LETTERS The T-box transcription factor Eomesodermin acts upstream of Mesp1 to specify cardiac mesoderm during mouse gastrulation Ita Costello1, Inga-Marie Pimeisl2, Sarah Dräger2, Elizabeth K. Bikoff1, Elizabeth J. Robertson1,3 and Sebastian J. Arnold2,3 Instructive programmes guiding cell-fate decisions in the region of the primitive streak (APS), in close proximity to the developing mouse embryo are controlled by a few so-termed cardiovascular progenitors10,11. master regulators. Genetic studies demonstrate that the T-box Mesoderm formation and patterning along the proximodistal axis transcription factor Eomesodermin (Eomes) is essential for of the primitive streak is known to be regulated by dose-dependent epithelial-to-mesenchymal transition, mesoderm migration and Nodal/Smad2/3 activities7. The highest level of Nodal/Smad2/3 specification of definitive endoderm during gastrulation1. Here signalling is required to specify APS derivatives, namely the definitive we report that Eomes expression within the primitive streak endoderm, node and notochord5,6,8. The transcription factor Smad4, marks the earliest cardiac mesoderm and promotes formation and its DNA-binding partner the forkhead transcription factor Foxh1, of cardiovascular progenitors by directly activating the bHLH also play essential roles in APS specification6,12,13. Furthermore, (basic-helix-loop-helix) transcription factor gene Mesp1 the T-box transcription factor Eomes acts cooperatively with upstream of the core cardiac transcriptional machinery2–4. In the Nodal/Smad2/3 pathway to promote delamination of nascent marked contrast to Eomes/Nodal signalling interactions that mesoderm and specification of APS fates1. cooperatively regulate anterior–posterior axis patterning and Eomes expression is initiated in the prospective posterior aspect allocation of the definitive endoderm cell lineage1,5–8, formation of the epiblast at embryonic day 5.75 (E5.75; ref. 14). During of cardiac progenitors requires only low levels of Nodal activity gastrulation expression is maintained in the distal primitive streak14,15, accomplished through a Foxh1/Smad4-independent encompassing the same region where cranial, cardiac and paraxial mechanism. Collectively, our experiments demonstrate that mesodermal subcell populations are generated10. Inactivation of Eomes governs discrete context-dependent transcriptional Eomes in the epiblast results in a severe block in EMT and arrests programmes that sequentially specify cardiac and definitive development at gastrulation1. To further characterize Eomes functional endoderm progenitors during gastrulation. contributions within the mesodermal cell lineages we generated an EomesCre reporter allele. Cre messenger RNA expression recapitulates Much has been learned about the coordinated activities of key endogenous expression (Supplementary Fig. S1a), enabling derivation regulatory networks of transcription factors and growth-factor of a fate map of Eomes-expressing cells in later-stage embryos (Fig. 1). signalling pathways governing cell-fate decisions during gastrulation9. EomesCre=C males were mated to females carrying the ROSA26 R Nascent mesoderm is induced as epiblast cells ingress through the reporter allele16 and the resulting embryos stained for LacZ activity primitive streak and undergo epithelial-to-mesenchymal transition (Fig. 1d,e and Supplementary Fig. S1b). Surprisingly, we found in (EMT). Distinct mesodermal subpopulations become allocated E8.5 and E9.5 EomesCre ; ROSA26 R embryos that LacZ-expressing according to the timing and order of these cell movements. Thus cells are mostly absent from the somites, intermediate and lateral extra-embryonic mesoderm arises from the posterior primitive plate mesoderm and largely restricted to the head mesenchyme, streak, whereas cardiac, paraxial and lateral plate precursors emerge cardiac mesoderm and vasculature (Fig. 1d,e). As expected1, the sequentially as the primitive streak elongates towards the distal definitive endoderm and gut tube exclusively consist of LacZ-marked tip of the embryo. Fate-mapping experiments demonstrate that EomesCre -positive descendants. At later stages endodermal but not definitive endoderm progenitors are specified in the most anterior mesodermal components of developing organs derived from the gut 1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. 2University Medical Centre, Renal Department, Centre for Clinical Research, Breisacher Strasse 66, 79106 Freiburg, Germany. 3Correspondence should be addressed to E.J.R. or S.J.A. (e-mail: [email protected] or [email protected]) Received 26 January 2011; accepted 23 June 2011; published online 7 August 2011; DOI: 10.1038/ncb2304 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 © 2011 Macmillan Publishers Limited. All rights reserved. LETTERS a 15.2 kb b Wild type 15.2 kb Eomes RV H SE HRV locus (wild type) Targeted 8.0 kb Targeting construct PGK-neo TK Cre RV Targeted RV H H RV neo allele PGK- Cre c 8.0 kb Wild type 15.2 kb RV Reporter RV H Targeted H RV 8.0 kb allele Reporter Cre 6.2 kb 6.2 kb de Hm Hm He E8.5 He Hm Gt Gt He Gt E9.5 Eomes Cre × ROSA26R Eomes Cre × ROSA26R Figure 1 Fate mapping of Eomes Cre -expressing cells reveals selective the phosphoglycerate kinase–neomycin (PGK-neo) selection cassette and contributions to definitive endoderm and cardiovascular cell lineages. generate the reporter allele, as shown by Southern blot. (d,e) Fate-mapping (a) Targeting strategy used to generate the Eomes Cre reporter allele. Cre experiments demonstrate that descendants of Eomes Cre -expressing cells recombinase coding sequences were inserted into exon 1 of the Eomes contribute to the myocardium and endocardium of the heart (He), the locus. RV, EcoRV; H, HpaI; S, SphI; E, EagI; Cre, Cre recombinase; head mesenchyme (Hm), vasculature and the endoderm of the primary TK, thymidine kinase. (b) Embryonic stem cell clones were screened gut tube (Gt), but rarely colonize other mesoderm tissues formed from by Southern blot on EcoRV-digested DNA using a 30 probe (red line paraxial and lateral plate mesoderm. Sections were counterstained with in a) to detect wild-type and targeted alleles. (c) Correctly targeted eosin to highlight non-labelled cells. The black lines indicate the plane embryonic stem cells were transiently transfected with Cre to excise of section. tube are LacZ positive (Supplementary Fig. S1c). Eomes-expressing Brachyury, Fgf8 and Snail (ref. 1). However, in marked contrast, cells thus give rise to two discrete progenitor cell populations during expression of the early cardiac marker genes Myl7 (also known as gastrulation, namely the prospective cranial and cardiac mesoderm Mlc2a), Wnt2 and Nkx2.5 was absent (Fig. 2a). Moreover, we observe that emerge from the primitive streak at early stages, and APS severely reduced expression of early vascular marker genes such as derivatives giving rise to the definitive endoderm, node and notochord. Agtrl1, Rasgrp3 and Klhl6 (Fig. 2a). In marked contrast, EomesC cells are excluded from the majority To test whether loss of cardiac-gene expression reflects a cell- of mesodermal tissues derived from the paraxial and lateral plate autonomous Eomes requirement we examined the developmental mesoderm. These observations indicate that a discrete subpopulation potential of Eomes-null embryonic stem cells in the context of chimaeric of cells within the pregastrulation epiblast preferentially ingress and embryos. Eomes-null embryonic stem cells1 were injected into wild-type migrate anteriorly as a cohort to form the cardiac crescent and blastocysts carrying the ROSA26LacZ allele17 and the resulting embryos prospective head mesoderm. analysed by LacZ staining (Fig. 2b). As expected1, at E8.5 and E9.5 To directly examine whether Eomes function is required for Eomes-null cells efficiently contribute to the majority of mesodermal specification of cardiovascular progenitors, we analysed E7.5 embryos tissues but are entirely excluded from the forming gut tube. Notably, in carrying an epiblast-specific Eomes deletion (EomesCA=N ;Sox2.Cre; all cases examined (n D 10), the myocardium and endocardium of the ref. 1) by whole-mount in situ hybridization. Embryos lacking developing heart also exclusively consisted of wild-type LacZ-positive Eomes function strongly express mesodermal marker genes, including cells (Fig. 2c,d). Thus we conclude that Eomes plays an essential 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION © 2011 Macmillan Publishers Limited. All rights reserved. LETTERS a E7.5 E7.5 E7.5 E7.5 E7.5 E7.75 b c Hm Wild type Gt Gt Eomes –/– embryonic stem cells E7.5 E7.5 E7.5 E7.5 E7.5 E7.75 He Eomes mutant d ROSA26 LacZ blastocyst / wild type Hm E7.75 E7.75 E7.75 E9.5 E9.5 E8.5 Gt Wild type He Myl7 Wnt2 Nkx2.5 Agtrl1 Rasgrp3 Klhl6 Figure 2 Eomes functional loss disrupts specification of cardiovascular for generation of chimaeric embryos. Eomes-null embryonic stem progenitors. (a) Whole-mount in situ hybridization analysis of cells were introduced into wild-type ROSA26LacZ blastocysts. cardiac mesoderm (Myl7, Wnt2, Nkx2.5) and vascular (Agtrl1, (c,d) Histological sections of two independent LacZ -stained E9.5 Rasgrp3, Klhl6) markers in control and Eomes N=CA;Sox2.Cre-mutant chimaeric embryos were counterstained with eosin to identify embryos. Eomes mutants entirely lack expression of cardiac Eomes-mutant cell populations (pink). The myocardium and endocardium marker genes and show significantly reduced expression