Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development John E. Pimanda*†‡, Katrin Ottersbach*, Kathy Knezevic*, Sarah Kinston*, Wan Y. I. Chan*, Nicola K. Wilson*, Josette-Rene´ e Landry*, Andrew D. Wood*, Anja Kolb-Kokocinski§, Anthony R. Green*, David Tannahill§, Georges Lacaud¶, Valerie Kouskoff¶, and Berthold Go¨ ttgens*‡ *Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom; †Lowy Cancer Research Centre and Prince of Wales Clinical School, University of New South Wales, Sydney NSW 2052, Australia; §The Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom; and ¶Paterson Institute for Cancer Research, Manchester M20 4BX, United Kingdom Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved August 28, 2007 (received for review July 26, 2007) Conservation of the vertebrate body plan has been attributed to the up the subcircuit. Subcircuits that perform essential functions in evolutionary stability of gene-regulatory networks (GRNs). We de- building body parts have been termed the kernels of the GRN (16). scribe a regulatory circuit made up of Gata2, Fli1, and Scl/Tal1 and Disruption of TFs that make up the kernel often results in loss of their enhancers, Gata2-3, Fli1؉12, and Scl؉19, that operates during the body part, and hence the basic architecture of kernels is highly specification of hematopoiesis in the mouse embryo. We show that conserved through evolution (16). the Fli1؉12 enhancer, like the Gata2-3 and Scl؉19 enhancers, targets Scl, Gata2, and the Ets factor Fli1 are required for normal hematopoietic stem cells (HSCs) and relies on a combination of Ets, hematopoiesis in mice (reviewed in ref. 17) and are expressed in Gata, and E-Box motifs. We show that the Gata2-3 enhancer also uses avian hematopoietic clusters (18) and hematopoietic mesodermal a similar cluster of motifs and that Gata2, Fli1, and Scl are expressed precursors in frog and zebrafish embryos (19, 20). In this article we in embryonic day-11.5 dorsal aorta where HSCs originate and in fetal describe a GRN kernel composed of Gata2, Fli1, and Scl, and their liver where they multiply. The three HSC enhancers in these tissues respective cis-regulatory modules. Using transgenic mice and in vivo and in ES cell-derived hemangioblast equivalents are bound by each ChIP assays of embryonic hematopoietic tissues, we demonstrate of these transcription factors (TFs) and form a fully connected triad that this GRN kernel operates during key stages of mouse HSC that constitutes a previously undescribed example of both this net- specification in the aorta–gonad-mesonephros (AGM) region and work motif in mammalian development and a GRN kernel operating in the midgestation fetal liver (FL). during the specification of a mammalian stem cell. Results hemangioblast ͉ hematopoiesis ͉ hematopoietic stem cell ͉ The Fli1؉12 Hematopoietic Enhancer Targets Long-Term Repopulating network motif ͉ transcription factor network Blood Stem Cells in the Embryo. We have previously established that the Fli1ϩ12 enhancer directs lacZ expression to blood and endo- ematopoietic stem cells (HSCs) represent the best-character- theliuminF0 transgenic embryos (21). However, it was not known Hized adult multipotent stem cell population, with the availabil- whether the Fli1ϩ12 enhancer targeted expression to HSCs per se. ity of a plethora of markers and biological (clonogenic and trans- To evaluate the in vivo activity of the Fli1ϩ12 enhancer in detail, plantation) assays to identify long-term repopulating HSCs and we established stable transgenic lines (L5760 and L5754) with distinguish them from multipotent progenitors with only limited founders generated using a 500-bp fragment of the human self-renewal potential (1). Transcriptional regulation is a key mech- FLI1ϩ12 locus cloned downstream of the SV minimal promoter anism controlling the formation and subsequent behavior of HSCs and lacZ reporter (SV/lacZ/FLI1 ϩ 12 in Fig. 1Ai). Whole-mount (2). Both gain and loss of function studies have identified a number analysis of gastrulating embryonic day-7.5 (E7.5) embryos showed of transcription factors (TFs), including Scl/Tal 1 (3, 4), Gata2 (5) expression of the transgene in the region of extraembryonic me- and Runx1 (6, 7), as critical regulators of HSC development. soderm that characterizes primitive hematopoiesis in the early yolk Significantly, disruption of many of these TFs contributes to the sac (compare Fig. 1Aiii with Fig. 1Aii). This finding is consistent pathogenesis of hematological malignancies, emphasising the im- with expression in hematopoietic tissues and the vasculature during portance of transcriptional regulation in both normal and leukae- later stages of development (Fig. 1A iv–xiii). mic stem cells. To establish whether the Fli1ϩ12 enhancer-targeted expression Biological complexity does not correlate with gene number but to FL HSCs, we first characterized the surface phenotype of FL rather with the intricacy of gene regulation (8). Enhancers and cells targeted by the SV/lacZ/FLI1ϩ12 transgene. One of every 5.7 other cis-regulatory elements play a central role in the coordinated expression of genes and a number of tissue-specific regulatory elements of key HSC TFs, such as Scl and Gata2, have been Author contributions: J.E.P. and B.G. designed research; J.E.P., K.O., K.K., S.K., W.Y.I.C., identified (9–13). However, it is clear from studies in nonvertebrate N.K.W., J.-R.L., A.D.W., A.K.-K., G.L., and V.K. performed research; J.E.P., K.O., K.K., S.K., W.Y.I.C., N.K.W., J.-R.L., A.K.-K., A.R.G., D.T., G.L., V.K., and B.G. analyzed data; and J.E.P. model organisms, such as Drosophila and the sea urchin, that to and B.G. wrote the paper. comprehend developmental processes, it is necessary to move The authors declare no conflict of interest. beyond the study of individual genes and determine how regulatory This article is a PNAS Direct Submission. genes interact to form functional gene regulatory-networks (GRNs) (reviewed in ref. 14). Abbreviations: AGM, aorta–gonad-mesonephros; DA, dorsal aorta; En, embryonic day n; EB, embryoid body; FDG, fluoro-deoxy-D-glucose; FL, fetal liver; GRN, gene-regulatory Comprehensive analysis of the sea urchin endomesoderm GRN network; HSC, hematopoietic stem cell; TF, transcription factor. showed that they consist of assemblies of subcircuits made up of TF ‡To whom correspondence may be addressed. E-mail: [email protected] or genes and their target cis-regulatory modules, with each subcircuit [email protected]. performing a distinct regulatory function during development (15). This article contains supporting information online at www.pnas.org/cgi/content/full/ The linkages of these subcircuits are highly recursive with each 0707045104/DC1. cis-regulatory module receiving inputs from multiple TFs that make © 2007 by The National Academy of Sciences of the USA 17692–17697 ͉ PNAS ͉ November 6, 2007 ͉ vol. 104 ͉ no. 45 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0707045104 Downloaded by guest on September 25, 2021 Fig. 1. The FLI1ϩ12 hematopoi- etic enhancer targets blood stem A +12 B L5760 E12.5 Fetal Liver 10kb FLI1 Non transgenic 0.034% (i) cells. (A) The FLI1ϩ12 enhancer di- (i) rects reporter activity to blood and (i) SV-lacZ 500bp lacZ/FLI1 99.3% blood vessels. (i) Schematic diagram SV/ +12 of the human FLI1 locus. A fragment (ii)E7.5 (iii) E7.5 (iv) E9.5(v) E11.5 (vi) E12.5 (vii) of DNA corresponding to the FLI1ϩ12 region was used to gener- 0.003% (ii) 0 5 ate transgenic mice. (ii–vii) E7.5– C 1D S E12.5 X-Gal-stained whole-mount S FLI1 SV/lacZ +12/ C ϩ WT and L5760 SV/lacZ/FLI1 12 em- Non-TG TG (ii) (iii) bryos. (ii) E7.5 WT embryo with no (viii) (ix) (x) (xi) (xii) (xiii) 95.5% staining. (iii) E7.5 transgenic em- DA YS 0.245% (iii) Heart bryo showing staining within the DA extra-embryonic hematopoietic re- FL FL gion. (iv) E9.5 transgenic embryo 3.1552% showing staining of the heart x20 x40 x20 x40 x10 x20 lacZ CD48/41 chambers (boxed) and vitelline ves- sels (arrow). (v) E11.5 transgenic embryo showing staining within the FL (arrow) and blood vessels. (vi) E12.5 transgenic embryo showing staining in the vitelline vessels (arrow). (vii) Magnified view of the boxed area in vi showing FL staining (arrow). (viii–xiii) Paraffin sections of a E11.5 SV/lacZ/FLI1ϩ12 embryo. (viii) Sagittal section of the DA showing staining in the endothelium. (ix) High-power view of the DA showing staining in a hematopoietic cluster (arrow). (x) Section through the FL showing staining in hematopoietic cells. (xi) High-power view of x. (xii) Section through the yolk sac showing staining in the wall of a blood vessel. (xiii) Section through the heart showing endocardial staining. (B) Flow cytometry of FDG treated nontransgenic and transgenic E12.5 FLs from a litter of SV/lacZ/FLI1ϩ12 (L5760) ϫ WT crosses. CD150ϩ/CD48Ϫ/CD41Ϫ cells are enriched in the population targeted by the transgene. YS, yolk sac. CD150ϩ/CD48Ϫ/CD41Ϫ FL cells gives long-term multilineage re- hematopoietic regulatory elements of the Scl and Lyl1 genes (17, constitution in irradiated mice (22). FL cell suspensions were 23). Clustering of Ets and Gata sites [two Ets and one Gata site with prepared from E12.5 embryos and stained with the fluorescent defined spacing and orientation constraints constitutes the Ets/Ets/ ␤-galactosidase substrate, fluoro-deoxy-D-glucose (FDG). As Gata (E/E/G) signature] can be exploited in genome-wide compu- shown in Fig. 1B, Ϸ3% of FL cells from SV/lacZ/FLI1ϩ12 trans- tational screens to identify new hematopoietic stem/progenitor genic embryos express the transgene.