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 BIOLOGY during streak formation. Target gene and mRNA-sequencing cor- divergent pathways. We further extended these studies to examine DEVELOPMENTAL 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. www.pnas.org/cgi/doi/10.1073/pnas.1402612111 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 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