Charting Brachyury-Mediated Developmental Pathways During Early Mouse Embryogenesis

Charting Brachyury-mediated developmental pathways during early mouse embryogenesis

Macarena Lolasa,b, Pablo D. T. Valenzuelab, Robert Tjiana,c,1, and Zhe Liud,1

dJunior Fellow Program, aJanelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147; bFundación Ciencia para la Vida, Santiago 7780272, Chile; and cLi Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine Center of Excellence, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

Contributed by Robert Tjian, February 11, 2014 (sent for review January 14, 2014) To gain insights into coordinated lineage-specification and mor- cells play diverse and indispensable roles in early mouse phogenetic processes during early embryogenesis, here we report development. a systematic identification of transcriptional programs mediated In mouse ES cell-based differentiation systems, Brachyury is by a key developmental regulator—Brachyury. High-resolution widely used as a mesoendoderm marker, and Brachyury-positive chromosomal localization mapping of Brachyury by ChIP sequenc- cells were able to differentiate into mesodermal and definitive ing and ChIP-exonuclease revealed distinct sequence signatures endodermal lineages, such as cardiomyocytes and hepatocytes enriched in Brachyury-bound enhancers. A combination of genome- (8–10). In a previous study, we found that depletion of a core wide in vitro and in vivo perturbation analysis and cross-species promoter factor, the TATA binding protein-associated factor 3, evolutionary comparison unveiled a detailed Brachyury-depen- in ES cells leads to significant up-regulation of Brachyury and dent gene-regulatory network that directly links the function of mesoderm lineages during ES cell differentiation (11). Here, we Brachyury to diverse developmental pathways and cellular house- systematically characterized the molecular function of Brachyury keeping programs. We also show that Brachyury functions pri- during ES cell differentiation by genomic, single-cell imaging, marily as a transcriptional activator genome-wide and that an and biochemical approaches. We then contrasted our results unexpected gene-regulatory feedback loop consisting of Brachyury, with published zebrafish and Xenopus Brachyury binding site Foxa2, and Sox17 directs proper stem-cell lineage commitment mapping data (12, 13) to compare evolutionarily convergent or during streak formation. Target gene and mRNA-sequencing cor- divergent pathways. We further extended these studies to examine relation analysis of the Tc mouse model supports a crucial role of the direct impact of Brachyury loss of function during early de- Brachyury in up-regulating multiple key hematopoietic and muscle- velopment with the Tc mouse model. Together, our data provide fate regulators. Our results thus chart a comprehensive map of a systematic and comprehensive view of Brachyury-mediated the Brachyury-mediated gene-regulatory network and how it regulation and also reveal unique mechanistic insights into how influences in vivo developmental homeostasis and coordination. this gene network likely contributes to early mouse embryogenesis.

primitive streak | early development | mesoendoderm differentiation Results and Discussion Characterization of in Vitro Primitive Streak Induction. We used the – n the past decade, significant insights have been gained in well-established Activin-A mediated differentiation protocol that Iunderstanding gene-regulatory programs responsible for em- efficiently drives ES cells to form primitive streak cells in vitro (9, bryonic stem (ES) cell pluripotency and self-renewal. However, 14) (SI Appendix,Fig.S1A). We successfully generated two transcriptional control mechanisms underlying finely balanced highly specific polyclonal antibodies directed against the Brachyury lineage-segregation and morphogenetic processes during early protein. At day 4 after differentiation, we detected strong induction of primitive streak markers (Brachyury and Gsc) mammalian embryogenesis remained elusive. During early mouse (SI Appendix,Figs.S1B–D and S2A), indicating a successful gastrulation, Brachyury (T), a classical enhancer-binding transcrip- differentiation of primitive streak cells. To comprehensively tion factor (TF), has been reported to be required for the proper development of primitive streak, allantois, axial, and posterior Significance mesoderm (reviewed in ref. 1). Specifically, mouse embryos that are homozygous for the − − Brachyury (T) deletion die at midgestation (2). T / mutant The gene-regulatory mechanisms for finely balanced cell-fate determination and morphogenesis during early animal de- epiblast cells are compromised in their ability to migrate away velopment remain largely elusive. Here, we combine genomic, from the primitive streak and, therefore, are unable to undergo single-cell imaging and biochemical approaches to chart the the morphogenetic movements carried out by their wild-type molecular pathways mediated by a key developmental regu- (WT) counterparts during gastrulation. In addition to defects in lator—Brachyury. Our results shed light on mechanistic insights the primitive streak, the notochord is absent in posterior portions into the ultrafine organization of Brachyury-bound enhancers of the embryo. Although the anterior portions of T mutant mice and link Brachyury function to cellular differentiation and contain notochordal precursor-like cells, they fail to undergo housekeeping processes critical for coordinating early mouse normal terminal differentiation (3, 4). The embryonic pattern embryogenesis. − − posterior to the forelimb region of T / animals is also disturbed, with somites posterior to the seventh pair absent or abnormal. Author contributions: M.L., P.D.T.V., R.T., and Z.L. designed research; M.L. and Z.L. per- Although neural folds fuse to form the neural tube, they are formed research; M.L. and Z.L. analyzed data; and M.L., R.T., and Z.L. wrote the paper. severely kinked in the caudal region, and the surface ectoderm The authors declare no conflict of interest. tends to form fluid-filled blisters (2, 4, 5). In addition to these Freely available online through the PNAS open access option. well-documented defects, numerous other phenotypic abnormali- Data deposition: The data reported in this paper have been deposited in the Gene Ex- −/− pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE54985). ties have been reported in T and T mutant embryos, including 1 – To whom correspondence may be addressed. E-mail: [email protected] or tjianr@ left right patterning defects and morphological abnormalities in hhmi.org. heart development (6, 7). Together, these genetic and phenotypic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. analyses strongly suggest that Brachyury and Brachyury-expressing 1073/pnas.1402612111/-/DCSupplemental.

4478–4483 | PNAS | March 25, 2014 | vol. 111 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1402612111 Downloaded by guest on October 2, 2021 characterize gene expression changes upon differentiation, we In line with these findings, Brachyury-bound regions were also performed mRNA sequencing (mRNA-seq) with differentiated enriched around genes involved in endoderm development embryoid bodies (EBs; EB day 4) and then compared gene ex- (P < 3.9E-09) and formation (P < 2.6E-05). pression levels to ES cell data. We detected 2,464 up-regulated and 1,329 down-regulated genes (statistics in Dataset S1). Con- Evolutionary Conservation of Brachyury-Mediated Regulation. With sistent with a successful epiblast transition and primitive streak recent Brachyury homolog Ntl ChIP-on-chip (zebrafish) and XBra induction, ES cell ground-state specific genes (Tbx3, Stra8, Pecam1, ChIP-seq (Xenopus tropicalis) data available (12, 13), we next Esrrb, Gbx2,andKlf4) were dramatically down-regulated. However, asked whether a similar set of target genes identified in these mesoderm and definitive endoderm (DE) markers (Cer1, Eomes, studies also bound Brachyury in the mouse. Intriguingly, we Wnt3, Lhx1, Fgf8, Cxcr4, Foxa2, Sox17,andGsc)(15)werestrongly found that ∼10∼13% of the Ntl (zebrafish) and Xbra (X.tropicalis) up-regulated (SI Appendix,Figs.S1C and S2B). We also confirmed target genes (zebrafish, 28/218; X. tropicalis, 113/1,040) have a the expression of Brachyury, Foxa2, and Sox17 at the protein level corresponding homologous gene bound by Brachyury in mouse by immunofluorescence staining (SI Appendix, Fig. S1D). (Fig. 1E and Dataset S3). For example, nine genes (Dusp6, Mesp2, Hoxd8, Cdx2, Irx3, Lefty1, Msgn1, Dhrs3,andSp5)were Brachyury Selectively Binds Key Developmental Genes. We applied bound by Brachyury in all three species (Fig. 1F). We also ob- ChIP sequencing (ChIP-seq) to our in vitro primitive streak served Brachyury-bound genes such as Fgf8, Sox17, Foxa2, Tbx3, formation system. To ensure specific detection of Brachyury Wnt9b, Wnt5b, and Rest that were common targets in mouse and binding, we used two independent anti-Brachyury antibodies for X. tropicalis but not in zebrafish (Fig. 1F and Dataset S3). Con- ChIP-seq experiments. By only calling peaks detected by both versely, Igf1r, Brf1, Gata2, Zfand5, Sox11, Mycn, Notch3,etc.are antibodies, we identified 3,160 high-confidence Brachyury-bound common targets in mouse and zebrafish but not in X. tropicalis.Itis regions in the mouse genome (Dataset S2). These sites display worth noting that the work performed in zebrafish used the pro- genome-wide biased association with transcription start sites moter-restricted ChIP-on-chip technique (12 kb around the TSS (TSS; 61% are within 5 kb of a TSS vs. 9% of random control of ∼11,000 genes) to map what is likely to be a relatively in- regions) (Fig. 1 A and B). Intriguingly, we also observed a strong complete set of Brachyury-binding sites. We would therefore biased localization of Brachyury bound at regions immediate expect to observe larger function overlaps between mouse and downstream of the TSS, particularly the 5′ UTR (Fig. 1C and SI zebrafish targets when more sensitive binding-site mapping

Appendix,Fig.S1E and F). As expected, many key developmental techniques were used. BIOLOGY

genes (Fgf8, Foxa2, Foxj1, Sox17, and Dusp6) were directly tar- DEVELOPMENTAL geted by Brachyury (Fig. 1A and SI Appendix, Fig. S8). We Map High-Resolution Brachyury-Binding Sites by ChIP-Exonuclease. correlated Brachyury-binding site information with annotated Previous in vitro studies suggested that Brachyury selectively genes genome-wide (<10 kb from TSS) and identified 1,942 genes binds a 7-bp DNA motif (TCACACCT) (17–19). However, sur- bound by Brachyury (Dataset S3). To calculate overrepresented prisingly, this motif was not evident from Brachyury ChIP-on-chip Gene Ontology terms associated with Brachyury binding, we data in a recent report (20) and in our ChIP-seq datasets. To next performed Genomic Regions Enrichment of Annotations resolve this discrepancy, we applied the much-higher-resolution Tool (GREAT) analysis (16) (Fig. 1D). We found that Brachyury- ChIP-exonuclease (ChIP-exo) technique to our in vitro primitive bound regions were significantly enriched around genes involved streak formation system. Exonuclease treatment steps in the ChIP- in the formation of primary germ layers (P < 1.4E-9), mesoderm exo protocol remove extraneous flanking DNA from the cross- cell migration (P < 3.0E-7), and mesoderm formation (P < 1.2E-5). linked protein–DNA complexes (21), allowing a more precise de- Recently, Brachyury was found to express in DE progenitors, and lineation of DNA sequences recognized and bound by Brachyury Brachyury-positive cells were able to efficiently differentiate (SI Appendix, Fig. S3A). Indeed, after peak calling by GeneTrack into definitive endodermal cell types in vitro and in vivo (8, 9). (22), we were able to detect 11,503 bifurcated ChIP-exo peaks

Fig. 1. Brachyury targets the core promoters and proximal enhancers (PEs) of key developmental genes. (A) Representative tracks for Brachyury-bound seq- regions associated with key developmental genes (the annotated gene length as indicated by the arrow: Upper Left, Fgf8, 6.087 kb; Upper Right, Foxa2, 3.028 kb; Lower Left, Foxj1, 4.561kb; and Lower Right, Dusp6,4.259kb).y-axis limit is from 3 to 50 reads for chip-seq data. Auto-scale was used for displaying mRNA-seq tracks. Two independent antibodies (Ab1 and Ab2) against Brachyury were used. Preimmune IgG served as a negative control. mRNA-seq data from control EBs at day 4 are also displayed. (B)Absolute TSS proximity fraction curve of Brachyury-bound regions (blue) and that of randomly selected genomic regions (gray). (C) TSS proximity fraction curve of Brachyury-bound regions (green). Brachyury-bound regions display a biased localization downstream of the TSS. (D) Enriched Annotation terms associated with Seq-regions (see Dataset S2 for more information; calculated by GREAT). (E) Venn diagram of evolution conservation analyses of common Brachyury target genes in mouse, zebrafish, and X. tropicalis.(F)Lists of selected common Brachyury target genes in all three species (M&Z&X) or in two species (M&Z; M&X). M, mouse; X, X. tropicalis;Z,zebrafish.Seealso Datasets S2 and S3.

Lolas et al. PNAS | March 25, 2014 | vol. 111 | no. 12 | 4479 Downloaded by guest on October 2, 2021 [11,000 (11k) dataset]. The average size of the binding sites was observed extensive double- and triple-motif colocalization (11k ∼11 base pairs (bp), compared with 823 bp for a typical ChIP-seq dataset) (Fig. 2D and SI Appendix, Fig. S5B), and the frequencies binding region. This significant improvement in resolution (∼76- of colocalization were significantly higher (up to sixfold) than fold) provided us with a more accurate view of Brachyury– values calculated for random control genomic regions (Fig. 2D). enhancer DNA interactions (SI Appendix,Fig.S3B). ChIP-exo These sequence signatures identified here provided us with valu- allowed us to successfully identify an overrepresented DNA able information to further dissect complex molecular trans- motif (TCACA) (1k dataset, top 10% of 11k dataset) that matched actions at Brachyury-bound enhancers. well with the Brachyury consensus sequence (P < 2.22E-16) (SI Appendix, Fig. S3 C and D). Genome-wide scanning (11k data- Brachyury Functions as an Activator. To probe how Brachyury set) further confirmed that 37% of the Brachyury exo-peaks (4,299 regulates its target genes, we performed mRNA-seq to analyze of 11,503) contain this motif (Fig. 2D). As expected, DNA probes gene-expression changes after Brachyury depletion by two in- containing these 5 bp were sufficient to interact specifically dependent lentivirus-mediated shRNAs (SI Appendix, Fig. S6A). with Brachyury in EMSAs (SI Appendix,Fig.S4A and B). Super- Compared with nontarget shRNA control, we detected 567 down- shift assays confirmed that this binding is Brachyury-specific (SI regulated and 806 up-regulated genes common to the two distinct Appendix, Fig. S4C). Strikingly, we observed that there are fre- Brachyury shRNA-mediated knockdowns. Next, we set out to test quently multiple Brachyury-binding sites within each ChIP-seq– whether there is a biased association of Brachyury sites with par- defined enhancer region. For example, Foxa2, Cer1,andWnt8b ticular gene categories (up, down, and unchanged) upon Brachyury proximal enhancers (PEs) contain as many as eight Brachyury depletion. To do so, we first assigned Brachyury-bound regions exo-peaks (SI Appendix,Fig.S3E). On average, there are ∼2.4 that were within 5 kb of the TSS to genes genome-wide. Then, we Brachyury-binding sites within each ChIP-seq region (Fig. 2A). analyzed the association between Brachyury-bound regions with each gene category (SI Appendix,Fig.S6B). Notably, we only de- Unique Sequence Signatures in Brachyury-Bound Enhancers. An im- tected a biased association of Brachyury sites with genes that were portant finding of the ENCODE (Encyclopedia of DNA Ele- down-regulated upon loss of Brachyury, whereas the opposite ments) project was that many eukaryotic genes are regulated by correlation was seen for up-regulated genes. This genome-wide multiple TFs cobinding to neighboring sites in the genome (23). biased association strongly favors the notion that, upon induction, To test whether this characteristic is also the case for Brachyury- Brachyury binds to the promoter and enhancer regions of target bound enhancers, we performed a motif search within 50 bp genes and activates transcription. Consistent with this model, rep- around Brachyury ChIP-exo peaks (1k dataset). We recovered resentative Brachyury-bound regions and, likewise, a synthetic DNA five overrepresented motifs (Fig. 2B and SI Appendix, Fig. S5A). element containing four repeats of the Brachyury DNA-binding Importantly, four of the five identified motifs were confidently motif efficiently elevated the gene expression of a firefly lucif- matched to known TF binding sequences (Brachyury, Myf fam- erase reporter in a Brachyury-dependent manner in human 293T ily, E2F1, and Sox17) (Fig. 2C). In agreement with bona fide TF cells (SI Appendix, Figs. S6C and S7). binding events, a positional preference plot confirmed the enrichment for these motifs only at Brachyury exo-peaks but not Brachyury Coordinates Gene Expression During in Vitro Streak within their flanking extended genomic regions (±200 bp) (Fig. Formation. To dissect how Brachyury regulates gene expression 2E). Consistent with the notion of combinatorial regulation, we at the single-cell level, we focused on two Brachyury direct target

Fig. 2. Unique sequence signatures at Brachyury- bound enhancers. (A, Left) The distribution of ChIP- exo peaks per ChIP-seq region. (Right) The modular binding of Brachyury at single enhancers. (B, Left) Raw sequencing reads (forward, blue; reverse, green) of 200-bp genomic regions containing DNA motifs 2, 3, and 4 that were de novo recovered by the gimmemotifs program (40) using the 1k dataset, centered at the motif midpoint. (Right) Color chart representa- tion of 20 bp of sequence located around the midpoint of motif 1 ordered as in Left.(C) Motifs 1, 2, 3, and 4 were confidently matched to the known TF binding sequences (T, Myf, E2F1, and Sox17) by Jaspar. (D, Left) Number of 50-bp genomic regions around exo-peaks (11k dataset) that contain either single or double motifs. (Right) Fold enrichment when comparing numbers in Left with numbers that were calculated with randomized control genomic regions (SI Appendix, SI Methods). (E, Left) Probability-density distribution of motifs 1–5 across 400-bp genomic regions surrounding exo-peaks (centered by the peak midpoint). (Right) Schematic diagram illustrating combinatorial regulation by colocalization of multiple transfactors at Brachyury-bound enhancers.

4480 | www.pnas.org/cgi/doi/10.1073/pnas.1402612111 Lolas et al. Downloaded by guest on October 2, 2021 genes, Foxa2 and Sox17 (Fig. 1A and SI Appendix, Fig. S8B). To further test this model in vivo, we first asked whether Interestingly, the target gene analysis comparing divergent species Brachyury, Sox17, and Foxa2 also displayed gene-expression indicated that both Foxa2 and Sox17 were also targeted by the patterns that spatially overlap during early mouse development. Brachyury homolog (XBra), suggesting that the regulatory link We first compared publicly available in situ hybridization (ISH) between Brachyury, Foxa2, and Sox17 might be evolutionarily gene expression data (24). Indeed, we detected coexpression of conserved (Fig. 1 E and F). Furthermore, Brachyury depletion Brachyury and Foxa2 at node/DE regions and coexpression of decreased Foxa2 and Sox17 expression at both protein and mRNA Foxa2 and Sox17 at DE regions (SI Appendix,Fig.S9E). Consis- levels (Fig. 3A and SI Appendix, Fig. S9 A and B). After double tent with our in vitro results, we found that Brachyury and Sox17 or triple immunofluorescence staining, we performed single-cell expression was negatively correlated in the definite endoderm region. If the model derived from our in vitro experiments is analysis to test the correlation of expression between genes (Fig. 3B medium medium and SI Appendix,Fig.S9C and D). Collectively, our single-cell correct, we would expect to detect a Brachyury Sox17 imaging analysis revealed several important correlations regarding cell population, suggesting an intermediate transition state the relationship between Brachyury, Foxa2, and Sox17 (Fig. 3B wherein Brachyury first activates the expression of Sox17 and in- and SI Appendix, Fig. S9C). Firstly, Foxa2high and Sox17high cells creasing levels of Sox17 gradually represses the expression of Brachyury. To further probe the coregulated gene expression at were mutually exclusive, as highlighted by the negative correlation the single-cell level, we performed confocal imaging experiments between their expression levels. We also identified a negative cor- of coimmunostained 7.0∼7.5 mouse embryos (Fig. 3 D and E, SI relation between Sox17 and Brachyury. In contrast, the expres- Appendix,Fig.S10,andMovies S1–S4). As expected, we detected sion of Foxa2 was always positively correlated with Brachyury a BrachyurymediumSox17medium cell population in the DE region, expression (Fig. 3B). We also observed a population of cells with but not at other regions, in embryonic day 7.5 (E7.5) embryos high levels of Brachyury and medium-to-low levels of Sox17 and (Fig. 3 D and F, SI Appendix, Fig. S10A, and Movie S1). Inter- Foxa2 (SI Appendix,Fig.S9D), suggesting coexpression of estingly, BrachyurymediumSox17medium cells are generally found Brachyury, Foxa2, and Sox17 within the same individual cells. located more distal to the midline of the notochord-primitive Together with genomic data, our single-cell imaging results streak compared with BrachyuryhighSox17low cells, suggesting favor a model in which, as differentiation occurs, Brachyury first that this population of cells might have a distinct morphogenetic targets and promotes the expression of Foxa2 and Sox17. How- mode during early embryogenesis (Fig. 3F Right). Consistent

ever, high levels of Sox17 would, in turn, directly or indirectly with the ISH results, we also detected coexpression of Brachyury BIOLOGY repress the expression of Brachyury (Fig. 3H). This potential

and Foxa2 within the same individual cells within node and DE DEVELOPMENTAL negative feedback may explain the Sox17highBrachyurylowFoxa2low regions (Fig. 3E, SI Appendix, Fig. S10 B and C, and Movies S2– cell population. To directly test this model, we overexpressed Sox17 S4). Brachyury-positive cells express Foxa2 at significantly higher after 2 days of differentiation when cells are at the epiblast stage. levels than Brachyury-negative cells (Fig. 3G). This finding is in Indeed, we observed significant down-regulation of Brachyury good agreement with our in vitro results showing that Brachyury at EB day 4 (Fig. 3C). directly targets and activates the transcription of Foxa2 and that

Fig. 3. A gene-regulatory system of Brachyury, Foxa2, and Sox17.(A) Western blot analysis of Foxa2 and Sox17 protein levels after Brachyury knockdown. ES cells and EB day 4 without Activin-A induction served as negative controls. (B, Left) Sin- gle-cell gene-expression correlation analysis. Seg- mented cell areas in the Sox17 channel were determined by proper computational steps (binary contrast enhancing and particle size/circularity fil- tering). Then, the mean fluorescent intensities from the two channels were plotted and tested for correlation. (Right) Negative correlation between the protein levels of Sox17 and Foxa2 in single cells. There is a negative correlation between the protein levels of Sox17 and Brachyury when segmented cell areas were determined by the Sox17 channel. There is a positive correlation between the protein levels of Brachyury and Foxa2 in single cells when segmented cell areas were determined by the Foxa2 channel. A.U., arbitrary units. (C) Sox17 represses the expression of Brachyury during in vitro streak formation. GFP served as transfection control. EBs (day 2; SI Appendix, Fig. S1A) were first dissociated by trypsin into single cells, and then electroporation was performed with either Sox17 or GFP over- expressing plasmid. Cells were reaggregated, and Western blot analysis of Brachyury, Sox17, and GFP was performed at EB day 4. (D) Whole-mount Brachyury and Sox17 Immunostaining confocal analysis of E7.5 embryos. DE region was selected to show. For other regions, see Movie S1. Cells with medium expression levels of Brachyury and Sox17 are indicated by gray arrows. (Magnification: Upper,10×; Lower,40× oil; scale bars: Upper, 100 μm; Lower,25μm.) (E) Whole-mount Brachyury and Foxa2 Immunostaining confocal analysis of E7.5 embryos. Cross-section covering both primitive streak and DE was selected to show. For other regions, see Movie S2. (Magnification: Upper,10×; Lower,40× oil; scale bars: Upper, 100 μm; Lower,25μm.) (F, Left) Three populations of cells were observed in Brachyury and Sox17 embryo Immunostaining analysis. High expression of Brachyury but low expression of Sox17 (B++/S−) is shown. Medium expression of Brachyury and medium expression of Sox17 (B+/S+), as indicated by arrows in D. is shown Low expression of Brachyury but high expression of Sox17 (B−/S++) is shown. (Right)B+/S+ cells are generally further away from the primitive streak midline than B++/S− cells. (G) At the node/DE region, Brachyury-positive cells generally have higher Foxa2 levels than Brachyury-negative cells (also see SI Appendix, Fig. S10 B and C and Movies S2–S4). (H) A diagram illustrating the gene-regulatory rela- tionships between Brachyury, Foxa2, and Sox17.

Lolas et al. PNAS | March 25, 2014 | vol. 111 | no. 12 | 4481 Downloaded by guest on October 2, 2021 BrachyuryhighFoxa2high and BrachyurylowSox17high cells are mutually exclusive. Collectively, these results support the model derived from in vitro experiments and suggest that that the regulation of Foxa2 and Sox17 by Brachyury might be region- specific. Specifically, we note that Sox17 and Foxa2 are also expressed in some embryonic tissues, such as the primitive endoderm, where Brachyury is largely absent (Fig. 3 D and E and Movies S1–S4) (25). Thus, it is likely that these two genes can be regulated by distinct factors and mechanisms at different developmental stages. It appears that, at least during streak formation, the expression of both genes displays significant reliance on Brachyury (Fig. 3A and SI Appendix,Fig.S9A and B).

Probing in Vivo Brachyury Function. To identify Brachyury target genes in vivo and elucidate how Brachyury-mediated regulation contributes to early mouse developmental homeostasis and coordination, we made use of the reported Brachyury mutant Tc (Curtailed) mouse model (26). Importantly, a single copy of the WT Brachyury transgene rescues the Tc mutant phenotype (27). Thus, the Tc mouse line should be a useful model for studying Brachyury loss of function. As reported, we found that, by E8.0, Tc/Tc embryos become severely shortened compared with WT embryos (SI Appendix, Fig. S11A), and by E10.0, Tc/Tc embryos showed dramatic axial and neural fold closure defects (SI Ap- pendix, Fig. S11B). We next performed mRNA-seq to compare gene-expression profiles of WT and Tc/Tc embryos at both E7.5 ∼8.0 and E10.0 ∼10.5 (SI Appendix, Fig. S11C). The Brachyury gene becomes highly expressed at E7.5 ∼8.0 (Fig. 4A), suggesting that these are reasonable stages at which to study the direct impact of Brachyury. Next, we correlated Brachyury target gene information with up-/down-regulated genes in E7.5 ∼8.0 Tc/Tc embryos (Fig. 4B and Dataset S3). We detected 70 down-regulated and 53 up-regulated functional Brachyury target genes. Thus, it is likely that, although Brachyury targets a large number (1,942) of genes in the genome, only a small fraction of targeted genes showed gene-activation dependence on Brachyury, consistent with the previous observation that only 81 genes of ∼1,400 targeted genes are dependent of Xbra in Xenopus (12).

Brachyury-Mediated Regulation in Early Development. We next ex- amined whether our integrated genome-wide data are consistent with previous genetic studies and whether our data could link the molecular function of Brachyury to unique developmental and cellular processes. For this analysis, we only considered genes that are directly targeted and up-regulated by Brachyury in vitro and/or in vivo. We also scored each target gene according to its evolutionary conservation (Fig. 4C and Dataset S4). Indeed, consistent with the positive feedback loop between T and Fgf8 (28–30), we found that Fgf8 is under the control of four Brachyury- bound enhancer regions (Fig. 1A) and that Fgf8 is down-regulated Fig. 4. Brachyury-mediated gene-regulatory network. (A) ISH assay map- in both in vitro and in vivo perturbation experiments (Dataset S4). ping Brachyury gene expression in E7.75 WT embryo. PS, primitive streak. Fgf8 is also a common Brachyury target in mouse and X. tropicalis The publicly shared data were obtained from the EMAGE gene expression (Fig. 1F). In parallel, we found that Brachyury targets the Dusp6 database under the citation agreement (EMAGE ID 104; ref. 24). (B) Tc/Tc gene, a negative regulator of Fgf signaling (31) (Fig. 1A). Impor- embryo differential gene-expression data (Dataset S1) are correlated with tantly, this regulatory link between Brachyury and Dusp6 was ob- Brachyury-target gene information (Dataset S3) to identify functional in vivo Brachyury targets. A total of 70 up-regulated (P = 1.14E-6) and 53 down- served in all three species (Fig. 1F), suggesting that this pathway is regulated (P = 0.036) Brachyury target genes were identified (listed in highly conserved. Thus, it is likely that, after first potentiating the Dataset S4). (C) Functional Brachyury target genes and their known func- Fgf8 signaling, Brachyury also initiates a negative feedback control tions. Selected genes directly targeted and activated by Brachyury are listed. to ensure the proper attenuation of signaling. Previous genetic A confidence score is assigned to each gene by weighting in vitro and in vivo studies showed that both Foxa2-null and Brachyury-mutant mice perturbation and evolution conservation results. See Dataset S4 for calcu- displayed node/notochord defects that manifest between E8 and lation details. A value of 4 denotes the highest confidence. (D) Differential E10 (27, 32). Here, we found that Foxa2 is a functional and direct gene-expression analysis to characterize downstream developmental con- sequences of Brachyury loss-of-function with mRNA-seq to identify up-/down- Brachyury target gene (Figs. 1A and 3A and SI Appendix,Fig.S9A ∼ and B), suggesting that loss of Foxa2 expression in Brachyury- regulated genes in Tc/Tc embryos at E10.0 10.5. The log2 scale values of fragments per kilobase per million (FKPM) for genes in both WT and Tc/Tc positive cells might partially explain some of the Brachyury mutant samples are plotted in the graph. Dark green, twofold down; light green, phenotypes. These results are also consistent with the proposed eightfold down; orange, twofold up; red, eightfold up. The hematopoiesis and regulation between Brachyury and Foxa2 in organizer derivatives skeletal muscle markers (Hba-x, Hbb-y,andMyog) are highlighted in red circles. (node/notochord) described by Tamplin et al. (33). See also SI Appendix,Fig.S11and Datasets S1 and S4.

4482 | www.pnas.org/cgi/doi/10.1073/pnas.1402612111 Lolas et al. Downloaded by guest on October 2, 2021 We identified the Mab21l2 gene (SI Appendix, Fig. S8A), a genes (Hba-x and Hbb-y) were significantly down-regulated (Fig. potential bone morphogenetic protein (BMP) antagonist (34), as 4D). Importantly, these results agree well with previous observa- a Brachyury target gene. Although the regulation of Mab21l2 by tions that hemangioblast commitment is initiated by Brachyury- Brachyury is only found in the mouse, the Mab21l2 gene is down- positive cells in the primitive streak of the mouse embryo (39) and regulated in both in vitro and in vivo Brachyury loss-of-function thus further establish a molecular link between Brachyury and he- experiments (Dataset S4). Our results further suggest that matopoietic lineage specification. Brachyury might coordinate primary cilia formation and left– Consistent with the role of Brachyury in directing mesoderm right axis patterning via regulation of Foxj1, Cdh2,andTbx6— lineages, Brachyury targets and promotes the expression of muscle all of which have been shown to be critical for the establishment and chondrocyte regulators (Msgn1, Pax7, My17, Mfhx4,and – – of early embryo left right asymmetry (35 37). Consistent with Sox9). As expected, skeletal muscle development (Myog) was the function of Brachyury in regulating morphogenetic cell move- compromised in the E10.0∼10.5 Tc/Tc embryos (Fig. 4D), con- – ments, Brachyury up-regulates the expression of key epithelial sistent with its reported regulatory function in zebrafish (13). mesenchymal transition (EMT) and cell-mobility regulators (Sox4 and Bves). The functional link between Brachyury and Sox4 also Methods offers mechanistic insights into recent observations that Brachyury Mouse D3 (ATCC) ES cells were used for in vitro differentiation experiments. The promotes the EMT and metastatic behavior of human tumor cells Tc/J mouse line was obtained from the Jackson Laboratory. Custom-made Bra- (38). We also identified other cancer-related genes (Tpd52l1, Ybx1, chyury antibodies were used for ChIP-seq/exo experiments. Deep-sequencing and Irf1) as direct targets of Brachyury. was performed with the illumina HiSeq system. Detailed methods are available Strikingly, we found that Brachyury directly activates a set of in SI Appendix, SI Methods. Primer information is available in Dataset S5. hematopoietic regulators (Mixl1, Mllt3, NKX2-3, Sox6, POU4F1, and CD93). Most of these regulatory pathways are not conserved ACKNOWLEDGMENTS. We thank M. Haggart, S. Zheng, C. Morkunas, in zebrafish and Xenopus, suggesting that these are evolutionarily S. Moorehead, and Katie McDole for assistance. M.L. was a visiting PhD divergent networks (Dataset S4). Tc/Tc embryo mRNA-seq re- student at Janelia Farm Research Campus and was also supported partially ∼ by Conicyt Program PFB16. Z.L. is a Janelia junior fellow and was also supported sults at later stages of development (E10.0 10.5) confirmed down- as a predoctoral California Institute for Regenerative Medicine fellow at stream defects in hematopoiesis. Specifically, the fetal hemoglobin University of California, Berkeley.

1. Showell C, Binder O, Conlon FL (2004) T-box genes in early embryogenesis. Dev Dyn 22. Albert I, Wachi S, Jiang C, Pugh BF (2008) GeneTrack—a genomic data processing and BIOLOGY

229(1):201–218. visualization framework. Bioinformatics 24(10):1305–1306. DEVELOPMENTAL 2. Chesley P (1935) Development of the short-tailed mutant in the house mouse. J Exp 23. Bernstein BE, et al.; ENCODE Project Consortium (2012) An integrated encyclopedia of Zool 70(3):429–459. DNA elements in the human genome. Nature 489(7414):57–74. 3. Yanagisawa KO (1990) Does the T gene determine the anteroposterior axis of 24. Richardson L, et al. (2010) EMAGE mouse embryo spatial gene expression database: a mouse embryo? Jpn J Genet 65(5):287–297. 2010 update. Nucleic Acids Res 38(Database issue):D703–D709. 4. Gruneberg H (1958) Genetical studies on the skeleton of the mouse. XXIII. The de- 25. Niakan KK, et al. (2010) Sox17 promotes differentiation in mouse embryonic stem velopment of brachyury and anury. J Embryol Exp Morphol 6(3):424–443. cells by directly regulating extraembryonic gene expression and indirectly antago- 5. Gluecksohn-Schoenheimer S (1938) The development of two tailless mutants in the nizing self-renewal. Genes Dev 24(3):312–326. house mouse. Genetics 23(6):573–584. 26. Searle AG (1966) Curtailed, a new dominant T-allele in the house mouse. Genet Res 6. Herrmann BG, Kispert A (1994) The T genes in embryogenesis. Trends Genet 10(8): 7(1):86–95. 280–286. 27. Stott D, Kispert A, Herrmann BG (1993) Rescue of the tail defect of Brachyury mice. – 7. King T, Beddington RS, Brown NA (1998) The role of the brachyury gene in heart Genes Dev 7(2):197 203. development and left-right specification in the mouse. Mech Dev 79(1-2):29–37. 28. Ciruna B, Rossant J (2001) FGF signaling regulates mesoderm cell fate specification – 8. Kubo A, et al. (2004) Development of definitive endoderm from embryonic stem cells and morphogenetic movement at the primitive streak. Dev Cell 1(1):37 49. in culture. Development 131(7):1651–1662. 29. Tam PP, Loebel DA (2007) Gene function in mouse embryogenesis: Get set for gas- – 9. Gouon-Evans V, et al. (2006) BMP-4 is required for hepatic specification of mouse trulation. Nat Rev Genet 8(5):368 381. 30. Sun X, Meyers EN, Lewandoski M, Martin GR (1999) Targeted disruption of Fgf8 embryonic stem cell-derived definitive endoderm. Nat Biotechnol 24(11):1402–1411. 10. Kita-Matsuo H, et al. (2009) Lentiviral vectors and protocols for creation of stable causes failure of cell migration in the gastrulating mouse embryo. Genes Dev 13(14): 1834–1846. hESC lines for fluorescent tracking and drug resistance selection of cardiomyocytes. 31. Ekerot M, et al. (2008) Negative-feedback regulation of FGF signalling by DUSP6/ PLoS ONE 4(4):e5046. MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a conserved site 11. Liu Z, Scannell DR, Eisen MB, Tjian R (2011) Control of embryonic stem cell lineage within the DUSP6/MKP-3 gene promoter. Biochem J 412(2):287–298. commitment by core promoter factor, TAF3. Cell 146(5):720–731. 32. Ang SL, Rossant J (1994) HNF-3 beta is essential for node and notochord formation in 12. Gentsch GE, et al. (2013) In vivo T-box transcription factor profiling reveals joint mouse development. Cell 78(4):561–574. regulation of embryonic neuromesodermal bipotency. Cell Rep 4(6):1185–1196. 33. Tamplin OJ, et al. (2008) Microarray analysis of Foxa2 mutant mouse embryos reveals 13. Morley RH, et al. (2009) A gene regulatory network directed by zebrafish No tail novel gene expression and inductive roles for the gastrula organizer and its de- accounts for its roles in mesoderm formation. Proc Natl Acad Sci USA 106(10): rivatives. BMC Genomics 9:511. 3829–3834. 34. Baldessari D, Badaloni A, Longhi R, Zappavigna V, Consalez GG (2004) MAB21L2, 14. Gadue P, Huber TL, Paddison PJ, Keller GM (2006) Wnt and TGF-beta signaling are a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and required for the induction of an in vitro model of primitive streak formation using interacts with SMAD1. BMC Cell Biol 5(1):48. – embryonic stem cells. Proc Natl Acad Sci USA 103(45):16806 16811. 35. Tamakoshi T, et al. (2006) Roles of the Foxj1 and Inv genes in the left-right de- 15. Tesar PJ, et al. (2007) New cell lines from mouse epiblast share defining features with termination of internal organs in mice. Biochem Biophys Res Commun 339(3):932–938. – human embryonic stem cells. Nature 448(7150):196 199. 36. García-Castro MI, Vielmetter E, Bronner-Fraser M (2000) N-Cadherin, a cell adhesion 16. McLean CY, et al. (2010) GREAT improves functional interpretation of cis-regulatory molecule involved in establishment of embryonic left-right asymmetry. Science – regions. Nat Biotechnol 28(5):495 501. 288(5468):1047–1051. 17. Naiche LA, Harrelson Z, Kelly RG, Papaioannou VE (2005) T-box genes in vertebrate 37. Hadjantonakis AK, Pisano E, Papaioannou VE (2008) Tbx6 regulates left/right pat- development. Annu Rev Genet 39:219–239. terning in mouse embryos through effects on nodal cilia and perinodal signaling. 18. Kispert A, Hermann BG (1993) The Brachyury gene encodes a novel DNA binding PLoS ONE 3(6):e2511. protein. EMBO J 12(12):4898–4899. 38. Fernando RI, et al. (2010) The T-box transcription factor Brachyury promotes epi- 19. Bruneau BG, et al. (2001) A murine model of Holt-Oram syndrome defines roles of the thelial-mesenchymal transition in human tumor cells. J Clin Invest 120(2):533–544. T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106(6):709–721. 39. Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G (2004) Haemangioblast commit- 20. Evans AL, et al. (2012) Genomic targets of Brachyury (T) in differentiating mouse ment is initiated in the primitive streak of the mouse embryo. Nature 432(7017): embryonic stem cells. PLoS ONE 7(3):e33346. 625–630. 21. Rhee HS, Pugh BF (2011) Comprehensive genome-wide protein-DNA interactions 40. van Heeringen SJ, Veenstra GJ (2011) GimmeMotifs: A de novo motif prediction detected at single-nucleotide resolution. Cell 147(6):1408–1419. pipeline for ChIP-sequencing experiments. Bioinformatics 27(2):270–271.

Lolas et al. PNAS | March 25, 2014 | vol. 111 | no. 12 | 4483 Downloaded by guest on October 2, 2021