C 2014 Wiley Periodicals, Inc. V

genesis 52:967–975 (2014)

TECHNOLOGY REPORT

Two New Targeted Alleles for the Comprehensive Analysis of Meis1 Functions in the Mouse  nica Gonza  lez-La  zaro,1* Alberto Rosello  -Dıez,1* Irene Delgado,1 Laura Carramolino,1 Mo 2 Marıa Angeles Sanguino, Giovanna Giovinazzo,2* and Miguel Torres1* 1

Department of Cardiovascular Development and Repair, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain

2

Pluripotent Cell Technology Unit, Centro Nacional de Investigaciones Cardiovasculares, CNIC, Madrid, Spain

Received 22 July 2014; Revised 27 October 2014; Accepted 27 October 2014

Summary: Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor, and therefore, plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood, and vasculature. In addition, Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and nonhomeodomain transcription factors. During postnatal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with predisposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1-expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1-expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and

disease.

C 2014 Wiley Periodigenesis 52:967–975, 2014. V

cals, Inc.

Key words: Meis1 knockin; ECFP reporter protein expression; tamoxifen-inducible CreERT2; lineage tracing

INTRODUCTION Myeloid ecotropic viral integration site 1 (Meis1) (Moskow et al., 1995) belongs to the Three-Amino acid Loop Extension (TALE) family of homeodomain proteins. The TALE homeodomain family comprises four classes, two of which (Meis and PBC) are cofactors of Hox proteins (Mukherjee and B€ urglin, 2007). By themselves, Hox proteins have low DNA-binding activity and specificity; however, in complexes with PBC and/or Meis class proteins they exhibit specific target regulation (Penkov et al., 2013; Xu et al., 2008). Meis proteins contain two conserved domains; the Meinox domain through which they interact with the PBC class proteins, and the homeodomain, through which they bind Hox proteins and DNA. Meis1 was identified as a common viral integration site in myeloid leukemia in a mouse model (Moskow et al., 1995). During embryonic development, Meis1 is

*Current address for Alberto Rosell o-Dıez:: Developmental Biology Program, Sloan Kettering Institute, 1275 York Avenue, 10065, New York. * Correspondence to: Giovanna Giovinazzo, Miguel Torres, Centro Nacional de Investigaciones Cardiovasculares, CNIC, 3, Melchor Fernandez Almagro, 28029 Madrid, Spain. E-mail: [email protected] Contract grant sponsor: Spanish Ministry of Economy and Competitiveness, Contract grant number: BFU2012–31086 M onica Gonzalez-Lazaro and Alberto Rosell o-Dıez contributed equally to this work. Published online 11 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22833

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expressed in various structures in a regionally restricted manner, such as the developing limbs (Mercader et al., 1999), eyes, neural tube and others (Zhang et al., 2002). Meis1 is highly conserved in evolution and its human counterpart is frequently found up-regulated in human leukemias (Argiropoulos et al., 2007) and neuroblastoma (Geerts et al., 2003). Genome-wide association studies have also found an association of Meis1 with Restless Legs Syndrome (Spieler et al., 2014; Winkelmann et al., 2007) and cardiac conduction defects (Butler et al., 2012; Pfeufer et al., 2010; Smith et al., 2011). Recently, Meis1 was found to be implicated in cardiomyocyte differentiation (Wamstad et al., 2012) and in the control of proliferation of post-natal myocardium (Mahmoud et al., 2013). Several mouse models with targeted mutations in the Meis1 locus have been described. These mouse models showed that Meis1 homozygote mutants die in utero and present extensive hemorrhages and hematopoietic defects (Azcoitia et al., 2005; Hisa et al., 2004) as well as cardiac malformations (Stankunas et al., 2008). Importantly, none of the Meis1 mutants described so far completely lacks Meis1 protein expression. The first reported allele (Meis1tm1Ngc) contained a deletion of the homeodomain-coding exon 8, leading to the overexpression of a potentially dominant-negative truncated Meis1 protein containing the conserved Meinox domain (Hisa et al., 2004). In the second reported allele (Meis1tm1Mtor), inactivation of the Meis1 protein was achieved by knocking-in the modified estrogen receptor hormone-binding domain ERT2 (ERT2; Feil et al., 1997) to the full-length Meis1a protein isoform (Azcoitia et al., 2005). A third allele (Meis1tm2Ngc) was produced by floxing exon 8, which led to the same truncated protein overexpression as the constitutive exon-8 deletion (Kocabas et al., 2012; Mahmoud et al., 2013; Unnisa et al., 2012). To further our understanding on the functions of this transcription factor, we generated two new Meis1 knockin alleles in which the expression of the Meis1 protein is completely abolished. The Meis1ECFP line reports the endogenous expression of Meis1 by means of a cyan fluorescent reporter, while the Meis1CreERT2 line allows tracing the fate of cells expressing Meis1 mRNA. RESULTS AND DISCUSSION Generation of the Meis1ECFP and Meis1CreERT2 Knockin Mouse Lines and Phenotypes Observed In contrast to the previously reported strategies, we inserted DNA cassettes in the first Meis1 exon, fusing the exogenous coding regions in-frame with the first six nucleotides of Meis1 coding region (Fig. 1a). This approach results in the loss of the last 6 nucleotides from exon 1 and the first 51 nucleotides from intron 1. We produced two knockin alleles, one carrying the

ECFP coding region, which allows the live detection of Meis1 mRNA expression, and a second one carrying the CreERT2 coding region (Fig. 1b), which allows Tamoxifen-inducible Cre-mediated recombination (Feil et al.,1997). This strategy predicts the production of the exogenous proteins fused to the first two amino acids of Meis1. The drug-selections cassettes were removed in both cases, as confirmed by PCR analysis (Fig. 1b,c). In the case of the Meis1ECFP knockin the drug-selection cassette was the self-excision ACN cassette (Bunting et al., 1999) which is self-eliminated through germ line transmission of the targeted allele (Fig. 1b,c). In the case of Meis1CreERT2, we used a frt-flanked NEO cassette that was excised in F1 mice by ubiquitous Flpe-induced recombination achieved by breeding chimeric males to ACTB:Flpe females (Rodriguez et al., 2000) (Fig. 1b,c). As indicated in Fig. 1a,b, the SalI restriction site in the 50 homology arm of the targeting vector was mutated to allow the cloning strategy. The incorporation of this mutated SalI restriction site in Meis1ECFP or Meis1CreERT2 allele has not been determined (Fig. 1a,b). Western blot analysis detects the Meis1 protein and several nonspecific bands in WT and Meis1ECFP/1 embryos but does not detect any alternative protein production in appreciable levels. The loss of the Meis1 protein in the Meis1ECFP homozygous embryos without any obvious alteration of the non-specific bands or any presence of alternative proteins suggests the strategy used results in Meis1 complete loss of function (Fig. 1d). The Meis1ECFP/ECFP and Meis1CreERT2/CreERT2 embryos die at around day E14.5 and present generalized pallor and extensive hemorrhaging in the trunk (Fig. 2c,d). These defects are similar to those observed in the Meis1tm1Mtor embryos (Fig. 2b) and are the result of a failure in the separation of the lymphatic and blood vasculatures, due to the absence of megakaryocytes in Meis1-deficient embryos (Carramolino et al., 2010). Accordingly, Meis1ECFP/ECFP embryos are also defective in megakaryocyte production (Fig. 2e,f). These aspects of the mutant phenotype are also present in Meis1tm1Ncg/tm1Ncg and Meis1tm1Mtor/tm1Mtor mutants (Azcoitia et al., 2005; Hisa et al., 2004), suggesting that the Meis1 proteins expressed from these alleles are not functional in megakaryocyte development. In addition, the cardiac defects reported in Meis1tm1Ncg/tm1Ncg mutants were also present in the Meis1ECFP/ECFP indicating functional equivalence of these alleles during cardiac development (Fig. 2g,h). In contrast, some differences were found regarding eye development in embryos carrying the different alleles. While in the Meis1tm1Ncg/tm1Ncg mutants, partial retina duplication and a reduced lens were reported, the Meis1CreERT2/CreERT2, Meis1ECFP/ECFP, and Meis1aERT2/ERT2 embryos presented a clear microphthalmia at E13.5 (Fig. 2b–d). These differences in the eye phenotype could be due to the truncated Meis1 protein

FIG. 1. Generation of the Meis1ECFP and Meis1CreERT2 alleles. (a) Diagram showing the Meis1 locus and the 50 region involved in homologous recombination. Filled boxes represent the coding region and the open boxes the 50 and 30 UTRs. Black arrows delimit the 50 and 30 homology arms used in both constructs, also highlighted by a thicker line. Oblique red lines indicate the position of the insertion cassette in both constructs. A red horizontal bar shows the position of 30 probe used for Southern blot screening in ES cell genomic DNA. The arrowed line above the scheme indicates the 19.2 Kb fragment resulting from EcoRV digestion used for screening. A general scheme of the targeting vectors is shown below the represented Meis1 Genomic DNA. (b) Side-by-side representation of the Meis1ECFP and Meis1CreERT2 targeting strategies. The Meis1ECFP insertion cassette (left) contains the ECFP ORF followed by the ACN cassette (Bunting et al., 1999). The Meis1CreERT2 insertion cassette (right) contains the CreERT2 ORF followed by the FRT-PGK-gb2-Neo-FRT-loxP cassette (from Gene Bridges). Note that both insertion cassettes introduce RV restriction sites that upon homologous recombination will generate 9.0 Kb and 6.7 Kb EcoRV fragments in the Meis1ECFP and Meis1CreERT2 alleles, respectively. Black and red arrows indicate the position of the primers for wild type and knockin allele amplification after removing NEO selection cassettes. (c) Screening of positive ES cell clones, and genotyping of F2 mutant mice. As shown in the Southern blot analysis, the wild type and knockin alleles EcoRV fragments (19.2 Kb in WT allele, 9.0 Kb or 6.7 Kb in targeted alleles) identify targeted clones in Meis1. To genotype F2 Meis1ECFP embryos, PCR analysis was performed on yolk sac DNA using the primers indicated on the scheme by black (WT) and red1black (KI) primers. A common set of primers flanking the CreERT2 cassette (black arrows) were used for PCR genotyping of Meis1CreERT2 mice. Internal primers (red arrows) to CreERT2 cassette were also used in routine genotyping. (d) Western blot of Meis1CFP embryos showing that the Meis1 protein expression has been completely abolished without the overexpression of shorter protein versions. (e) Schematic representation of the Meis1 locus expression in the mutant mice generated here and in previous studies. aa indicates the number of Meis1 amino acids retained in each fusion protein or truncated forms. RV, EcoRV; S; SalI; N, NotI; A, ApaI; B, BstXI. Wt, wild-type; Tg, targeted allele. The crossed S on the targeting vectors indicates a deleted SalI site. The question mark over the crossed S site in the targeted alleles indicates uncertainty on whether this mutation has been targeted together with the insertion cassettes.

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FIG. 2. ECFP Phenotypic comparison of E13.5 Meis1 mutants generated by different transgenic strategies. (a) Wild type, (b) Meis1tm1Mtor/tm1Mtor, (c) Meis1 /ECFP, and (d) Meis1CreERT2/CreERT2 null embryos. Similar to previous observations in Meis1tm1Mtor/tm1Mtor embryos (Azcoitia et al., 2005), E13.5 Meis1tm1Mtor/tm1Mtor, Meis1ECFP/ECFP, and Meis1CreERT2/CreERT2 null embryos show extensive hemorrhaging in the dorsal trunk region (arrowheads) as well as general pallor and microftalmia (asterisks). Immunofluorescence for CD61 in sections of E12.5 liver sections detects megakaryocytes in wild type (e) but notMeis1ECFP/ECFP mutant (f) embryos. Hematoxylin and eosin staining in histological sections shows the abnormal communication between ventricles in Meis1ECFP/ECFP mutants (arrowheads, g, h).

that is overexpressed from the Meis1tm1Ncg allele. Caution should then be called when interpreting the phenotypes observed in the Meis1 alleles that produce this truncated form. Characterization of the ECFP Expression Pattern in the Meis1ECFP Knockin Allele ECFP

Live imaging of endogenous ECFP in E10.5 Meis1 embryos showed dose-dependent differences in expression. While null Meis1ECFP/ECFP embryos showed a strong fluorescent expression (Fig. 3a,b), Meis1ECFP/1 showed a similar expression pattern but with a reduced fluorescence intensity. As expected, no ECFP expression was seen in wild-type individuals (Fig. 3a,b). ECFP fluorescence in E10.5 Meis1ECFP/ECFP embryos recapitulates the expression of the endogenous Meis1 transcript. As seen in Fig. 3c, ECFP is strongly detected in the eye primordium, which coincides with a strong expression of the Meis1 transcript in this structure (Fig. 3c,d). Additional regions of common expression of the ECFP and Meis1 transcripts at E10.5 in the head are the hindbrain, the posterior forebrain, the region where the maxillary and nasal processes fuse and the second branchial arch (Fig. 3c,d). In addition, ECFP is expressed in the proximal domain of the limb (Fig. 3f), in coincidence with the region where Meis1 transcripts are detected at that stage (Fig. 3e), as previously reported (Mercader et al., 1999, 2009). Additionally, in the trunk region, ECFP also reveals the extensive expression of

Meis1 in the lateral and paraxial mesoderm and spinal cord (Fig. 3e,f). In histological sections the ECFP native signal was lost; however, the Meis1 expression pattern could be recognized using anti-GFP immunofluorescence (Fig. 3g,h). Labeling of Meis1-expressing Cell Lineages by Inducible Cre Recombination We characterized by in situ hybridization the expression of CreERT2 mRNA at several developmental stages and compared it with Meis1 expression in age-matched embryos. While CreERT2 expression reproduced faithfully the expression of the endogenous gene, we noted that the expression levels seemed to be much lower (Fig. 4a–b0 ). To test the utility of the Meis1CreERT2 allele for tracing the Meis1-expressing cells we combined this allele with the Cre-recombinase reporter allele Rosa26RLacZ (Soriano, 1999). We then administered tamoxifen (Tx) during the gestation of embryos carrying this allele combination (Fig. 4c). Following administration at E8.25 we observed LacZ-positive cells in forelimbs and the cardiac and pericardiac region (Fig. 4d). Labeled cells were found at any position of the limb proximo-distal axis (Fig. 4d,e) in accordance with the expression of Meis1 in the whole limb-forming regions at early stages (Mercader et al., 2009). Following Tx administration at E9.5 we found strong LacZ labeling in the visceral area and lateral plate-derived structures including both limbs. Clear LacZ staining was also observed in facial regions including the

COMPREHENSIVE ANALYSIS OF MEIS1 FUNCTIONS

FIG. 3. The expression of cyan fluorescent protein (ECFP) reporter protein recapitulates the Meis1 transcription pattern. (a, b) ECFP fluorescence in E10.5 wild type (1/1), Meis1ECFP/1 (ECFP/ 1) and Meis1ECFP/ECFP (ECFP/ECFP) embryos and corresponding brightfield images. (c) Detail of the head region from a wholemount in situ hybridization of E10.5 WT embryos using a probe for the Meis1 transcript and (d) corresponding ECFP expression in E10.5 Meis1ECFP/1 embryos. Arrowheads point to different regions of Meis1 expression. e, eye; fb, forebrain; hb, hindbrain; II, second branchial arch. (e) Detail of the trunk-limb region from a wholemount in situ hybridization of E10.5 WT embryos using a probe for the Meis1 transcript and (f), corresponding ECFP expression in E10.5 Meis1ECFP/1 embryos. Dotted lines depict limb contour and arrowheads, the distal limits of Meis1 expression in the limb. (g) Immunofluorescent detection of ECFP expression in an E12.5 Meis1ECFP/1 embryo transverse section using by anti-GFP. ECFP-positive tissues are detected in green (Alexa-488). (h) Magnification of boxed zone in “g” showing megakaryocyte detection in the fetal liver (Compare with Meis1 protein detection in Fig. 2e).

eye (Fig. 4h,i). Expression in limbs was restricted to the proximal region in accordance with the proximally restricted expression pattern of Meis1 at E9.5-E10.5 (Mercader et al., 2009 and Fig. 4a,b,g). In the facial region strong recombination was detected in the frontonasal mesenchyme but not in the majority of 1st branchial arch-derived tissues, both in accordance with

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FIG. 4. Lineage labeling of Meis1-expressing cells. (a–b0 ) In situ hybridization for Meis1 (a, b) and CreERT2 (a0 , b0 ) in trunk-forelimb (a, a0 ) and tail-hindlimb regions (b, b0 ) of age-matched Meis1CreERT2/1 embryos. (c) Scheme for the administration of Tamoxifen and analysis of Meis1CreERT2; Rosa26RLacZ embryos. Tamoxifen was administered at either E8.25 or E9.5 (color-coded) and fetuses were retrieved and analyzed for ß-Gal activity at E13.5. Representative specimens showing the overall distribution of Crerecombined cells in E13.5 embryos are shown in d and f. Recombination induced at E8.25 results in unrestricted labeling along the proximo-distal limb axis (e), while at E9.5 results in labeling of only the proximal limb (g, arrowhead), in addition to the eye (i) and facial mesenchyme (h, arrowhead). Close-up examination of the eye recombination pattern shows positive cells in the lens and periocular ectoderm (i) and histological analysis demonstrates recombination in the retina (l and magnified inset) and the lens (m). NR, Neural retina; PR, pigmented retina; L, lens.

Meis1 expression pattern at E10.5. Neck areas derived from the 2nd branchial arch were however strongly labeled in accordance with Meis1 expression at E10.5.

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Within the eye, we detected labeled cells in both the lens and periocular ectoderm as well as in the neural retina (Fig. 4i–m). Faithfull Meis1-expressing cell tracing was thus observed in lateral plate-derive structures and in branchial arch and eye primordium-derived structures. In contrast, no labeling was evident outside the eyes in the Central Nervous System, despite expression of Meis1 in areas of the forebrain and hindbrain at E10.5. Increasing tamoxifen dose or injecting at earlier (E6.5– 7.5) and later stages (E10.5) did not change these observations (data not shown). This result might indicate inefficiency of this allele in driving CreERT2 expression in neural tissues. In part this finding could be related to a general low expression of the CreERT2 driven by the Meis1 endogenous promoter (Fig. 4a0 ,b0 ). In fact CreERT2 ISH hardly detected any signal in the E10.5 neural tube (data not shown). In addition, we found that in most cases recombination was only affecting a subpopulation of cells within the labeled areas. This was especially evident in the eye, were only a few dispersed cells were found in each of the labeled tissues. The fact that the highest recombination is found in lateral plate-derived tissues and that this is the region with strongest Meis1 expression in the embryo (as determined by the time required for detecting Meis1 in situ mRNA hybridization signal; not shown) also suggests that the levels of CreERT2 produced by the Meis1CreERT2 allele are limiting for the detection of Meis1 lineage. This allele will thus be useful for the genetic tracing of Meis1-expressing lineages, although the ability to label comprehensively all Meis1-expressing cells is limited by the low recombination efficiency. MATERIALS AND METHODS Generation of the Meis1ECFP and Meis1CreERT2 Alleles Meis1ECFP and Meis1CreERT2 knockin mice were generated by gene targeting and both targeting vectors assembled following standard cloning procedure. The long arm homologous sequence (LAm1) is an 8.01-Kb fragment going from 28010 to 1 5 relative to the ATG of the gene. The short homology arm (SAm1) is a 1.69-Kb fragment extending from 1 63 to 11748 position. Both fragment were amplified by PCR using isogenic genomic DNA as template and the Expand Long Template PCR System (Roche). The long arm was amplified using the following primers: 50 -CTCGAG-AATAATCATTCCCCACTACCA-30 (sense, underlined the XhoI restriction site) and 50 -GTC GAC-GCCATCGGCCTCTCTGGCT-30 (antisense, underlined SalI restriction site), and the short arm amplified 50 TGGCTGAAAGACACTGCTACTAAG-30 and 50 -TAAAGTTG AGCGTTCTGTTGTAA-3 (sense and antisense, respectively). Both PCR products were firstly cloned into pGEMTeasy (Promega) and sequences verified. They then were

separately cloned into NotI restriction site of pBluescript II KS (1), (Stratagene) creating two intermediate vectors, pBSLAm1 and pBSSAm1. The internal SalI site at 12.27 Kb was eliminated from the long homologous arm. For the generation of Meis1ECFP construct, we created a pACN-ECFP vector where the coding region of the enhanced cyan florescent protein (ECFP) and the SVpolyA signal was excised SalI/AflII from the pECFPN1 vector (Clontech) and cloned into the modified pACN vector (Azcoitia et al., 2005, Bunting et al., 1999), after adding an adequate synthetic adaptor. The short arm (SA) was cloned into PmlI/NotI sites of the pACN-ECFP vector and the whole fragment containing the ECFP gene, the ACN cassette-and the SA was cloned SalI/NotI in pBSLAm1 intermediate vector. For the generation of Meis1CreERT2 targeting vector the FRT-PGK-gb2-Neo-FRT-loxP cassette from a commercial vector (GeneBridges) was PCR-amplified and cloned Sal I-Cla I into pBluescript KS, yielding pFNF. The coding region of CreERT2 (Feil et al., 1997) was Sal I/Xho I digested and cloned into the SalI restriction site of pFNF, creating the intermediate vector pCFNF. SAm1 was cloned into the Not I restriction site of pCFNF and finally LAm1 was cloned into the Apa I/Sal I restriction sites. Due to differences in the cloning strategy, an adaptor sequence had to be inserted in the Sal I site after LAm1 (Fig. 1c) to recover the proper reading frame. The backbone of both Meis1ECFP and Meis1CreERT2 targeting vectors was eliminated by NotI or ApaI-BstXI digestion, respectively (see Fig. 1a). Gene targeting were performed as previously described in Torres (1998), using R1 embryonic stem cells (Nagy et al., 1993). Positive clones were screened for homologous recombination by Southern blot of EcoRV digested DNA, using an external probe 30 to the short arm. The 30 probe was amplified by PCR using the following primers: 50 -TAGGTATAATGGGCAGGAGTT-30 (sense), and 5-GCAAATCTTAGGGAGGCAGTA-30 (antisense). Heterozygous targeted clones were microinjected into C57Bl/6 blastocysts embryos and chimeric males were bred to females to achieve germline transmission [the females were ACTB:Flpe in the case of the Meis1CreERT2 line (Rodriguez et al., 2000)]. Mice were housed in accordance with Spanish bioethical regulations for laboratory animals. PCR Genotyping Germline transmission of Meis1ECFP allele was confirmed by PCR using genomic DNA isolated from yolk sacs of F2 embryos. Primers were designed to amplify the region where the ECFP DNA was inserted. Sense-50 ACAAATAAAGCAATAGCATCACA-30 and antisense-50 TTCTCAGCTGTTCATTTTCATTA-30 primers gave a 508 bp long PCR product in the targeted locus. PCR reactions using sense 50 -GTTTGCATATTTGTTTCTTTTCA-30

COMPREHENSIVE ANALYSIS OF MEIS1 FUNCTIONS

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and the above antisense primer amplify a 460 bp long fragment in the wild type allele. Self- excision of the ACN cassette in the F1 embryos (Bunting el al., 1999) was confirmed by a 1.5 Kb long PCR product amplified using the same wild type primers. Meis1CreERT2 mice were genotyped using the following primers: sense-50 -CCCTCCCACTGCTGTCTT-30 ; antisense50 -AAGACAAGGGGGAAGATGCT-30 . The WT allele was identified as a 521 bp long PCR product and the knockin allele, after NEO removal, was identified by a 2,758 bp long PCR product. To confirm the knockin genotype CreERT2 internal primers were used: 50 0 0 TGACGGTGGGAGAATGTTAAT-3 5 -GCCGTAAATCAATCGATGAGT-30 , sense and antisense, respectively.

Both Meis1ECFP and Meis1CreERT2 mouse lines will be available to the research community upon acceptance of the manuscript.

Western Blot

ACKNOWLEDGMENTS

Individual Wild type, heterozygous and knockin/out E10.5 embryos were homogenized and lysed in RIPA buffer and the protein concentration quantified using a BioRad DC protein assay. Approximately, 50 mg of protein were separated by SDS-PAGE electrophoresis and transferred to a membrane using standard procedures. A polyclonal rabbit anti-Meis1 antibody was used at a 1:500 dilution (Azcoitia et al., 2005) Histological Analysis Whole mount in situ hybridization was performed using standard procedures (Wilkinson and Nieto, 1993). For detection of cyan fluorescent protein (ECFP) reporter protein expression, embryos were dissected in PBS. Detection of native ECFP fluorescence was performed using a Leica MZ FLIII fluorescence stereomicroscope. For histological sectioning, embryos were processed substituting xylene in the standard processing protocol for isopropanol to minimize any loss of LacZ staining. The blocks were sectioned on a Leica RM2245 microtome at 6 um. After rehydration the sections were counterstained in Nuclear Fast Red, dehydrated in alcohols, cleared in xylene and mounted using DPX. Immunohistochemistry and immunofluorescence were performed as previously described. (Azoitia et al., 2005; Carramolino et al., 2010). Anti-GFP rabbit polyclonal (Living Colors 632460) diluted 1/200 was used, in combination with a secondary antibody (Alexa-fluorconjugated goat anti rabbit from Life Technologies), to detect ECFP protein on fixed samples. Lineage Tracing Assays Tamoxifen (Sigma-Aldrich; 20 mg/ml) was diluted in corn oil. For CreERT2 induction at E.8.25 and E9.5, pregnant females were administered 3 and 4 mg of Tamoxifen, respectively by oral gavage. Embryos were obtained at E13.5 and fixed in 0.25% glutaraldehyde at room temperature for 60 min. Embryos were washed twice with

PBS and stained in LacZ staining solution (0.1 M phosphate buffer pH 7.3 (72.9 ml 1 M Na2HPO4 1 27.1 ml 1 M NaH2PO4 for 1,000 ml), 2 mM MgCl2, 0.11% sodium deoxycholate, 0.2% Igepal, 20 mM Tris HCL pH 7.3, 1 mg/ml X-gal, 5 mM K4[Fe(CN)6], and 5 mM K3[Fe(CN)6]). Staining was carried out at room temperature for 24–36 hours with shaking. After staining, embryos were washed three times with PBS and postfixed overnight at 4 C with 4% paraformaldehyde. Availability Declaration

The authors wish to thank Andras Nagy for R1 embryonic stem cell line, Luis Miguel Criado for blastocysts injection, Roisin Doohan for histological section. Virginia Garcıa and Ana Belen Ricote for mouse care and Vanessa Cadenas for mice genotyping. This work was supported by the Spanish Ministry of Economy and Competitiveness (grant BFU2012-31086 to M.T.). M.G-L was supported by a grant from the Mexican Council for Science and Technology (CONACyT – “Estancias Posdoctorales y Sabaticas al Extranjero para la Consolidaci on de Grupos de Investigaci on”, Fase 3). The CNIC is supported by the Ministry of Economy and Competitiveness and the Pro-CNIC Foundation. LITERATURE CITED Argiropoulos B, Yung E, Humphries RK. 2007. Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis. Genes Dev 21:2845–2849. Azcoitia V, Aracil M, Martinez AC, Torres M. 2005. The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Dev Biol 280:307–320. Bunting M, Bernstein KE, Greer JM, Capecchi MR, Thomas KR. 1999. Targeting genes for self-excision in the germ line. Genes Dev 13:1524–1528. Butler AM, Yin X, Evans DS, Nalls MA, Smith EN, Tanaka T, Li G, Buxbaum SG, Whitsel EA, Alonso A, Arking DE, Benjamin EJ, Berenson GS, Bis JC, Chen W, Deo R, Ellinor PT, Heckbert SR, Heiss G, Hsueh WC, Keating BJ, Kerr KF, Li Y, Limacher MC, Liu Y, Lubitz SA, Marciante KD, Mehra R, Meng YA, Newman AB, Newton-Cheh C, North KE, Palmer CD, Psaty BM, Quibrera PM, Redline S, Reiner AP, Rotter JI, Schnabel RB, Schork NJ, Singleton AB, Smith JG, Soliman EZ, Srinivasan SR, Zhang ZM, Zonderman AB, Ferrucci L, Murray SS, Evans MK, Sotoodehnia N, Magnani JW and Avery CL. Butler AM, Yin X, Evans DS, Nalls MA, Smith EN, Tanaka T, Li G,

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