). synthesis, and the protein preferentially localized to the EB outer Threefold overexpression of miR-205 in WT ES cells accel- layer in lieu of E-cadherin. In contrast, another family of Cdh erated ExEn formation without affecting differentiation of other genes (Cdh4, 5, 6, and 11) was induced by miR-205, as were many germ-cell lineages. Notably, our goal was not to try to identify other genes implicated in regulating cell motility and adherence. mRNA targets regulated by miR-205 in ES cells, but, instead, to Given that qualitative differences in cadherin signaling dictate test whether miR-205, acting as a p19Arf–p53 signaling response patterns of mutual adhesive binding and help to define mechan- element, might “rescue” Arf deficiency and promote ExEn dif- ical polarization boundaries (39), the segregation of external ferentiation. Strikingly, introduction of miR-205 into Arf-null ES ExEn tissue from internal ES cells within EBs likely depends cells was sufficient to induce complete ExEn differentiation in on many such factors, and not simply one. In agreement with EBs after LIF was withdrawn. Given that p19Arf regulates miR- observations that activation of β-catenin signaling is required 205 levels and vice versa, a feed-forward mechanism likely drives for retinoic acid-induced differentiation of ES cells into ExEn ExEn formation, after which miR-205 is without observed effects cells (35), Wnt pathway components, including Wnt3, Wnt5a, and in mature ExEn cells. Unlike the vast majority of microRNAs, Lef1, were also induced. Together, these findings indicate that inactivation of miR-205 results in early embryonic lethality (38), regulation of miR-205 by p19Arf–p53 signaling affects the ex- whereas physiological up-regulation of miR-205 might compen- pression of genes required for late stages of ExEn differentiation, sate for Arf loss in the embryo proper. including many that govern cell migration and sorting within EBs. PCR analysis, immunofluorescence imaging, and gene profiling Differential adhesion plays well-documented roles not only in pointed to widespread miR-205–induced effects on the expression embryogenesis but also in malignancy. Notably, some of the out- of genes that govern cell migration and adhesion. Introduction comes observed during ExEn formation in EBs were inconsistent of miR-205 into Arf-null ES cells acutely triggered Cdh1 down- with the reported role of miR-205 in maintaining epithelial fates regulation even before LIF was withdrawn, and elevated expres- and E-cadherin expression and in forestalling the epithelial to sion of the Cdh1 corepressors Snail and Zeb2 was observed as mesenchymal transition (EMT) during tumor progression (29, 33, miR-205-expressing Arf-null EBs emerged and formed ExEn 34). The few reported targets of miR-205 in tumor cells include

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1302184110 Li et al. Downloaded by guest on September 29, 2021 rtTA vector plus 2.5 mL of medium containing the STEMCCA-tetO-4F vector PNAS PLUS 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–ERTAM plasmid is described in SI Materials and Methods. The p53ERTAM 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 Fig. 9. miR-205 drives vimentin synthesis and relocalization of E-cadherin with EB culture medium, consisting of 90% DMEM, 10% FBS (both vol/vol), within Arf-null EBs. Immunoﬂuorescence staining of Arf-null day-4 EBs de- 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine (all from rived from cells infected with a control vector (Ctl; Left) and a vector Invitrogen), and 0.1 mM mercaptoethanol (Gibco). The medium was changed expressing miR-205 (205; Right) is shown. Confocal images of EBs stained every other day. To generate chimeric EBs, ES and ExEn cells were trypsinized, with antibodies to E-cadherin or vimentin and with DAPI to visualize nuclei mixed 1:1, and dropped onto ultralow attachment Petri dishes at a density of are merged. (Scale bars, 100 μm.) 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.

Zeb1/2 corepressors, which down-regulate Cdh1. However, when BIOLOGY

miR-205 expression was enforced in Arf-null ES progenitors that RT-PCR. Total RNA was extracted from cells by using TRIzol (Invitrogen) and DEVELOPMENTAL were induced to form EBs, Zeb2 expression was unexpectedly transcribed into cDNA by using a high-capacity reverse-transcription kit (ABI), increased, and Cdh1 levels fell. We can only presume that other which included oligo(dT)15 and ReverTra Ace reverse transcriptase. For as-yet-undefined mechanisms intercede in preventing Zeb2 miRNA reverse transcription, universal PCR Master Mix, No AmpErase UNG mRNA down-modulation by miR-205 during EB formation. (Applied Biosystems) was used. For quantitative RT-PCR, raw data were Despite these differences, our data argue that miR-205 ex- obtained on PRISM 7900 (ABI) by using SYBR Green I double-stranded DNA- pression is positively regulated by Arf–p53 signaling and that Arf binding dye chemistry (Applied Biosystems) as described (44). For detection of loss compromises miR-205 expression in at least two different miR-205 and endogenous miRNA RNU6B, TaqMan MicroRNA Assay kits physiological settings—ExEn differentiation and spermatogen- (Applied Biosystems) were used. See Table S1 for primers. esis. Down-regulation of miR-205 in tumor cells promotes the Immunofluorescence and Immunoblotting. Staining was performed as de- EMT and metastasis in several forms of cancer (29, 33, 34, 40, scribed (49). Primary antibodies listed in Table S2 were diluted in blocking 41). A parsimonious hypothesis is that a role for Arf as a tumor buffer and incubated with sections overnight at 4 °C. Fluorescently coupled suppressor in somatic cells is mediated, at least in part, by its secondary antibodies, including Alexa anti-rat 488, anti-mouse 488, anti- regulation of EMT in response to oncogene activation. This rabbit 488, anti-rat 555, anti-mouse 555, and anti-rabbit 555 (all from Invi- finding is consistent with a plethora of studies indicating that Arf trogen), were incubated for 1 h at room temperature. Representative images expression is induced by activated oncogenes and that sub- were captured by confocal microscopy (Zeiss LSM 510 NLO Meta). Immu- sequent Arf inactivation facilitates late stages of tumor pro- noblotting was performed as described (50). gression and metastasis. Affymetrix Microarray Analysis. RNA from Arf-null iPS cells and EBs infected Materials and Methods with a control vector expressing GFP alone or with another vector coex- Animals and Cell Lines. Work with mice was performed under established pressing GFP and miR-205 were collected and subjected to hybridization by guidelines and supervision by the St. Jude Children’s Research Hospital’s using Affymetrix Mouse Gene Chips (Version 1.0). Data presentation and (SJCRH) Institutional Animal Care and Use Committee, as required by the US analysis are described in SI Materials and Methods. Expression of selected Animal Welfare Act and National Institutes of Health policy to ensure proper genes was validated by qPCR using the PCR primers listed in Table S1. care and use of laboratory animals for research. We previously generated RNA sequencing experiments performed on whole mouse testes of WT Arf-null (10), Arf–GFP (14), Arf–Flox, and Arf–Cre mice (6). Mouse strains and Arf-null mice by Kaja Wasik and Gregory Hannon (Cold Spring Harbor deficient for Ink4a (42) and Ink4a–Arf (43) were derived by others. Geneti- Laboratory) first revealed down-regulation of miR-205 in response to Arf cally engineered mice were back-crossed nine or more times onto a C57BL/6 inactivation. These insights provided the impetus for studying miR-205 ex- background to create isogenic strains. C57BL/6 mice deficient for p53 were pression during extraembryonic development. Relevant RNA sequencing fi purchased from Jackson Laboratories (stock no. 2101), and immunode cient data were generously provided for our own use. 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 ACKNOWLEDGMENTS. Relevant RNA sequencing data were generously fl described. E3.5 blastocysts were ushed and cultured in suspension until provided by Kasa Wasik and Gregory Hannon. We thank Gregory Hannon E4.5 before analysis by immunofluorescence. ES cells (Arf154) expressing an and Scott Lowe for providing vectors encoding miR-205; Konrad Hoched- shRNA directed to Arf mRNA (30) were generously provided by Prem Pre- linger for lentiviruses encoding four reprogramming factors and rtTA; msrirut (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Mirimus, Gerard Evan for p53–ERTAM; Prem Premsrirut for Arf154 ES cells; Ronald A. Cold Spring Harbor, NY). Early passage primary MEF strains were generated DePinho for Ink4a-null mice; Hiroshi Koide for H-Ras mutants; Nadine as described (10). Hachouche and Rebecca Singleterry for excellent technical assistance; Fred- erique Zindy for providing mice and cell lines; core resources of St. Jude A single inducible lentiviral vector containing a Dox-responsive element Comprehensive Cancer Center CA-21765 for imaging, flow cytometric anal- coregulating Oct4, Klf4, Sox2, and cMyc (STEMCCA-tetO-4F) and another yses, synthesis of oligonucleotides, and gene profiling analysis; and members vector expressing the reverse tetracycline activator (rtTA) were used to of the C.J.S./Martine F. Roussel laboratory for critical comments and encour- produce iPS cells (45, 46). Approximately 1 × 105 MEF cells were seeded in agement. This work was supported in part by ALSAC of SJCRH. C.J.S. is an 60-mm culture plates and infected with 2.5 mL of medium containing the Investigator of the Howard Hughes Medical Institute.

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