Liu F et al. (2008), Fli1 acts at the top of the transcriptional net...

XB-ART-38293

Curr Biol 2008 Aug 26;1816:1234-40. doi: 10.1016/j.cub.2008.07.048.

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Fli1 acts at the top of the transcriptional network driving blood and endothelial development.

Liu F , Walmsley M , Rodaway A , Patient R .

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Blood and endothelium arise in close association during development, possibly from a common precursor, the hemangioblast [1-4]. Genes essential for blood and endothelial development contain functional ETS binding sites, and binding and expression data implicate the transcription factor, friend leukaemia integration 1 (Fli1) [5-10]. However, loss-of-function phenotypes in mice, although suffering both blood and endothelial defects, have thus far precluded the conclusion that Fli1 is essential for these two lineages [11, 12]. By using Xenopus and zebrafish embryos, we show that loss of Fli1 function results in a substantial reduction or absence of hemangioblasts, revealing an absolute requirement. TUNEL assays show that the cells are eventually lost by apoptosis, but only after the regulatory circuit has been disrupted by loss of Fli1. In addition, a constitutively active form of Fli1 is sufficient to induce expression of key hemangioblast genes, such as Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1. Epistasis assays show that Fli1 expression is induced by Bmp signaling or Cloche, depending on the hemangioblast population, and in both cases Fli1 acts upstream of Gata2, Scl, Lmo2, and Etsrp. Taken together, these results place Fli1 at the top of the transcriptional regulatory hierarchy for hemangioblast specification in vertebrate embryos.

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Species referenced: Xenopus laevis
Genes referenced: aplnr bmp4 cadm1 etv2 fli1 gata2 hba4 kdr kit lmo2 mpo myod1 nkx2-5 pax2 pax8 prdm1 runx1 spib st14 tal1
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	Figure 1. Fli1 Is Required Upstream of Gata2 for the Primitive Hemangioblast Program(A–L) Fli1 requirement for the primitive hemangioblast. Xenopus embryos injected with Fli1 MO in the DMZ at the 4-cell stage were processed for in situ hybridization at stage 17, either as whole mounts (WISH) (A–H, J, K) or as sections (I, L).(M–X) Gata2 morphants and rescue experiments confirm that Fli1 is at the top of the primitive hemangioblast hierarchy. Gata2 MOs were injected into the DMZ at the 4-cell stage and embryos were processd for WISH at stages 16/17. Expression of SpiB (S–U) and Runx1 (V–X) was partially rescued by coinjection of mRNA for Scl, Lmo2, and Gata2.Arrows mark the primitive hemangioblast population. Proportion of embryos with phenotype shown indicated in top right corner.
	Figure 2. Fli1 Is Required Upstream of Gata2 for the Definitive Hemangioblast Program(A–L) Fli1 requirement for the definitive hemangioblast. Xenopus embryos injected with Fli1 MO in the VMZ at the 4-cell stage were processed for WISH at stage 26. Note remaining DLP signal in (B) marks the pronephric duct (PND). Red outlines hemangioblasts, black outlines PND. Black arrows, DLP; red arrows, pVBI.(M–T) Gata2 morphants confirm that Fli1 is at the top of the definitive hemangioblast hierarchy. Embryos injected with Gata2 MOs in the VMZ at the 4-cell stage were processed for WISH for expression in the DLP (arrows) at stages shown.
	fli1 (Fli-1 proto-oncogene, ETS transcription factor) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	kdr (kinase insert domain receptor (a type III receptor tyrosine kinase)) gene expression in Xenopus laevis embryo, assayedvia in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	gata2 (GATA binding protein 2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	lmo2 (LIM domain only 2 (rhombotin-like 1)) gene expression in Xenopus laevis embryo, NF stage 24, assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	aplnr (apelin receptor) gene expression in Xenopus laevis embryos, NF stage 26, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	Figure S2. Titration of Gata2 MOs. A 1:1 mix of Gata2A and Gata2B MOs was injected into two ventral blastomeres at the 8 cell stage, the embryos were grown to stage 32 and probed for αT4globin expression by in situ hybridisation. A total of 10, 25 and 50 ng was used. 25 ng was optimal (C,F) compared to controls (A,D) and 50ng was toxic (data not shown). A-C, lateral views, D-F ventral views.
	Figure S3. Loss of gene expression in the pVBI and PND in Fli1 morphants. 40 ng Fli1 MO was injected into the VMZ at the 4 cell stage, a region containing the precursors of the pVBI and PND as well as the DLP. Runx1 and αT4globin (blood genes not expressed in the DLP) were down regulated in Fli1 morphants (red arrows, A-D) along with PND markers Pax2 and Pax8 (blue arrows, E-H), although the PND genes were less sensitive to the MO than the blood genes. Partial down regulation of genes in the aVBI results from leakage of MO from posterior to anterior at time of injection.
	Figure S4. Control tissues and genes abutting the anterior hemangioblast are unaffected in Fli1 morphants. 40 ng Fli1 MO was injected into the DMZ at the 4/8 cell stage and embryos were grown to stage 17. Rohon Beard neuron expression of Runx1 (A,B) and somite expression of Lmo2 (C,D) was unchanged in Fli1 morphants. MyoD, Nkx2.5, Blimp1 and Bmp4 expression abuts the anterior hemangioblast but in no case does expression of these genes increase in the hemangioblast territory (asterisks) in Fli1 morphants (E-L), indicating that in the morphants re-specification is not the reason for lost hemangioblast gene expression.
	Figure S5. Down regulation of hemangioblast markers occurs before apoptosis in the primitive and definitive hemangioblast populations. (A-D) TUNEL assay using in situ cell death assay kit. At st14, Scl expression is downregulated (A,B red circles) but there is no apoptosis, whereas at st17, apoptosis is clear (D, white dots within white circle) and little Scl expression remains (D, black arrows) compared with controls (C, red circle). (G-J) TUNEL assay using Apoptag kit. At st24, although Scl expression is absent in Fli1 morphants (E,F) [apparent staining just above the black arrow is trapping], no apoptosis is seen in the DLP (G,H, arrows), but some is seen in the somites of both control and morphant embryos. At st26, TUNEL signals are detected in Fli1 morphants (J, arrow) which includes the DLP and PND.
	tal1 st17
	tal1 (T-cell acute lymphocytic leukemia 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	hba4 (alpha-T4 globin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	prdm1(PR domain containing 1) gene expression in stage 17 Xenopus laevis embryos as assayed by in situ hybridization.