HoxB5 is an upstream transcriptional switch for differentiation of the vascular endothelium from precursor cells

Abstract

Endothelial cells differentiate from mesoderm-derived precursors to initiate the earliest events in vascular development. Although the signaling events that regulate the successive steps of vascular development are known in some detail, the transcriptional processes that regulate the first steps in vasculogenesis are not well defined. We have studied the regulatory mechanisms of flk1 expression as a model to understand the upstream events in endothelial cell differentiation, since flk1 is the earliest marker of endothelial precursors. Using a variety of biochemical approaches, we identified a cis-acting element in the first intron of the flk1 gene that is required for endothelium-dependent expression in transgenic reporter gene assays. Using the yeast one-hybrid system, we identified HoxB5 as the transcription factor that binds this cis-acting element, the HoxB5-binding element (HBE). HoxB5 mRNA colocalized with flk1 expression in differentiating embryoid bodies, and HoxB5 potently transactivated the flk1 promoter in an HBE-dependent fashion in transient-transfection assays. Overexpression of HoxB5 led to expansion of flk1(+) angioblasts in differentiating embryoid bodies and increased the number of PECAM (platelet-endothelial cell adhesion molecule)-positive primitive blood vessels. HoxB5 is necessary and sufficient to activate the cell-intrinsic events that regulate the differentiation of angioblasts and mature endothelial cells from their mesoderm-derived precursors.

FIG. 1.

Identification of cis-acting elements in the mouse flk1 first-intronic enhancer. (A) Scheme of mouse flk1 genomic locus and transgenic construct. A total of 940 bp (positions from −640 to +299) of the 5′-flanking sequence of the mouse flk1 gene and 510 bp of the first intronic enhancer are sufficient to target lacZ expression to the vascular endothelium in transgenic mice. (B) Diagram of the general approach undertaken to identify cis-acting elements in the mouse flk1 first intronic enhancer. (C) Protein-binding site identification by in vitro DNase I footprinting. A single footprint, located between bp 150 and 195 in the intronic enhancer, was detected on both sense and antisense strands. The dark line denotes the putative binding site for DNA-binding proteins. (D) Analysis of cell type differences in DNase I footprinting patterns. The labeled probe was incubated with nuclear extracts from MECs, Py-4-1, C166, bEnd.3, and C2C12 cells, or BSA prior to digestion with DNase I.

FIG. 2.

Identification of the minimal DNA sequences in the flk1 intronic enhancer necessary for nuclear protein interactions. (A) Nuclear protein interactions with the flk1 cis-acting element detected by EMSA. The probe corresponding to bp 150 to 195 of the mouse flk1 first-intronic enhancer was used to characterize nuclear protein interactions with endothelial and nonendothelial nuclear extracts. The arrow denotes a rapidly migrating complex, while the asterisk denotes a more intense, slowly migrating complex. (B) Mutant oligonucleotides used in EMSA. Residues that differ from the wild-type sequence are in boldface. (C) EMSA with C166 nuclear extract with mutant probes described in panel B. (D) Comparison of partial sequences of the mouse and human flk1 first introns. Identical residues are highlighted, and the nuclear protein-binding site identified by DNase I footprinting and EMSA is boxed.

FIG. 3.

The nuclear protein-binding site is required for in vivo activity of the mouse flk1 promoter in transgenic mouse embryos. (A) Representative whole-mount β-galactosidase-stained E11.0 embryos. Transgenic mouse embryos expressing β-galactosidase under control of the flk1 promoter-enhancer stain the vascular endothelium diffusely and strongly. In contrast, a 5-bp mutation within the nuclear protein-binding site identified by footprinting resulted in the loss of β-galactosidase expression in most transgenic embryos. (B) Summary of in vivo activity of the wild-type and mutant transgenic constructs.

FIG. 4.

Identification of HoxB5 binding to the flk1 enhancer in a yeast one-hybrid screen. (A) β-Galactosidase assays of clones transfected with the indicated plasmids grown on synthetic complete medium lacking uracil to test for specific DNA-protein interactions. (B) Yeast transfectants were grown under growth-resistant conditions (−His, −Leu, +3-amino-1,2,4-triazole). Growth was detected only in yeast transfected with both the wild-type flk1 intronic enhancer motif (HBE) and the HoxB5 expression plasmid. (C) EMSA examining the binding of the labeled HBE with nuclear proteins in C166 cell extract in the presence of excess unlabeled HBE (+) or with mutant or wild-type HoxB5-binding element LP2 as marked. (D) EMSA with recombinant GST-HoxB5 (or GST alone) indicating specific binding to the HBE that is competed away by excess specific (+) competitor but not by a mutant sequence (NS). In addition, migration of this specific activity is shifted with a GST antibody and not by an isotypic control antibody (NS). SB, specific binding; SS, supershift.

FIG. 5.

Transactivation of the mouse flk1 promoter-enhancer by HoxB5 in transient-transfection assays. (A) Dose-response analysis of transactivation of the flk1 promoter-enhancer construct pflk1/en3 by HoxB5. pflk1/en3 (1 μg) was transiently transfected with the indicated doses of pECH/HoxB5 or vector pECH in MECs. pCMV-βgal was cotransfected to normalize transfection efficiency. The ratio of luciferase activity to β-galactosidase activity in each sample served as a measure of the normalized luciferase activity. The normalized luciferase activity was expressed as a percentage of pflk1/en3. (B) The ability of HoxB5 to transactivate the flk1 promoter was examined in the presence or absence of an intact HBE. HoxB5 (500 ng) was transfected along with wild-type pflk1/en3 (□) or with a mutant containing a 4-bp mutation in the HBE (▪). The results were normalized by cotransfection with pCMV-βgal. (C) Comparison of transactivation of the flk1 promoter-enhancer by HoxB5 and its adjacent cluster mate HoxB6.

FIG. 6.

Expression of flk1 and HoxB5 mRNA in differentiating embryoid bodies. (A) To assess the time course of HoxB5 and flk1 mRNA expression, embryoid bodies were differentiated for 2, 4, 6, 8, or 10 days, and total RNA was extracted from the cells at the indicated times. The specific genes were detected by RT-PCR. GAPDH served as an RNA integrity and normalization control. (B) To determine whether HoxB5 is expressed in flk1⁺ populations, embryoid bodies were differentiated for 4 days and then sorted into flk1⁺ and flk1⁻ populations. Total RNA was isolated from 10⁶ sorted cells from each population. RT-PCR analysis demonstrated expression and relative enrichment of HoxB5 mRNA in the flk1⁺ cell population.

FIG. 7.

Increased flk1⁺ cells in embryoid bodies stably expressing HoxB5. (A) Single-cell suspensions were prepared from wild-type or stably transfected embryoid bodies at day 4, and the expression of flk1 was analyzed by flow cytometry with phycoerythrin-conjugated rat anti-mouse flk1 antibody. The y axis represents the relative cell number; the x axis represents the fluorescence intensity. Gray lines indicate negative control, light lines represent results obtained with the addition of specific antibody, and dark lines represent the flk1⁺ population. The percentages of cells that fall within the indicated gate are noted. Values of <2% represent background staining. A significantly increased number of cells expressing the mouse flk1 gene were observed in the HoxB5-transfected embryoid bodies. (B) Comparison of flk1⁺ cells in HoxB5- and HoxB6-overexpressing embryoid bodies.

FIG. 8.

HoxB5 is a positive regulator of endothelial cell differentiation and primitive blood vessel formation. (A) Immunostaining for PECAM. The indicated stably transfected embryonic stem cells were differentiated, fixed on day 8, and stained with antibody to PECAM, followed by secondary staining with an Alexafluor 488-conjugated antibody. Magnification, ×20. (B) PECAM sorting was performed to determine the absolute number of endothelial cells in embryoid bodies at day 8 of differentiation. The y axis represents relative cell number, and the x axis represents fluorescence intensity. Gray lines indicate negative control (secondary antibody only), light lines represent results obtained with the addition of the specific antibody, and dark lines represent the PECAM⁺ cell population. The percentages of cells that fall with the indicated gate are noted.