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. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Genesis. 2013 Aug 30;51(10):725–733. doi: 10.1002/dvg.22417

Generation and Characterization of a Tamoxifen-Inducible EomesCreER Mouse Line

Inga-Marie Pimeisl 1, Yakup Tanriver 1,2, Ray A Daza 3, Franz Vauti 4, Robert F Hevner 3, Hans-Henning Arnold 4, Sebastian J Arnold 1,5,*
PMCID: PMC4112203  NIHMSID: NIHMS602511  PMID: 23897762

Summary

Transgenic mouse lines expressing inducible forms of Cre-recombinase in a tissue-specific manner are powerful genetic tools for studying aspects of development and various processes in the adult. The T-box transcription factor eomesodermin (Eomes) plays critical roles for maintenance and differentiation of different pools of stem and progenitor cells from early embryonic stages to adulthood. These include trophoblast stem cells, epiblast cells during the generation of the primary germ layers, neurogenic intermediate progenitor cells in embryonic and adult cortical neurogenesis, and maturing natural killer and T cells. Here, we report on the generation and analysis of an EomesCreER-targeted allele by placing the tamoxifen-activatable Cre-recombinase (CreER) under the control of the Eomes genomic locus. We demonstrate that CreER expression recapitulates endogenous Eomes transcription within different progenitor cell populations. Tamoxifen administration specifically labels Eomes-expressing cells and their progeny as demonstrated by crossing EomesCreER animals to different Cre-inducible reporter strains. In summary, this novel EomesCreER allele can be used as elegant genetic tool that allows to follow the fate of Eomes-positive cells and to genetically manipulate them in a temporal specific manner.

Keywords: mouse, Eomesodermin, Tbr2, tamoxifen-activatable CreER, lineage tracing

T-box proteins (Tbx factors) comprise a family of 17 transcription factors defined by their highly conserved, DNA-binding domain, the so-called T-box. Tbx factors frequently show highly restricted spatiotemporal expression patterns and loss-of-function mutants exhibit profound developmental phenotypes (Naiche et al., 2005). The T-box gene Eomesodermin (Eomes, also called Tbr2) becomes first expressed in cells of the trophectoderm lineage from early postimplantation stages onward marking the population of undifferentiated, multipotent trophoblast stem cells (TSCs) (Niwa et al., 2005; Russ et al., 2000; Strumpf et al., 2005). Eomes-positive TSCs are found in the extraembryonic ectoderm (ExE) at early pregastrulation stages and become later restricted to the chorion until extraembryonic expression ceases around embryonic day 9.5 (E9.5). Eomes is required to maintain TSCs in an undifferentiated state and to allow for the expansion of TSCs, which constitute the cellular source for the embryonic part of the placenta (Russ et al., 2000; Strumpf et al., 2005). During onset of gastrulation starting from E6.25, a second Eomes expression domain arises in the epiblast at the site of primitive streak initiation where it is maintained for a short time period until E7.5 (Ciruna and Rossant, 1999; Russ et al., 2000). The conditional deletion of Eomes in the epiblast has uncovered crucial functions for specification of two of the germ layers, namely, for the formation of definitive endoderm (Arnold et al., 2008a; Teo et al., 2011) and the cranio-cardiac mesoderm (Costello et al., 2011). Accordingly, the deficiency of Eomes in the epiblast leads to developmental arrest at the primitive streak stage shortly after gastrulation onset due to combined defects of mesoderm and endoderm formation (Arnold et al., 2008a; Costello et al., 2011).

During cerebral corticogenesis, Eomes expression is first found in Cajal-Retzius cells of the developing fore-brain vesicles (Bulfone et al., 1999; Russ et al., 2000). From E12.5 Eomes marks the pool of neurogenic intermediate progenitor cells (IPCs) located in the embryonic subventricular zone (SVZ) that gives rise to cortical projection neurons (Englund et al., 2005; Hevner et al., 2006; Hodge et al., 2008). Deletion of Eomes function in the developing cortex results in deficits of early and late neurogenesis due to a gross reduction of IPC proliferation and major disturbances of neuronal differentiation and cortical patterning (Arnold et al., 2008b; Elsen et al., 2013; Sessa et al., 2008). Similarly, human cases of a chromosomal translocation 2215 kb upstream to the Eomes locus were identified that leads to severe microcephaly in homozygously affected individuals. Most likely, this translocation leads to the loss of regulatory elements for Eomes expression during cortical neurogenesis (Baala et al., 2007). During adult murine neurogenesis, Eomes is expressed in intermediate progenitors of the mature brain and is essential for glutamatergic neurogenesis (Brill et al., 2009; Hodge et al., 2008; Hodge et al., 2012).

Additional sites of adult Eomes expression include central memory CD8+ T cells (Intlekofer et al., 2005; Pearce et al., 2003) and natural killer (NK) cells (Gordon et al., 2012; Tayade et al., 2005) of the adaptive and innate immune system, respectively. Maintenance of both cell types depends on Eomes function, explaining deficiencies in the immune responses of Eomes conditional mutants (Banerjee et al., 2010; Gordon et al., 2011).

In conclusion, numerous studies using constitutive or conditional gene deletion approaches have established central functions of the transcription factor Eomes during lineage specification, differentiation, and maintenance of various stem and progenitor cell populations. To characterize Eomes-expressing cells and their progeny, mouse lines harboring reporter genes in the endogenous Eomes locus were previously established, such as insertions of eGFP (Arnold et al., 2009) or the Cre-recombinase (Costello et al., 2011). These reporter alleles allow for studies of cells during the time of Eomes expression (EomesGFP) or for lineage tracing of progeny of Eomes-expressing cells by combining the EomesCre allele with Cre-inducible reporter alleles.

To study roles of Eomes-expressing cells and their progeny at defined developmental stages, we generated a novel Eomes allele by integrating the tamoxifen (Tx)-activatable version of Cre (CreERT2, hereafter simply referred to as CreER) (Feil et al., 1997) into the Eomes locus via homologous recombination in embryonic stem (ES) cells. We inserted the cDNA of Cre recombinase that is fused to a mutated ligand-binding domain of the human estrogen receptor (ER) into the translational start site of the endogenous Eomes locus, thereby deleting ~500 bp of the exon 1 coding region (Fig. 1a). For positive and negative ES cell selection, a loxP-flanked neomycin cassette and a thymidine kinase cassette (pMCI.TK) were integrated into the targeting vector (Fig. 1a). ES cell clones were screened by Southern blot (Fig. 1b) and correctly targeted clones were used for the generation of chimeras by morula aggregation. Following germline transmission, F1 progeny and subsequent generations were genotyped by polymerase chain reaction (PCR) resulting in a 327-bp band for the wild-type and a 202-bp band for the targeted allele (Fig. 1c). Heterozygous EomesCreER mice are viable, fertile, and show no obvious abnormalities.

FIG. 1.

FIG. 1

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Generation of the EomesCreER allele. (a) Strategy to introduce CreER into exon 1 of the Eomes locus by homologous recombination in ES cells. The CreER cassette was placed into the transcriptional start site of the endogenous Eomes locus, followed by a removable, LoxP-flanked neomycin selection cassette (PGK.neo). (b) Drug-resistant ES cell clones were screened by Southern blot of EcoRV-digested genomic DNA using a 3′ external probe [indicated as red bar in (a); wild-type allele (wt, 15 kb), targeted allele (T, 7 kb)]. (c) PCR genotyping distinguishes wild-type (wt, 327 bp) and CreER reporter allele (CreER, 202 bp). Genotyping primers are indicated as purple arrowheads in (a). E, EagI; H, HindIII; neo, neomycin-positive selection cassette; S, SphI; RV, EcoRV, TK, thymidine kinase negative selection marker.

To evaluate the novel EomesCreER allele, we first tested if CreER expression resembles the patterns of endogenous Eomes mRNA. In situ hybridizations were performed using probes for Eomes and CreER on E7.25 EomesCreER/+ whole-mount embryos and on E14.5 brain sections. Comparative expression analysis shows that CreER expression recapitulates the spatiotemporal distribution of endogenous Eomes transcription (Fig. 2). Thus, Eomes and CreER expression is found in the primitive streak and ExE of E7.25 embryos (Fig. 2a,b) and in cells of the SVZ of the developing cerebral cortex at E14.5 (Fig. 2c,d). We conclude that CreER expression faithfully recapitulates the patterns of Eomes transcription, suggesting that the insertion of cDNA for CreER and a polyA-signal into the transcriptional start site of exon 1 does not disturb transcriptional regulation from the locus. Similar to previously established Eomes reporter alleles (Arnold et al., 2009; Costello et al., 2011) the EomesCreER allele acts as a null allele leading to a heterozygous genetic background for Eomes. Accordingly, no homozygous EomesCreER/CreER embryos could be recovered from EomesCreER/+ intercrosses at E7.5 (n=43, data not shown) owing to requirements of Eomes function in the trophectoderm lineage (Russ et al., 2000; Strumpf et al., 2005). No obvious phenotypic alterations indicative for haploinsufficiency could be observed in heterozygous EomesCreER/+ embryos and adult animals.

FIG. 2.

FIG. 2

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CreER mRNA expression recapitulates the endogenous Eomes transcription pattern. (a and b) Whole-mount in situ hybridization of E7.25 EomesCreER/+ embryos using probes for (a) Eomes and (b) CreER. Expression of Eomes and CreER is similarly found at the primitive streak (asterisk) and in the ExE (arrow). (c and d) In situ hybridization of coronal sections from the cerebral cortex of an E14.5 EomesCreER/+ embryo. Note the almost identical distribution of CreER and endogenous Eomes mRNAs in (a and b) the E7.25 embryo and in (c and d) cells of the embryonic ventricular (VZ) and subventricular zone (SVZ). Scale bar: 200 μm.

To test for recombination activity of the Tx-activated CreER during gastrulation stages, EomesCreER/+ mice were first crossed with animals carrying a Creactivatable mGFP-reporter allele (Muzumdar et al., 2007). Pregnant females were intraperitoneally (i.p.) injected with either a single dose of Tx at E5.5 or injected on two consecutive days at E5.5 and E6.5. Treatment before gastrulation onset induces reporter expression in a few cells in the ExE and rarely in cells of the primitive streak when embryos are analyzed at E6.5 (Fig. 3a). Dual injection at E5.5 and E6.5 results in mGFP expression in the ExE and in cells of the primitive streak (Fig. 3b). In a second approach, EomesCreER/+ males were crossed with females carrying the Rosa26 LacZ-reporter allele (R26R) (Soriano, 1999) and pregnant females injected with Tx at E6.5 or twice at E6.5 and E7.5. At E8.5 resulting embryos show LacZ-positive cells in the developing heart, the aorta, the primitive gut tube, and in the visceral yolk sac after single injections (Fig. 3c – f). Dual application of Tx at E6.5 and E7.5 significantly increases the number of labeled cells, indicating the dependancy on concentration and timing of Tx administration for activation of the CreER recombinase (Fig. 3g – j). Thus, the EomesCreER allele can be used to follow Eomes-expressing cells during gastrulation at the level of single cells in a close-to-clonal analysis. Alternatively, higher numbers of Eomes-expressing cells can be marked using repeated, high-dose injections of Tx.

FIG. 3.

FIG. 3

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Reporter activation by EomesCreER at gastrulation stages. (a and b) Females carrying the Cre-inducible mGFP reporter allele were mated to EomesCreER/+ males and pregnant females received Tx at (a) E5.5 and (b) E5.5 plus E6.5. Resulting embryos were analyzed at E6.5 and E7.5 by cryosections for mGFP expression and DAPI counterstaining. mGFP labeling is found in cells of the ExE (red arrowhead) and in the primitive streak (asterisk). Dual application at E5.5 and E6.5 resulted in increased number of labeled cells. (c–j) EomesCreER/+ males were crossed with females carrying the R26R LacZ-reporter allele and pregnant females received Tx at (c–f) E6.5 and (g–j) E6.5 plus E7.5. Embryos were recovered at E8.5, stained for LacZ expression, and used for histological analysis at the indicated plane of section. (c–f) Single-dose injections of Tx at E6.5 leads to reporter activation in a limited number of cells in the visceral endoderm (c, blue arrow), in the primary gut tube (e, white arrowhead), the aorta (e, asterisk), and in the developing myocardium (f, black arrowhead). (g–j) Dual injections at E6.5 and E7.5 result in significantly increased recombination activity in a larger number of cells. LacZ-reporter activity is present in the visceral yolk sac (h, arrow), in the myocardium (j, black arrowhead), in head mesenchyme (j, grey arrowhead), and gut endoderm (j, white arrowhead). a, anterior; p, posterior; r, right side; l, left side. Scale bar: 200 μm.

During development of the brain, Eomes is expressed dynamically in neurogenic regions, such as the embryonic cerebral cortex and cerebellum (Arnold et al., 2008b; Englund et al., 2005; Hodge et al., 2008). Additionally, Eomes is also found in the neurogenic niches of the adult brain including the olfactory bulb and the adult dentate gyrus (Hevner et al., 2006; Hodge et al., 2008). To test for recombination in neurogenic brain regions during development we first crossed EomesCreER/+ animals to R26R LacZ-reporter mice and injected Tx on days E14–E16. Dorsal views of resulting whole-mount brains at E17 show activation of the LacZ-reporter gene in the hemispheres of EomesCreER-positive brains, but not in control brains (Fig. 4a,c). Coronal sections reveal LacZ-positive cells that range from the SVZ toward the cortical plate in the hemisphere of EomesCreER/+ embryos, but not in sections of control brains (Fig. 4b,b′,d,d′).

FIG. 4.

FIG. 4

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Detection of EomesCreER-mediated cell labeling in the developing and adult brain. (a and c) EomesCreER/+; R26R, and control embryos were induced by repeated Tx injections of the pregnant mother from E14 to E16 and analyzed for reporter activation in the brain by LacZ staining at E17. Dorsal views of control (a) and EomesCreER/+ (c) brains and corresponding coronal sections (b, b′d, and d′) dem-onstrate EomesCreER-induced reporter-activation accurately labeling cells in the embryonic subventricular zone (SVZ) and their progeny in the cortical plate (CP). (e–i′) Analysis of induction of the Ai14 reporter that produces the red fluorescent protein, tdTomato, following EomesCreER-mediated recombination in developing and adult brains. (e) Sagittal section of a neonatal (P0.5) mouse brain treated with Tx on E14 showing tdTomato expression in the cerebral cortex (Ctx) and associated axon pathways, and in cerebellum (Cb). (e′) Within the cortex, tdTomato-positive cells are seen in layer 3 (L3) but not L2 or marginal zone (MZ), consistent with differentiation of Eomes-expressing intermediate progenitor cells into cortical projection neurons. (f) In the cerebellar vermis, tdTomato is detected in cells migrating through the cer-ebellar white matter, consistent with unipolar brush cells (UBCs). (g and g′) In the adult hippocampus tdTomato-expressing cells can be observed in the subgranular layer (SGL) of the dentate gyrus (DG) 2 days after Tx treatment. Double labeling with tdTomato and DCX (green), a marker of intermediate progenitors and new neurons, shows partially overlapping expression. (h, i, and i′) Cell labeling in the adult cerebellum (h) and olfactory bulb (i and i′ ) 5 months after Tx treatment. TdTomato-expressing cells are abundant in folia IX and X of the cere-bellum, matching the characteristic distribution of unipolar brush cells. In the olfactory bulb, reporter-expressing cells are seen in the peri-glomerular layer. (i′) Double labeling using an Eomes-specific antibody and tdTomato expression demonstrates that in the olfactory bulb all reporter-positive cells (red) show Eomes protein (green), but only a subset of Eomes-expressing cells are marked with tdTomato. Eomes-, tdTomato-double positive cells are indicated with arrowheads in (i′).

In a second set of experiments, EomesCreER/+ mice were bred with Ai14-reporter mice, in which the red-fluorescent tdTomato reporter is expressed after Cremediated recombination (Madisen et al., 2012). Pregnant females were injected at E14 and offspring analyzed at postnatal day 0.5 (P0.5). Red-fluorescent tdTomato can be detected in middle to upper layer neurons of the cerebral cortex and in migrating unipolar brush cells of the cerebellum (Fig. 4e,e′,f). When adult animals were treated with Tx, neurogenic progenitor cells and resulting neurons are correctly labeled in the neurogenic niche of the dentate gyrus, in the cerebellum, and in the olfactory bulb (Fig. 4g,g′,h,i). Double immunofluorescence staining to detect tdTomato and Eomes protein shows that dtTomato is expressed in many but not all Eomes-positive cells, e.g., in the olfactory bulb (Fig. 4i′). Thus, administration of a single dose of Tx induces mosaic recombination by EomesCreER in a subset of Eomes-positive cells but does not lead to ectopic recombination. We estimate that recombination efficiencies in the developing cortex reach 60–80% of Eomes-expressing cells, whereas recombination rates are considerably lower during earlier stages of development, possibly reflecting reduced bioavailability of injected Tx during early pregnancies.

Next, we wanted to test whether the CreER allele also functions in Eomes-expressing cells of the innate immune system. Splenic NK cells are innate lymphocytes that can be identified based on the presence of the cell surface receptors NKp46 and CD49b. The liver harbors an additional NK cell population that expresses the death-ligand TRAIL but not CD49b. Previous studies suggested that Eomes is only expressed in CD49b+ NK cells but not in TRAIL+ NK cells (Gordon et al., 2012). To test if the EomesCreER allele is able to specifically label CD49b+ NK cells we fed female mice carrying the EomesCreER allele and a Cre-inducible GFP-reporter allele (Muzumdar et al., 2007) with Tx-containing food and analyzed splenic and hepatic NK cells by fluorescence activated cell sorting (FACS). In agreement with previous findings, only CD49b+ NK cells in spleen and liver show GFP expression after EomesCreER activation, whereas TRAIL+ NK cells of the liver do not exhibit GFP-reporter expression (Fig. 5). Thus, EomesCreER marks and distinguishes different NK cell lineages after administration of Tx in adult mice.

FIG. 5.

FIG. 5

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Labeling of NK cell subpopulations by the EomesCreER allele. EomesCreER/+ female mice carrying a Cre-inducible GFP reporter were fed with Tx-enriched food for 21 days and different NKp46+ NK cell populations from spleen and liver were analyzed by flow cytome-try for GFP-reporter expression. One representative result demonstrates that reporter activation by EomesCreER is only detected in Eomes+ CD49b+ NK cells, but not in Eomes− TRAIL+ NK cells.

In summary, we present a novel EomesCreER allele that allows for Tx-inducible activation of the Cre-recombinase in Eomes-expressing stem and progenitor cells during development and in adulthood. This genetic tool will be instrumental to resolve some of the eminent questions regarding the cellular fate and mechanisms of lineage decisions of Eomes-expressing progenitors. For example, limited-number cell labeling in combination with time-lapse imaging of early gastrulation-stage embryos will enable to delineate the origin and emergence of cardiac mesoderm and definitive endoderm. Additionally, this allele will allow to further resolve neurogenesis from embryonic and adult IPCs, e.g., with respect to temporal differences in neuronal subtype specification and contribution to the different layers of the embryonic cerebral cortex. In combination with Cre-inducible alleles that impact on cellular signaling or behavior, this allele will further enable studies toward mechanisms of lineage specification and lineage-specific behavior in descendants of Eomes-expressing progenitors. In conclusion, this EomesCreER allele will be a valuable tool for future genetic studies in different areas of progenitor cell research.

MATERIALS AND METHODS

Generation of the EomesCreER-Targeted Allele

The targeting vector for the generation of an EomesCreER-targeted allele comprises an 8.25-kb HpaI-HpaI fragment of the Eomes locus. A CreERT2.pA cassette and a LoxP-flanked neomycin resistance cassette were integrated between the SphI site at the translational start site and an EagI site, thereby deleting ~500 bp of the coding region of exon 1. The 3′ homology region of the targeting construct was flanked with a pMCI.TK negative selection cassette. Linearized targeting vector was electroporated into CCE ES cells, and neomycin and FIAU-resistant ES cell colonies were screened by Southern blot using a 3′ external probe on EcoRV-digested DNA (wt allele: 15 kb and targeted allele: 7 kb). Generation of chimeras was performed by morula aggregation. F1 progeny and subsequent generations were genotyped by multiplex PCR at 60° C annealing temperature to detect wild-type allele (wt, 327 bp) and CreER allele (202 bp) using the following primers: forward primer 5′-GAGGGAGGAAGGGGACATTA-3′; reverse primer1 5′-CAGGTTCTTGCGAACCTCAT-3′; reverse primer2 5′-CAGGTTCTTGCGAACCTCAT-3′. Animals carrying the EomesCreER allele are available to the research community and can be requested from the corresponding author.

Animals

R26R-reporter (Soriano, 1999), Ai14-reporter (Madisen et al., 2012), and mGFP-reporter mice (Muzumdar et al., 2007) were described previously. Tamoxifen (Tx) (Sigma-Aldrich, Taufenkirchen, Germany, T5648; 20 mg/ml) and progesterone (Sigma-Aldrich P0130; 10 mg/ ml) were diluted in corn oil (Sigma-Aldrich C8267). For CreER induction at E5.5, E6.5, and E7.5 pregnant females were injected i.p. with 4 mg of Tx and 2 mg of progesterone per injection. For analysis of embryonic brains, pregnant females received repeated i.p. injections of 1 mg of Tx/0.5 mg of progesterone at E14, E15, and E16. Adult mice (age 8 weeks) received a single dose of Tx (180 mg/kg, i.p.) followed by analysis after 2 days or 5 months. For labeling of NK cells, adult female mice were fed with Tx-enriched food (TAM400, Harlan Laboratories, Horst, Netherlands) for 21 days and ana-lyzed thereafter.

Histological Analysis

In situ hybridization on whole-mount embryos and paraffin sections was performed according to standard protocols (Nagy et al., 2003) using Eomes- and CreER-specific probes. LacZ staining was carried out as described (Nagy et al., 2003). For histological analysis of LacZ-stained embryos and brains, specimens were postfixed in 4% paraformaldehyd/phosphate-buffered saline, dehydrated through ethanol series, and embedded in paraffin before sectioning at 8 μm. Cryostat sections of embryos and Eosin counterstaining was performed according to standard protocols (Nagy et al., 2003).

For detection of red fluorescent protein (RFP)-reporter expression, animals were perfused and studied by detection of native RFP fluorescence or immunofluo-rescence to detect RFP (Clontech 632496, Mountain View, California, 1:1,000) in combination with DCX or Eomes/Tbr2 as described (Hodge et al., 2008). Images were collected on a Zeiss (Jena, Germany) Axiovision or Zeiss LSM710 microscope and processed for brightness and contrast using Photoshop.

Flow Cytometry

Spleen and liver lymphocytes were isolated as previously described (Gordon et al., 2012), followed by antibody staining as described (Klose et al., 2013). In brief, single-cell suspensions of purified cells were kept on ice and Fc receptors were blocked using CD16/CD32 antibody. NK cells in spleen and liver were identified as lineage-negative (CD4, CD8a, CD19, and Gr-1) CD45+ NKp46+ cells, which were further divided based on the expression of CD49b and TRAIL. All antibodies were purchased from eBioscience (Frankfurt, Germany). FACS data were acquired on a LSR Fortessa (BD Biosciences, Heidelberg, Germany) and analyzed using FlowJo software (TreeStar, Ashland, Oregon).

ACKNOWLEDGMENTS

The authors thank Rebecca Hodge and Yannick d’Hargues for experimental assistance, Dani Klewe-Nebenius at the Biozentrum Basel for valuable advice during generation of targeted ES cells and chimeric animals, and Mihael Pavlovic for excellent technical support and animal husbandry.

Contract grant sponsor: German Research Council (DFG, Emmy Noether Program); Contract grant numbers: AR 732/1-1, SFB850 project A3, TA 436/2-1

LITERATURE CITED

  1. Arnold SJ, Hofmann UK, Bikoff EK, Robertson EJ. Pivotal roles for eomesodermin during axis formation, epithelium-to-mesenchyme transition and endoderm specification in the mouse. Development. 2008a;135:501–511. doi: 10.1242/dev.014357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold SJ, Huang GJ, Cheung AF, Era T, Nishikawa S, Bikoff EK, Molnar Z, Robertson EJ, Groszer M. The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone. Genes Dev. 2008b;22:2479–2484. doi: 10.1101/gad.475408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arnold SJ, Sugnaseelan J, Groszer M, Srinivas S, Robertson EJ. Generation and analysis of a mouse line harboring GFP in the Eomes/Tbr2 locus. Genesis. 2009;47:775–781. doi: 10.1002/dvg.20562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baala L, Briault S, Etchevers HC, Laumonnier F, Natiq A, Amiel J, Boddaert N, Picard C, Sbiti A, Asermouh A, Attie-Bitach T, Encha-Razavi F, Munnich A, Sefiani A, Lyonnet S. Homozygous silencing of T-box transcription factor EOMES leads to microcephaly with polymicrogyria and corpus callosum agenesis. Nat Genet. 2007;39:454–456. doi: 10.1038/ng1993. [DOI] [PubMed] [Google Scholar]
  5. Banerjee A, Gordon SM, Intlekofer AM, Paley MA, Mooney EC, Lindsten T, Wherry EJ, Reiner SL. Cutting edge: The transcription factor eomesodermin enables CD8+ T cells to compete for the memory cell niche. J Immunol. 2010;185:4988–4992. doi: 10.4049/jimmunol.1002042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brill MS, Ninkovic J, Winpenny E, Hodge RD, Ozen I, Yang R, Lepier A, Gascon S, Erdelyi F, Szabo G, Parras C, Guillemot F, Frotscher M, Berninger B, Hevner RF, Raineteau O, Gotz M. Adult generation of glutamatergic olfactory bulb interneurons. Nat Neurosci. 2009;12:1524–1533. doi: 10.1038/nn.2416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bulfone A, Martinez S, Marigo V, Campanella M, Basile A, Quaderi N, Gattuso C, Rubenstein JL, Ballabio A. Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development. Mech Dev. 1999;84:133–138. doi: 10.1016/s0925-4773(99)00053-2. [DOI] [PubMed] [Google Scholar]
  8. Ciruna BG, Rossant J. Expression of the T-box gene Eomesodermin during early mouse development. Mech Dev. 1999;81:199–203. doi: 10.1016/s0925-4773(98)00243-3. [DOI] [PubMed] [Google Scholar]
  9. Costello I, Pimeisl IM, Drager S, Bikoff EK, Robertson EJ, Arnold SJ. The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation. Nat Cell Biol. 2011;13:1084–1091. doi: 10.1038/ncb2304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elsen GE, Hodge RD, Bedogni F, Daza RA, Nelson BR, Shiba N, Reiner SL, Hevner RF. The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map. Proc Natl Acad Sci USA. 2013;110:4081–4086. doi: 10.1073/pnas.1209076110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A, Kowalczyk T, Hevner RF. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J Neurosci. 2005;25:247–251. doi: 10.1523/JNEUROSCI.2899-04.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feil R, Wagner J, Metzger D, Chambon P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun. 1997;237:752–757. doi: 10.1006/bbrc.1997.7124. [DOI] [PubMed] [Google Scholar]
  13. Gordon SM, Carty SA, Kim JS, Zou T, Smith-Garvin J, Alonzo ES, Haimm E, Sant’Angelo DB, Koretzky GA, Reiner SL, Jordan MS. Requirements for eomesodermin and promyelocytic leukemia zinc finger in the development of innate-like CD8+ T cells. J Immunol. 2011;186:4573–4578. doi: 10.4049/jimmunol.1100037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gordon SM, Chaix J, Rupp LJ, Wu J, Madera S, Sun JC, Lindsten T, Reiner SL. The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation. Immunity. 2012;36:55–67. doi: 10.1016/j.immuni.2011.11.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hevner RF, Hodge RD, Daza RA, Englund C. Transcription factors in glutamatergic neurogenesis: Conserved programs in neocortex, cerebellum, and adult hippocampus. Neurosci Res. 2006;55:223–233. doi: 10.1016/j.neures.2006.03.004. [DOI] [PubMed] [Google Scholar]
  16. Hodge RD, Kowalczyk TD, Wolf SA, Encinas JM, Rippey C, Enikolopov G, Kempermann G, Hevner RF. Intermediate progenitors in adult hippocampal neurogenesis: Tbr2 expression and coordinate regulation of neuronal output. J Neurosci. 2008;28:3707–3717. doi: 10.1523/JNEUROSCI.4280-07.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hodge RD, Nelson BR, Kahoud RJ, Yang R, Mussar KE, Reiner SL, Hevner RF. Tbr2 is essential for hippocampal lineage progression from neural stem cells to intermediate progenitors and neurons. J Neurosci. 2012;32:6275–6287. doi: 10.1523/JNEUROSCI.0532-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Intlekofer AM, Takemoto N, Wherry EJ, Longworth SA, Northrup JT, Palanivel VR, Mullen AC, Gasink CR, Kaech SM, Miller JD, Gapin L, Ryan K, Russ AP, Lindsten T, Orange JS, Goldrath AW, Ahmed R, Reiner SL. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol. 2005;6:1236–1244. doi: 10.1038/ni1268. [DOI] [PubMed] [Google Scholar]
  19. Klose CS, Kiss EA, Schwierzeck V, Ebert K, Hoyler T, d’Hargues Y, Goppert N, Croxford AL, Waisman A, Tanriver Y, Diefenbach A. A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. Nature. 2013;494:261–265. doi: 10.1038/nature11813. [DOI] [PubMed] [Google Scholar]
  20. Madisen L, Mao T, Koch H, Zhuo JM, Berenyi A, Fujisawa S, Hsu YW, Garcia AJIII, Gu X, Zanella S, Kidney J, Gu H, Mao Y, Hooks BM, Boyden ES, Buzsaki G, Ramirez JM, Jones AR, Svoboda K, Han X, Turner EE, Zeng H. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat Neurosci. 2012;15:793–802. doi: 10.1038/nn.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L. A global double-fluorescent Cre reporter mouse. Genesis. 2007;45:593–605. doi: 10.1002/dvg.20335. [DOI] [PubMed] [Google Scholar]
  22. Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the mouse embryo: A laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2003. [Google Scholar]
  23. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE. T-box genes in vertebrate development. Annu Rev Genet. 2005;39:219–239. doi: 10.1146/annurev.genet.39.073003.105925. [DOI] [PubMed] [Google Scholar]
  24. Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi R, Rossant J. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell. 2005;123:917–929. doi: 10.1016/j.cell.2005.08.040. [DOI] [PubMed] [Google Scholar]
  25. Pearce EL, Mullen AC, Martins GA, Krawczyk CM, Hutchins AS, Zediak VP, Banica M, DiCioccio CB, Gross DA, Mao CA, Shen H, Cereb N, Yang SY, Lindsten T, Rossant J, Hunter CA, Reiner SL. Control of effector CD8+ T cell function by the transcription factor Eomesodermin. Science. 2003;302:1041–1043. doi: 10.1126/science.1090148. [DOI] [PubMed] [Google Scholar]
  26. Russ AP, Wattler S, Colledge WH, Aparicio SA, Carlton MB, Pearce JJ, Barton SC, Surani MA, Ryan K, Nehls MC, Wilson V, Evans MJ. Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature. 2000;404:95–99. doi: 10.1038/35003601. [DOI] [PubMed] [Google Scholar]
  27. Sessa A, Mao CA, Hadjantonakis AK, Klein WH, Broccoli V. Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex. Neuron. 2008;60:56–69. doi: 10.1016/j.neuron.2008.09.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71. doi: 10.1038/5007. [DOI] [PubMed] [Google Scholar]
  29. Strumpf D, Mao CA, Yamanaka Y, Ralston A, Chawengsaksophak K, Beck F, Rossant J. Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development. 2005;132:2093–2102. doi: 10.1242/dev.01801. [DOI] [PubMed] [Google Scholar]
  30. Tayade C, Fang Y, Black GP, VAP, Erlebacher A, Croy BA. Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells. J Leukoc Biol. 2005;78:1347–1355. doi: 10.1189/jlb.0305142. [DOI] [PubMed] [Google Scholar]
  31. Teo AK, Arnold SJ, Trotter MW, Brown S, Ang LT, Chng Z, Robertson EJ, Dunn NR, Vallier L. Pluripotency factors regulate definitive endoderm specification through eomesodermin. Genes Dev. 2011;25:238–250. doi: 10.1101/gad.607311. [DOI] [PMC free article] [PubMed] [Google Scholar]

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