Forward and reverse genetics through derivation of haploid mouse embryonic stem cells

Abstract

All somatic mammalian cells carry two copies of chromosomes (diploidy), whereas organisms with a single copy of their genome, such as yeast, provide a basis for recessive genetics. Here we report the generation of haploid mouse ESC lines from parthenogenetic embryos. These cells carry 20 chromosomes, express stem cell markers, and develop into all germ layers in vitro and in vivo. We also developed a reversible mutagenesis protocol that allows saturated genetic recessive screens and results in homozygous alleles. This system allowed us to generate a knockout cell line for the microRNA processing enzyme Drosha. In a forward genetic screen, we identified Gpr107 as a molecule essential for killing by ricin, a toxin being used as a bioweapon. Our results open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale.

Figure 1. Generation of haploid murine ES cell lines

A) Schematic overview of induction of parthenogenesis and the derivation of haploid ES cell lines. Mouse oocytes were activated with either 5% ethanol (or 25mM Strontium Chloride (SrCl₂)) and implanted into pseudopregnant females. ES cells were then generated from blastocysts and haploid cells subsequently sorted by FACS. Cultures were routinely resorted until we derived stable haploid cells. B) Flow cytometric analysis of DNA content in the control diploid ES cell line IB10/C and the haploid HMSc2 cell line. DNA content was determined using Hoechst33342. 1n and 2n chromosome sets for haploid and 2n and 4n chromosome sets for diploid ES cells are indicated. The histograms show data from cells at the 10^th sort. C) Representative chromosome spreads of control diploid ES cells and haploid HMSc1 and HMSc2 cells. Spreads from anaphase (1n) and prophase (2n) of mitosis are shown for haploid cells. As a control, anaphase (2n) and prophase (4n) spreads are shown for diploid ES cells. D,E) Sequence coverage relative to the common reference of parental in-house C57BL/6 and 129 strains is shown on a log2 scale. Haploid cells were derived from C57BL/6×129 crosses. Chromosomes are arranged in numerical order and separated by small gaps. See also Supplemental Figure S1.

Figure 2. Marker analysis and in vitro differentiation potential of haploid ES cell lines

A) Both haploid HMSc1 and HMSc2 cell lines exhibit a morphology characteristic of ES cell colonies (asterisk). Representative phase contrast images are shown. Note the feeder layer of mouse embryonic fibroblasts (MEF) (arrowheads). Haploid cells stain also positive for the ES cell marker alkaline phosphatase (blue, bottom panels). B) Expression of Oct4, Nanog, and Sox2, prototypical markers for murine embryonic stem cells. Phalloidin staining indicates the feeder cell layer. Haploid HMSc1 and HMSc2 cell were co-stained for Oct4 (FITC) and Nanog (TRITC). In both cases, stainings are shown separately in the red channel. Scale bars are 50μm. Data are from cells after the 4^th sort. C) Expression of prototypic ES cell marker genes in the haploid HMSc1 (blue) and HMSc2 (red) cells. mRNA expression was determined using qPCR and normalized to diploid IB10/C ES cells (black bars). D) Gata4 protein expression in embryoid bodies (EB, day 7) as a marker for endoderm. Representative EBs are shown for both haploid HMSc1 and HMSc2 ES cell lines counterstained with phalloidin (green). Scale bars are 50μm. E) qPCR revealed down-regulation of the ES cell markers Nanog, Rex1, Oct4, Sox2, Klf2, Klf4, and Sall4 in EBs (day 7) derived from the haploid ES cell line HMSc2 accompanied by expression of the indicated lineage commitment markers (see text). mRNA expression was normalized to the parental, undifferentiated haploid ES cells (set at 1). See also Supplemental Figure S2.

Figure 3. In vivo differentiation potential of haploid ES cell lines

A) Haploid ES cells can contribute to tissues in adult mice. Diploid cells from the Agouti⁺ clone HMSc2 were injected in into C57BL/6 blastocysts and coat color chimerism was observed (brown fur patches). B) Histological and immunohistological analysis of teratomas derived from control diploid IB10/C ES cells and the haploid ES cell lines HMSc1 and HMSc2. Haploid ES cells can contribute to all three germlayers, namely muscle cells (H&E), intestinal endoderm (mucin producing goblet cells stained by Alcian blue, counterstained with nuclear fast red), Tuj1⁺ neurons, and Cytokeratin 5 (K5) expressing ectoderm. Tuj1⁺ and K5⁺ cells were detected by immunohistochemistry (DAB, brown), counterstained with hematoxylin (blue). Scale bars: 100μm. See also Supplemental Figure S3.

Figure 4. Haploid ES cells have the intrinsic ability for stable growth and differentiation

A) Immunostaining for Oct4 protein expression (red) on three different subclones that were established by plating single haploid cells directly after FACS purification (top panels). The middle and bottom panels show immunostaining for Oct4 (red) and Tuj1 (green) expression and expression of the endodermal marker Gata4 (red, counterstained with DAPI) in attached embryoid bodies (EBs, day 10) derived from the indicated subclones. Data are from cells that were subcloned after > 30 passages of the parental line. Scale bars are 50μm. B) Proliferation rates and C) percentages of haploid cells in control cultures containing 100% diploid MHSc2-27 cells and cultures of HMSc2-27 cells seeded at 80:20 and 50:50 ratios of haploid:diploid cells. Multiplication rates and percent haploidy were determined every 24 hours using FACS analysis of Hoechst33342 stained cells. Note that for this experiment cells were continuously kept in culture for 7 passages (14 days). Based on this experiment, we estimate that ∼2-3% of haploid cells became diploid each day over the course of the experiment. D) Development of myoblasts from the haploid ES cell subclone HMSc2-27. The feeder cell free diploid ES cell line CCE was used as a control for this experiment. Representative phase contrast images are shown (see Suppl. movies to watch typical “beating” of these myoblasts). Scale bars are 100μm. E) GFP expression (green) in a GFP-tagged haploid ES cell subclone. Non-GFP labeled cells are shown as control (grey shaded histogram). Flow cytometry of DNA content (Hoechst33342) is shown for the same subclone demonstrating that both haploid and diploid cells express GFP. F) Differentiation of haploid and diploid HMSc2-27 cells into EBs (day 13) that contain Tuj1 neurons (green) and Gata4 expressing endodermal cells (red). Note downregulation of Oct4 expression (red, upper panel) and the presence of residual clusters of Nanog⁺ cells (green, bottom panels). In the top panels, cells were counterstained with DAPI to visualize nuclei. See also Supplemental Figure S4-S6.

Figure 5. Differentiation potential of haploid ES cells

A) Analysis of the haploid ES cell clone HMSc2-27 cultured under conditions to maintain an ES cell fate (ES cells), in vitro differentiated into Nestin⁺ neural stem cells (NS cells), and further differentiation into GFAP⁺ astrocytes by withdrawal of EGF and FGF2 in the presence of 1% serum (differentiated NS cell culture conditions). Immunofluorescence labeling of Oct4, Nestin, and GFAP are shown, counterstained for DAPI. Representative imagines are shown. Scale bar is 100μm. B) Flow cytometry analysis of DNA content in cells gated for Oct4 and Nestin expression and grown under ES cell (top), NS cell (middle), and differentiated NS cell (bottom panels) conditions. The gates used and percentages of cells are inserted in each plot. Haploid cells are prominent in Oct4⁺ fractions under all conditions while Nestin⁺ cells differentiated for 4 days are devoid of haploid cells. The red line in the top left panel shows the representative DNA content of the diploid control IB10/C ES cell line gated for Oct4 expression. C) Haploid cells exit the pluripotent state following the same dynamics as diploid cells. The left panel shows control Oct4 levels (mean intensity depicted) in haploid HMSc2-27, mixed (haploid and diploid) HMSc2-27, and control diploid CCE ES cells after 72h under control (plus LIF) conditions. Differentiation by LIF withdrawal leads to diminished Oct4 expression of diploid and haploid cells (middle panel). Differentiation induction using 0.5μM retinoic acid results in a rapid loss of Oct4 expression in both haploid and diploid cells, indicative of differentiation (right panel). The same results were obtained when we used 0.1μM retinoic acid (not shown). Data are shown as mean Oct4 fluorescence intensity +/- SEM analysing more than 50.000 cells per condition. 1way ANOVA (p>0.05) showed increased expression of Oct4 in diploid cells (consistent with increased nuclear area) in all conditions except the 48- and 72-hour retinoic acid treatments wherein Oct4 expression was at background levels. See also Supplemental Figure S7.

Figure 6. Reverse genetics in haploid ES cells

A) Analysis of virus integration sites after neomycin selection. 176,178 insertions were determined by deep sequencing. The retrovirus landed in 49% in intergenic and in 51% in intragenic regions, with a high frequency of integration into introns, especially the first intron. B) Graph shows percentage of genes with virus integrations following a single round of retroviral mutagenesis for different fractions of the total viral integration sites (X-axis). Genes with the 10% lowest expression (0-10%) showed the least integration efficiency while higher expressed genes (50-100%) show more efficient gene trapping. For increasing fractions of the total viral integration sites (X-axis) higher saturation is reached, up to 67% yet not reaching saturation, indicating that additional genes are trapped in the total library of 7.5 million independent insertions. C,D) PCR analysis using site specific primers for the indicated genes and a primer specific for the LTR of the inserted retrovirus. The location of the used PCR primers is schematically indicated on top of each panel. Of note, all primers were used for all 10 different genes showing (C) that the virus has indeed integrated into the site identified by initial sequencing and (D) that the integrations result in homozygous mutations of the respective loci. Lane 1 = Madcam1; lane 2 = Drosha; lane 3 = Retinoic acid receptor gamma (Rarg); lane 4 = Ap4s1; lane 5 = Arap1; lane 6 = Evx1; lane 7 = Bcl2l1; lane 8 = 2210012G02RIK; lane 9 = Titin; lane 10 = Chr2:50928851. Positive wild type (Wt) and negative H2O controls are shown. E) qPCR analysis of RARG mRNA expression in haploid HMSc2-27 cells that are wild type for rarg (Wt), HMSc2-27 cells that contain the splice acceptor in antisense orientation (AS), and HMSc2-27 cells that contain the splice acceptor in the sense orientation (S). mRNA expression was normalized to the parental HMSc2-27 cells. F) Representative images of cultures containing the indicated wild type, antisense, and sense RARG HMSc2-27 cells treated with 0.1μM retinoic acid for 10 days. Note the near complete absence of cells in the Wt and antisense cultures. Scale bars are 100μm. G) qPCR analysis of Drosha mRNA expression in haploid HMSc2-27 cells that are wild type for Drosha (Wt), HMSc2-27 cells that contain the splice acceptor in antisense orientation (AS), and HMSc2-27 cells that contain the splice acceptor in the sense orientation (S). mRNA expression was normalized to parental HMSc2-27 cells. H) Complete absence of cystic embryoid bodies in Drosha deficient HMSc2-27 cells as compared to Drosha expressing wild type HMSc2-27 cells and cells containing the splice acceptor in the antisense orientation. Representative images for embryoid bodies are shown on day 10 after EB induction. Of note, we did not observe a single cystic EB in Drosha mutant cells even in prolonged culture. Scale bars are 100μm. I) Histograms showing Venus reporter gene expression in wild type HMSc2-27 cells (Wt), HMSc2-27 cells that contain the splice acceptor in antisense orientation (AS), and HMSc2-27 cells that contain the splice acceptor in the sense orientation (S) transduced with pSENSOR-based miRNA constructs harboring a potent shRNA targeting Firefly Luciferase with (target) or without its target (no target) site in the 3′UTR of Venus. Cells were gated on shRNA expressing (dsRed⁺) cells and Venus expression levels were compared to nontransduced control cells (grey).

Figure 7. Forward genetic screen for ricin toxicity in haploid ES cells

A) Haploid HMSc2-27 with/without gene-trap mutagenesis was exposed to ricin from Ricinus communis for 3 weeks. Colonies only appeared in the mutagenized batch and were processed for deep sequencing. B) Top hits identified in the ricin toxicity screen. Sense (green) and antisense (red) insertions in Gpr107, Fut9, and Slc35c1 genomic loci. The vertical lines indicate the respective exons for each gene with the first exon always moved to the left side of each diagram. Insertions in antisense might disrupt gene function, sense integrations will do so in almost all cases. Note that nearly all insertions are in sense for the splice acceptor and that some antisense integrations map to exons, all of which should result in disruptive mutations. Considering that ∼ 50% of intragenic insertions are sense and ∼ 50% in antisense, these data also show that the screen has indeed strongly enriched for disruptive mutations (p>1.13E^-10 for Gpr107; p>3.95E^-6 for Fut9; p>0.000019 for Slc35c1). C) Genes identified in the ricin toxicity screen. The numbers of distinct retroviral insertions predicted to disrupt gene expression (either because intragenic regions containing the sense orientation of the splice acceptor, or sense and antisense integrations into exons) are indicated. Enrichment for sense mutations vs anti-sense integrations was assessed using a binomial test and the respective p values are indicated. Of note, anti-sense integrations can also lead to gene disruption. Assigned biochemical pathways and allocation to the Golgi apparatus are also indicated. D, E) Validation of Gpr107 in Ricin toxicity. HMSc2-27 ES cells and NIH3T3 cells were transduced with LMN-constructs expressing Gpr107 or control shRNAs together with GFP and then challenged with a lethal dose of ricin for 2 days. Images show representative cultures after 48 hours of ricin treatment (D). Scale bars are 100μm. (E) The ricin survival rate as ratio between recovered cells of ricin treated vs. ricin untreated cells is shown in % (as determined by quantitative FACS analysis of cells gated for viability by forward scatter, side scatter, and PI staining after 48 hours of ricin treatment). Cells were cultured in 10cm dishes in triplicates and average survival +/- SD was determined for eGFP negative (not transduced) and eGFP positive haploid HMSc2-27 ES cells and NIH 3T3 cells for each plate.