A loss-of-function and H2B-Venus transcriptional reporter allele for Gata6 in mice

The GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs. We have generated a Gata6H2B-Venus knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution. Expression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6H2B-Venus/H2B-Venus homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.

Background GATA-binding factor 6 (GATA6) is a member of the GATA family of zinc finger transcription factors that are characterized by their DNA binding domain [1]. GATA factors are highly conserved across vertebrate species, which include six members of the family [2,3]. They are also evolutionarily conserved among invertebrates (D. melanogaster and C. elegans) where they participate in heart and endoderm formation [4]. In humans, de novo mutations in the Gata6 locus cause haploinsufficiency that is associated with congenital heart malformations and neonatal diabetes due to pancreatic agenesis [5][6][7][8][9][10].
During pre-implantation embryo development in mice, Gata6 is required for formation of extra-embryonic tissues [11][12][13][14]. In the early mouse blastocyst (32-64 cell stage), GATA6 protein is uniformly expressed in the inner cell mass (ICM) and trophectoderm (TE) [12,15]. By the mid-blastocyst (64-100 cell) stage, expression of GATA6 in the ICM becomes restricted in a mosaic 'salt-and-pepper' pattern [15,16] and co-localizes with GATA4 [15,17]. At this stage, exclusive enrichment of GATA6 serves as the earliest known determinant of the primitive endoderm (PrE) lineage, which is the precursor to the parietal endoderm (ParE) and visceral endoderm (VE) [18][19][20]. GATA6 is necessary for PrE specification in the mouse embryo, and either GATA6 or GATA4 are sufficient to promote differentiation into extra-embryonic endoderm from embryonic stem (ES) cells [13,14,21,22]. Gata6 +/− heterozygotes have delayed PrE specification and a reduction in the number of cells that adopt a PrE fate at the late blastocyst stage [13,14].
Following implantation, strong Gata6 expression continues in extra-embryonic tissues; namely the ParE which deposits Reichert's membrane that lines the parietal yolk sac, and the allantois which will contribute blood vessels to the umbilical cord [18]. Weaker expression of Gata6 mRNA can be observed in the VE that gives rise to the visceral yolk sac and a fraction of the embryonic gut endoderm, although GATA6 protein levels in the VE are diminished by gastrulation stages [18,19,23,24]. Tetraploid embryo complementation, where only the embryonic tissue was null for Gata6, demonstrated that GATA6 is indispensible for embryonic liver development [25]. Heart development in contrast does not require embryonic expression of Gata6, likely due to compensation by Gata4, which shares 90 % amino acid sequence homology with the DNA-binding domain of GATA6 [26] and is also expressed in the myocardium [25]. When both factors are conditionally deleted, cardiac progenitors are specified even though the heart does not form [27]. Gata6 +/− ;Gata4 +/− compound heterozygotes die by E13.5 with cardiovascular anomalies [28].
After gastrulation, Gata6 is expressed in the cardiac crescent at the headfold stage (E7.75), as well as in the lateral plate mesoderm, primary and secondary heart fields, and heart tube [18,27]. By E9.5, Gata6 expression is restricted to the heart myocardium and gut endoderm where it persists throughout development [12,18]. Later onset of Gata6 expression during development is observed in arterial smooth muscle cells, the bladder, lung bronchi, and the urogenital ridge; none of which coexpress Gata4 [18]. However, both Gata6 and Gata4 are expressed throughout the pancreatic epithelium during early specification and expansion. Then, later in development their expression domains become mutually exclusive with Gata6 restricted to cells of the endocrine pancreas [29,30]. When either factor alone is conditionally ablated in the pancreas, only mild and nonpersisting defects are observed. However, tissue-specific deletion of both GATA6 and GATA4 factors results in pancreatic agenesis [31].
In adult organs, Gata6 expression continues in the heart, lung, stomach, small intestine, liver, bladder, pancreas, adrenal glands, ovaries, and skin [18,[32][33][34][35][36]. Developmental expression of GATA6 is extensive in the intestinal epithelium, but later becomes exclusive to the enteroendocrine lineage of adults [37,38]. GATA6 is also the only GATA family member that is expressed in adult vascular smooth muscle cells [32,39]. Misregulation of GATA6 has been linked to various tumor expression profiles. Loss of GATA6 is common in ovarian cancer and may lead to dedifferentiation of ovarian epithelial cancer cells and increased occurrence of aneuploidy [40]. Reduced GATA6 activity may directly impact metastatic progression of lung adenocarcinoma [41], while overexpression of GATA6 is associated with poor prognosis in esophageal adenocarcinoma [42]. In colorectal cancer, high levels of GATA6 predict the likelihood of metastasis to the liver [43], and overexpression may promote survival of oncogenic cells in gastric cancer [44]. GATA6 is also a useful marker of pediatric germ cell tumors [35].
Given the importance of Gata6 as a key regulatory factor during development as well as in particular adult organs, it would be useful to have a method of identifying and tracing the fate of Gata6 expressing cells. While other Gata6 transcriptional LacZ reporters exist [12,45], a nuclear-localized fluorescent reporter instead would be suitable for live imaging and cell sorting. In this report, we describe a new Gata6 H2B-Venus knock-in mouse line that acts as both a loss-of-function and transcriptional reporter allele. In mice, the bright nuclear-localized H2B-Venus yellow fluorescent protein reporter correlates well with endogenous GATA6 protein and recapitulates tissuespecific expression patterns from pre-implantation stages of embryonic development to adulthood. Gata6 H2B-Venus will be a useful reporter for live imaging the dynamics of transcriptional activation in individual cells that are expressing, or recently have expressed Gata6. It could also be used for studies and analysis of Gata6 expression in endoderm and mesoderm lineages in mice, including the isolation of these cell populations. Furthermore, expression of Gata6 H2B-Venus could be utilized to assess misregulation of the locus that may occur at the onset and/or as a consequence of disease states such as cancer.

Results and discussion
Generation of Gata6 H2B-Venus knock-in reporter mice An enhancer element located −8 kb from the Gata6 transcriptional start site is sufficient to drive expression of GATA6 in the heart and gut [46][47][48]. To obtain a reporter that recapitulates the full spectrum of Gata6 transcriptional control, we targeted the endogenous Gata6 locus by modification of a EUCOMM knockoutfirst construct [49,50]. Specifically, we targeted H2B-Venus to the first non-coding intron of the mouse Gata6 gene, upstream of two alternative translation initiation codons located 438 bp apart from one another within Exon 2 (E2, Fig. 1a) [48]. Targeted Gata6 H2B-Venus/+ ES cells were injected into mouse blastocysts to generate chimeric mice.
Expression of Venus was examined in ES cells, which normally do not express GATA6. Accordingly, the Venus reporter was not detected in pluripotent Gata6 H2B-Venus/+ ES cells (Fig. 1b). To determine if Gata6 H2B-Venus/+ performs faithfully as a reporter in cells, we directed the differentiation of ES cells into endoderm, which expresses Gata6, using three different methods. First, Gata6 H2B-Venus/+ ES cells were cultured under conditions to promote formation of embryoid bodies, which are suspended aggregates of cells capable of differentiating into all three germ lineages. Embryoid bodies did not initially express Venus, however Venus + cells were observed by Day 8 in both endoderm cells located on the surface of the bodies as well as in mesoderm progenitors located inside (Fig. 1c). Using growth factors, we also differentiated Gata6 H2B-Venus/+ ES cells into definitive endoderm. Upon treatment with Activin A, GSK3 inhibitor, and the BMP inhibitor Dorsomorphin [51], activation of Venus was seen in Gata6 H2B-Venus/+ cells starting at Day 3 and increasing in frequency up to Day 5 (Fig. 1d). Finally, Gata6 H2B-Venus/+ ES cells were directed to differentiate towards a PrE fate by transiently overexpressing GATA4-mCherry, making use of a single-copy Tet-ON system for inducible gene expression formed by ColA1 TetO-Gata4-mCherry/+ and R26 M2rtTA/+ alleles present in the background of reporter ES cells [50,52,53]. Upon treatment with Doxycycline for 2 days, induction of GATA4-mCherry was sufficient to activate expression of Venus in Gata6 H2B-Venus/+ ;ColA1 TetO-Gata4-mCherry/ + ;R26 M2rtTA/+ ES cells (Fig. 1e, [50]).
Expression of H2B-Venus reporter is restricted to primitive endoderm To assess Venus expression in live embryos, Gata6 H2B-Venus/+ blastocysts were collected at E3.5 and imaged over the course of 17 h by laser scanning confocal microscopy. At these stages, Venus was expressed in the ICM and TE at levels that were bright enough for live time-lapse imaging (Fig. 2a). Furthermore, differential levels of nuclear Venus signal within the ICM were detected. To assess differential expression of Venus in each blastocyst lineage, Gata6 H2B-Venus/+ blastocysts were fixed at E3.5 (n = 4 embryos) and E4.5 (n = 3 embryos) and stained for endogenous GATA6 (Fig. 2b). Overall, cells that expressed Venus also expressed GATA6, mainly in the PrE. However, Venus was present in GATA6-negative cells in the ICM, possibly due to translational repression mediated by sequences present in the wild-type Gata6 mRNA but not the reporter mRNA, or as a consequence of the longer half-life of the H2B-Venus reporter compared to GATA6 protein. For example, Notch signaling reporter mice also express an H2B-Venus reporter and, in these mice, the perdurance of the reporter protein acts as a short-term lineage tracer of cells receptive to Notch signaling [54].
Perdurance of Venus may explain low-level expression that continues in the epiblast (Epi) that may be prohibitive for its use as a PrE-only reporter in the blastocyst, but could remain useful as a short-term lineage tracer. Confocal z-stacks were segmented using the nuclear segmentation algorithm MINS (Modular Interactive Nuclear Segmentation) to quantify fluorescence intensity in single cells. PrE cells (GATA6 positive) displayed significantly higher levels of Venus than Epi cells at both blastocyst stages (p < 0.01, Fig. 2c). Furthermore, we observed a highly significant correlation between the level of GATA6 and Venus protein levels in PrE cells at both E3.5 (r = 0.6581, p < 0.001) and E4.5 (r = 0.3257, p < 0.001; Fig. 2d). At E3.5, we also observed a weak, although significant correlation (r = 0.2512, p < 0.05) between the levels of GATA6 and Venus in Epi cells (Fig. 2d). The absence of correlation in Epi cells at E4.5 (r = −0.1793, p = 0.957; Fig. 2d) suggests that the correlation at E3.5 may be due to residual GATA6 protein found in Epi cells at that stage.

Gata6 H2B-Venus is a loss-of-function allele
To confirm that Gata6 H2B-Venus is a loss-of-function allele for endogenous GATA6, Gata6 H2B-Venus/H2B-Venus blastocysts were immunostained for expression of GATA6 (labels the PrE) and NANOG (labels the Epi) at E3.5 (Fig. 3a). At this time, GATA6 and NANOG expression begin to resolve into a mutually exclusive 'saltand-pepper' expression pattern defining PrE and Epi precursors respectively. This occurs prior to lineage segregation in which PrE cells are sorted to the surface while Epi cells remain in the ICM. Gata6 H2B-Venus/H2B-Venus homozygous blastocysts did not express GATA6 protein and instead expressed NANOG in all ICM cells which fail to specify PrE, similar to what was observed in mutants made with other Gata6 null alleles (Fig. 3a, [13,14]. Venus continued to be robustly expressed in the ICM in the absence of GATA6 protein, possibly due to the presence of transcriptional machinery that normally activates the Gata6 locus despite the inability to produce GATA6 protein. Alternatively, it could mean that downstream factors that typically repress the Gata6 locus are dependent on GATA6 protein. Again, MINS was employed on confocal z-stacks to quantify fluorescence intensity in single cells. As expected, Gata6 H2B-Venus/+ heterozygous blastocysts had a reduced number of cells that adopted a PrE fate (Fig. 3b) [13]. The levels of Venus in Gata6 H2B-Venus/H2B-Venus homozygous A weak correlation between Venus and GATA6 levels was observed in the EPI of E3.5 but not E4.5 embryos. Pearson's correlation coefficient and p values are shown in the graphs next to the corresponding group. ** = p < 0.01 Scale bars = 20 μm embryos were higher compared to Gata6 H2B-Venus/+ heterozygous embryos (Fig. 3b). This could be due to biallelic expression of the reporter in homozygous embryos.
Gata6 H2B-Venus/+ embryos were morphologically indistinguishable from wild-type littermates except for their fluorescence. In wild-type embryos, GATA6 protein was expressed throughout the extraembryonic VE (exVE) and embryonic VE (emVE) at E5.5. Venus was also detected in the VE at E5.5, and optical sectioning confirmed that the reporter was not active in the Epi or extra-embryonic ectoderm (Fig. 4a). At E6.0, prior to formation of the primitive streak that is (referred to as pre-streak), expression of Venus continued in throughout the VE. Co-expression of GATA6 and Venus was observed in the emVE on the surface of the embryo, but neither were detected in the Epi or extra-embryonic ectoderm (Fig. 4b). However Venus was not activated in all GATA6+ cells of the VE, resulting in a mosaic pattern of Venus expression in the exVE (Fig. 4a) that was also evident at later stages (Figs. 4b, an Fig. 5a). One possible explanation for this observation could be that the Gata6 locus is subject to mono-allelic expression in certain tissues.

Activity of Gata6 H2B-Venus during gastrulation
Next, expression of Venus was characterized during gastrulation stages. Gastrulation begins at E6.25 when Epi cells in the proximal posterior portion of the embryo form the primitive streak (PS). The PS is a region characterized by an epithelial-to-mesenchymal transition whereby ingression of mesoderm and endoderm progenitors results in their migration anterolaterally to populate the space in between two apposed epithelia, the Epi and VE. At the early-streak (ES, E6.75) stage, Venus was observed in the ParE (Reichert's membrane) upon bisection of decidua that contained Gata6 H2B-Venus/+ embryos ( Fig. 6a-b). Expression of Venus was mosaic in the VE at E6.75 and mid-streak (MS, E7.0) stages of Gata6 H2B-Venus/+ embryos, and did not always correlate with expression of endogenous GATA6 (Fig. 5a). It is possible that expression of Gata6 mRNA and protein are dynamically changing in the VE at these stages. It is also possible that there are differences in translation regulation of Gata6 mRNA compared to the reporter mRNA which lacks the wild-type 3′UTR.
Sections through the PS region at E7.0 revealed Venus and GATA6 expression in cells that had left the Epi epithelium (Fig. 5a). These cells may represent the earliest cardiac mesoderm progenitors that will populate the primary and secondary heart fields. To visualize movement of ingressed cells in vitro, we performed PS explants that were cultured and time-lapse imaged. The posterior portion of the Epi was dissected from E6.5 embryos, treated with pancreatin/trypsin enzymatic digestion to remove the endoderm and wings of mesoderm, and then Fig. 3 Gata6 H2B-Venus/H2B-Venus homozygous blastocysts exhibit a Gata6 null phenotype. a Immunofluorescence for GATA6 and NANOG protein in Gata6 +/+ (+/+; wild-type) Gata6 H2B-Venus/+ (Venus/+; heterozygous) and Gata6 H2B-Venus/H2B-Venus (Venus/Venus; homozygous) blastocysts at E3.5. Gata6 H2B-Venus/H2B-Venus mutants did not express GATA6 protein, and instead expressed NANOG in all ICM cells even though the reporter is transcriptionally active. Nuclei are stained with Hoechst (blue). b Quantification of cells with epiblast (EPI, NANOG+) versus PrE (PRE, GATA6+) identity at E3.5. Cells that did not adopt a clear identity were marked as double positive (DP) for both NANOG and GATA6. Gata6 H2B-Venus/H2B-Venus homozygotes did not specify PrE, and Gata6 H2B-Venus/+ heterozygotes had a relatively reduced number of cells with PrE identity and increased numbers of double positive cells with undecided identity. Gata6 H2B-Venus/H2B-Venus embryos with both alleles of the reporter, and effectively a Gata6 null, had higher levels of Venus expression compared to Gata6 H2B-Venus/+ embryos with only one reporter allele. This would suggest that GATA6 either does not regulate, or negatively feeds back on, Gata6 gene expression. cultured on fibronectin-coated chamber slides [55,56]. Mesoderm precursors migrated away in a centrifugal fashion thus forming what is referred to as a mesodermal sheet that surrounded the original explant. When PS explants were cultured from Gata6 H2B-Venus/+ embryos, migrating cells expressed Venus, and individual nuclei could be identified and followed over time (Fig. 5b).
The next stage of development is defined by allantoic bud formation, axial extension from the node, and appearance of headfolds. At the late bud (LB, E7.5) and early headfold (EHF, E7.75) stages, Venus was expressed mosaically in the exVE and throughout the emVE. Venus, together with endogenous GATA6, was strongly expressed in the anterior definitive endoderm, cardiac crescent, and lateral plate mesoderm (Fig. 7a). Venus expression continued in the mesoderm, although it did not appear to coincide with BRACHYURY in the posterior embryonic and extra-embryonic mesoderm at the early bud (EB, E7.5) stage, indicating that Venus was labeling a specific sub-population of mesoderm and/or endoderm progenitors which was distinct from BRACHYURY labeled cells (Fig. 7b). Thus, as cells exit the primitive streak, they likely downregulate BRACHYURY and activate GATA6.
Exclusion of Venus + endoderm cells from the midline was evident in ventral, frontal, and posterior views of Gata6 H2B-Venus/+ embryos at headfold stages (E7.75) (Fig. 8a). This was confirmed by immunostaining for BRACHYURY and FOXA2, two transcription factors that are expressed along the midline at E7.75 [57]. Trace expression of Venus was detectable in the midline at early headfold (EHF) stages, likely due to perdurance. However, expression of Venus mostly did not coincide with that of BRACHYURY or FOXA2 in the midline (Fig. 8b) as well as in the streak (data not shown). At this stage (EHF), Venus was expressed in both the mesenchyme and endoderm. Expression in the endoderm was confirmed by colocalization with FOXA2 in the surface endoderm (Fig. 8c).  Gata6 H2B-Venus expression in later stage embryos and adults From E8.25 (4 somite stage, ss) to E9.5 (23ss), wholemount expression of Venus was observed in the heart and gut endoderm ( Fig. 9a-b). Sections through Gata6 H2B-Venus/+ embryos at E8.25 revealed strong expression of the reporter in all regions of the gut, the heart myocardium, sinus venosus, and yolk sac. Weaker expression of the reporter was observed in the pharyngeal mesenchyme. There were some discrepancies between endogenous GATA6 and Venus reporter expression. For example, the endocardium and the body wall, which derives from the lateral mesoderm, expressed low levels of GATA6, but did not express Venus. Conversely, GATA6 appeared mostly downregulated in the yolk sac, although Venus reporter expression was still robust (Fig. 9c). While this may reflect delays in the activation and/or downregulation of the reporter compared to the endogenous protein, it is also possible that the reporter may be retained more strongly in cells that are not actively dividing. Similarly, faster growing tissues may dilute the reporter more rapidly.
(See figure on previous page.) Fig. 5 Gata6 H2B-Venus reporter is expressed in nascent endoderm and mesoderm during gastrulation. a Wholemount immunofluorescence for endogenous GATA6 protein (red) on Gata6 H2B-Venus/+ embryos at early streak (ES, E6.75) and mid-streak (MS, E7.0). Venus is expressed in a mosaic pattern in the VE. The PS region is indicated in the posterior portion of the embryo (bracket). Transverse sections through the PS at E7.0 (level of section indicated by dotted line) are shown below. GATA6 and Venus are expressed in cells that have ingressed from the PS and are migrating laterally to the anterior of the embryo. Venus and GATA6 are not expressed in the Epi, weakly expressed in the VE, and heterogenously expressed in the intervening mesodermal wings (Mes). The red fluorescent signal in the apical VE that forms a ring around the tissue section is likely due to non-specific binding of the GATA6 antibody to the surface of the extraembyonic tissue. b PS explants to track movements of migrating Venus + cells in vitro. The PS region was dissected from the posterior portion of E6.5 Gata6 H2B-Venus/+ embryos and germ layers were separated by enzymatic treatment. The PS was plated on FIBRONECTIN-coated glass chamber slides. The PS explants (grey) attach to the glass and mesenchymal cells migrate outwards to form a mesodermal sheet (green) over the course of several days. Direction of migration is indicated by red dotted arrows. Right-to-left movement (red arrow) of Venus + cells is indicated as they move away from a Gata6 H2B-Venus/+ PS explant over the course of 3 h. Images were captured every 7 min. Proximal By E12.5, expression of Venus was tissue-specific in Gata6 H2B-Venus/+ embryos. Low-level expression of Venus was observed in the midgut epithelium whose derivatives express GATA6 later on, but not in the foregut or hindgut. The heart myocardium and outflow tract continued to strongly express Venus. Additional sites of expression detectable at this stage included the stomach epithelium, pancreas, kidneys, lung epithelia, liver, gall bladder, urogenital ridge, arterial endothelium, and umbilical vessels. Consistent with previous characterizations of endogenous GATA6 expression [18], Venus was not observed in certain foregut-derived organs such as the esophagus and trachea (Fig. 10).
In Gata6 H2B-Venus/+ adults, expression of Venus was observed in organs that have previously been reported to express Gata6 [18,[32][33][34]39]. These include the heart, lung, pancreas, liver, gall bladder, ovaries, adrenal glands, stomach, and bladder. Strong expression of Venus within the skin was also observed (Fig. 11a). Tissue sections of organs from Gata6 H2B-Venus/+ mice revealed Venus expression specifically in the mucosa and mesothelium of the corpus region of the stomach and small intestine, as well as expression within the pancreas and skin (Fig. 11b). Gata6 H2B-Venus/+ heterozygous adult mice were not recovered at Mendelian ratios (105 mice in total; 44 heterozygous, 61 wild-type), suggesting that adult Gata6 H2B-Venus/+ heterozygous mice may have reduced viability.

Conclusions
Our characterization of the Gata6 H2B-Venus mouse line suggests that it should serve as a useful tool for singlecell resolution imaging of Gata6 transcriptional activation in vivo. Since this is also a loss-of-function allele, it can, in principle, be used in combination with a conditional (floxed) Gata6 allele to trace the fate of Gata6  Co-expression of GATA6 and Venus was well correlated in the embryo from the pre-implantation blastocyst Fig. 9 Expression of Venus at somite stages. a Frontal and lateral views of wholemount Gata6 H2B-Venus/+ embryos merged with brightfield images at E8.25 (4 somite stage, ss). Venus is expressed in the heart, sinus venosus, and gut endoderm. b Lateral views of wholemount Venus expression at E9.5 (23ss) in Gata6 H2B-Venus/+ embryos. c Immunofluorescence for GATA6 (red) on transverse sections through a Gata6 H2B-Venus/+ embryo. Varying levels of GATA6 co-expression with Venus (green) can be seen in the gut endoderm and heart. Some tissues express GATA6 exclusively, for example the body wall and endocardium, while other tissues express Venus only, such as the yolk sac and weak expression in the pharyngeal mesoderm. Nuclei are stained with Hoechst (blue) to gastrulation and early somite stages of development. However, some differences were observed between the endogenous GATA6 protein versus the Venus-based transcriptional reporter in terms of levels and localization. For example, in Gata6 H2B-Venus/H2B-Venus homozygous null blastocysts, Venus continued to report transcriptional activity in the absence of GATA6 protein. These data suggest that GATA6 does not feedback on its own transcription, and identify this transcriptional reporter as a readout of Gata6 expression in the absence of protein function. However, it is also possible that some cells normally express Gata6 mRNA that is not translated into protein. In the future, this could be determined by quantitative real-time PCR or RNA in situ hybridization to detect Gata6 transcripts in specific cells in comparison with GATA6 protein levels. Alternatively, some of these differences may be due to stability of the Venus protein, which may pose challenges for certain experiments. For example, since Gata6 is activated in all ICM cells prior to the sorting of Epi and PrE lineage progenitors into two distinct layers, then live imaging of the PrE lineage may be difficult if low levels of Venus perdure within Epi progenitors.
In post-implantation embryos, mosaic activation of the Gata6 H2B-Venus reporter in the exVE at E5.5 may potentially reflect mono-allelic regulation of the Gata6 locus.
This characteristic of the reporter could be utilized to investigate dynamics of the expansion of cells in the exVE at early stages of post-implantation development that lead up to gastrulation. During gastrulation, Venus brightly labels both mesoderm and endoderm progenitors and continues to be expressed in some of their derivatives, such as the cells that populate the heart field and gut endoderm. During organogenesis, Venus is expressed in a tissue -specific manner in organ primordia that normally express endogenous GATA6.
In adult mice, the Gata6 H2B-Venus allele may prove useful for studies of disease states, such as cancer models, in which Gata6 activity may be aberrantly regulated. For example, the reporter may be silenced in tissues that normally express GATA6. Alternatively, the reporter may be ectopically activated and changes in its activity levels may potentially correlate with progression of disease. Gata6 H2B-Venus may also be used as a bright marker of specific cell populations within adult organs, such as in the skin, stomach and small intestine.
Overall, the Gata6 H2B-Venus allele should provide a useful tool for detecting transcriptional activation of the Gata6 locus that correlates well with endogenous GATA6 protein in both embryonic and adult mice, and can be used for the analysis and isolation of specific cell Fig. 10 Expression of Gata6 H2B-Venus reporter at E12.5. Lateral view of a wholemount Gata6 H2B-Venus/+ embryo at E12.5 merged with brightfield (top left). Transverse sections at E12. 5 showing Venus expression in various endoderm and mesoderm derived organs. Expression is observed in the midgut epithelium, but not the foregut or hindgut. Venus is also seen in the lung bud epithelium, throughout the heart and outflow tract, vascular endothelium, portions of the stomach epithelium, pancreas, kidney, liver, gall bladder, urogenital ridge and umbilical vessels. Venus was not expressed in the esophagus or trachea, Nuclei are stained with Hoechst (grey) populations that normally express Gata6. Noting the issue of Venus perdurance, it would be of interest to retarget the Gata6 locus with a destabilized fluorescent protein reporter in order to obtain an improved dynamic readout of Gata6 transcriptional activity, and not only detect when Gata6 is activated, but also to determine when the gene is turned off. A destabilized Gata6 transcriptional reporter might be expected to be dimmer, but accordingly may provide better concordance between Gata6 gene activity and reporter protein Fig. 11 Expression of Gata6 H2B-Venus reporter in adult organs. a In Gata6 H2B-Venus/+ adult mice at 3 months of age, Venus (green) is expressed in the heart, lung, pancreas, liver, gall bladder, ovaries, oviducts, uterus, adrenal glands, corpus region of the stomach, bladder, and skin. Right atrium (RA), left atrium (LA), right ventricle (RV), left ventricle (LV). b Tissue sections through organs from Gata6 H2B-Venus/+ adult mice at 3 months of age demonstrated expression of Venus in the mucosa and mesothelium of the corpus region of the stomach and small intestine, as well as expression within the pancreas and skin fluorescence. For example, the Venus-NLS-PEST (VNP) fusion protein reporter [58] has a short half-life and has been successfully used to monitor the transcriptional activity of genes, such as Nanog, which are dynamically expressed [59]. Similarly, if available, a Gata6 VNP allele could be useful for live quantitative imaging of the rapid changes in Gata6 expression that occur during various cell lineage specification events involving GATA6.

Image acquisition and processing
ES cells and embryoid bodies were imaged on a Zeiss Axio Vert.A1 inverted microscope with a black and white camera (Axiocam MRm). Raw data was processed using Axiovision software and dark field photos were pseudocolored green in Adobe Photoshop. Fixed blastocysts, wholemount embryos (E5.25-7.75), and tissue sections were imaged on a Zeiss LSM880 laser scanning confocal microscope. Blastocysts were imaged along the entire z-axis with 1 μm z-steps using an EC Plan-Neofluar 40×/1.30 oil immersion objective. For live imaging, blastocysts were imaged with 2 μm z-steps and with 15-min intervals. Blastocysts (on glass bottom dishes, MatTek) and PS explants (on 2 chamber coverglass, Lab-Tek) were imaged on the LSM880 inside a heated CO 2 incubation chamber. Raw data was processed in Zeiss ZEN Black software. Wholemount decidua (E6.5), embryos (E8.25, E9.5, E12.5), and adult organs were imaged on a Leica M165FC dissecting microscope with a color camera (Axiocam MRc) and raw data was processed using Axiovision software.

Quantitative analysis of reporter co-localization in pre-implantation stage embryos
Blastocyst images were segmented using the algorithm MINS (Modular Interactive Nuclear Segmentation) for fluorescence quantification (http://katlab-tools.org) [64]. Confocal z-stacks were processed with MINS for nuclear segmentation as described [65]. Fluorescence decay along the z-axis for each nuclei was corrected using a factor dependent on the position of the nucleus in z, as described [65]. PrE and Epi populations were identified manually based on the expression of GATA6 and NANOG, respectively. RStudio was the implementation of R used for all analyses. CSV file containing the raw data for Fig. 2 is provided in the Additional file 1. R-script used for analysis is available upon request. Pearson's product moment correlation was used to assess the correlation between GATA6 and Venus levels in the PrE at each blastocyst stage. Statistical differences in Venus expression between blastocyst lineages were tested performing analysis of variance (ANOVA) on the average fluorescence level for all cells in each lineage, in each embryo (plotted in Fig. 2c). Tukey's range test was used as the posthoc test to determine the groups responsible for the statistical difference.

Primitive streak explants
The posterior region encompassing the primitive streak (PS) and overlying visceral endoderm was dissected from E6.5 embryos following removal of Reichert's membrane. The tissue was then incubated for 5-10 min in 2.5 % pancreatin (Sigma) and 0.5 % trypsin (Calbiochem), and washed in PBS. The VE overlying the PS was removed by pipetting, and germ layers were further separated using Tungsten needles to remove the nascent mesodermal wings. PS explants were plated on 2-well chamber coverglass (Lab-Tek) that had been coated with FIBRONECTIN from