Etsrp/Etv2 is directly regulated by Foxc1a/b in the zebrafish angioblast

Abstract

Rationale: Endothelial cells are developmentally derived from angioblasts specified in the mesodermal germ cell layer. The transcription factor etsrp/etv2 is at the top of the known genetic hierarchy for angioblast development. The transcriptional events that induce etsrp expression and angioblast specification are not well understood.

Objective: We generated etsrp:gfp transgenic zebrafish and used them to identify regulatory regions and transcription factors critical for etsrp expression and angioblast specification from mesoderm.

Methods and results: To investigate the mechanisms that initiate angioblast cell transcription during embryogenesis, we have performed promoter analysis of the etsrp locus in zebrafish. We describe three enhancer elements sufficient for endothelial gene expression when place in front of a heterologous promoter. The deletion of all 3 regulatory regions led to a near complete loss of endothelial expression from the etsrp promoter. One of the enhancers, located 2.3 kb upstream of etsrp contains a consensus FOX binding site that binds Foxc1a and Foxc1b in vitro by EMSA and in vivo using ChIP. Combined knockdown of foxc1a/b, using morpholinos, led to a significant decrease in etsrp expression at early developmental stages as measured by quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization. Decreased expression of primitive erythrocyte genes scl and gata1 was also observed, whereas pronephric gene pax2a was relatively normal in expression level and pattern.

Conclusions: These findings identify mesodermal foxc1a/b as a direct upstream regulator of etsrp in angioblasts. This establishes a new molecular link in the process of mesoderm specification into angioblast.

Figure 1. Tg(−2.3etsrp:gfp) contains two evolutionarily conserved regions and faithfully recapitulates the endogenous expression pattern of etsrp

(A) The etsrp gene locus with conserved regions up1 and int2 along with the region corresponding to tg(−2.3etsrp:gfp) highlighted. The up1 region is approximately equidistant from the transcription start sites of etsrp and the adjacent fli1b gene and int2 is located within intron 2 of etsrp. Conserved regions between zebrafish and pufferfish were identified using the ECR Browser (http://ecrbrowser.dcode.org) and the locus image is adapted from this website. (B-S) Fluorescent images of tg(−2.3etsrp:gfp) panels B-D, H-J, N, P, and R and in situ hybridization for etsrp panels E-G, K-M, O, Q, and S demonstrating near perfect correlation of expression between the transgene and endogenous gene at different developmental stages. (B-G) 10-somite stage; (H-M) 18-somite stage; (N-S) 24 hours post fertilization (hpf).

Figure 2. Evolutionarily conserved regions up1 and int2 are sufficient but not necessary for tg(−2.3etsrp:gfp) expression in endothelial cells

(A and B) The conserved region up1 (A) or int2 (B) was placed in front of the minimal gata2 promoter driving GFP expression and germline transgenics generated. Both regions are sufficient to drive GFP expression in the developing vasculature as demonstrated by fluorescent images of whole embryo and trunk vasculature at 24hpf. (C) Up1 and int2 are not necessary for strong vascular expression from the etsrp promoter transgene. Deletion of both up1 and int2 from the GFP reporter lines does not eliminate vascular expression as demonstrated by tg(−1.8etsrpEx2:gfp), suggesting that more regulatory elements are present. The numbers in the whole embryo image represents the number of germline transgenic fish lines expressing vascular GFP versus the total number of lines examined. Note that up1 drives stronger expression in the dorsal aorta and its deletion results in decreased relative expression in the dorsal aorta (A and C).

Figure 3. An unconserved region at −110 to −75 accounts for the majority of the remaining promoter activity in endothelial cells

(A-C) Tg(−0.110etsrp:mcherry) containing only 110 base pairs of the etsrp promoter is sufficient for vascular expression at 24hpf. (D-F) Deletion of 35 base pairs from this site tg(−0.075etsrp:mcherry) abolishes expression in the vasculature. (A-F) mCherry was used as a reporter for the element being tested and GFP was driven by a constitutive cardiac promoter in the same transgene to identify transgenic germlines independently of the element being tested. (G-I) Deletion of up1, int2, and −0.110/−0.075, tg(−1.8Δ110/75Ex2:gfp), almost completely abolishes expression from the etsrp promoter, compare expression in H and I to Figure 2C, suggesting these three regions are critical for high levels of etsrp expression in the developing vasculature.

Figure 4. Up1 enhancer activity is present at −2266 to −2014

(A) Schematic of the up1 fragments tested for enhancer activity using the gata2 minimal promoter and GFP reporter. Number of lines demonstrating vascular expression out of total lines examined is noted. (B-I) Fluorescent images demonstrating that fragment AB (−2266 to −2014) is the minimal region necessary for vascular expression from the up1 enhancer. Fragments A (F and G) and BCD (H and I) exhibit some nonvascular expression presumably due to insertional enhancer trapping effects.

Figure 5. Multiple protein binding sites are present in the AB region of the up1 enhancer

(A) Evolutionarily conserved sequence between different fish species identified using clustalW analysis at the AB region. Overlapping EMSA probe sequences are underlined and labeled Up1-(1-5). (B) EMSA using the oligonucleotide probes defined in (A) and nuclear protein extracts, NE, from HUVEC or PAE cells. Unlabeled probe competition, Comp. +/-, was used to define specific binding protein complexes denoted with an asterisk (*). All oligos bound specific, well-defined proteins except for Up1-3. F, free probe.

Figure 6. Foxc1a/b binds to up1 in vitro and in vivo

(A) EMSA using Up1-1 probe and HUVEC nuclear protein extracts, NE, demonstrates that the FoxC1/2 consensus binding site oligo can compete for binding with the Up1-1 probe while Cepbα, Gata, and Evi1 consensus binding site oligos cannot. B, bound; NS, nonspecific; F, free probe. (B) EMSA demonstrating Up1-1 probe bound to in vitro synthesized Foxc1a and Foxc1b protein. (C) Chromatin immunoprecipitation, ChIP, from wild-type zebrafish embryos (−) or embryos expressing a foxc1a-myc. Up1 primers detect enrichment in embryos expressing foxc1a-myc while negative control rhodopsin, rho, primers do not. (D) The Up1-1 sequence, 5′-TGTTTGTTT-3′, contains a FoxC1 consensus binding site, 5′-(T/G)(G/C)(T/R)(T/Y)T(A/G)TTT-3′.

Figure 7. Morpholino knockdown of foxc1a/b results in decreased angioblasts and primitive erythrocytes

(A-L) Dorsal view flatmounts, anterior up, of 6-8 somite stage embryos injected with control morpholino, C-MO, or morpholinos to knockdown expression of both foxc1a and foxc1b, dMO. In situ hybridization for tg(−2.3etsrp:gfp) or the genes noted at the center of each pair of panels was performed to assay the affects of foxc1a/b knockdown. Representative images of more than 10 embryos per group. Note the decreased expression of tg(−2.3etsrp:gfp), etsrp, fli1a, scl, and gata1 in dMO treated embryos especially at the posterior lateral plate mesoderm (bracketed). Expression of pax2a in the intermediate mesoderm is not affected by foxc1a/b knockdown except at the pronephric primordia, arrow in L, and nervous system expression is unaffected. (M) Quantitative RT-PCR demonstrates reduced expression of etsrp, fli1a, scl, and gata1 in dMO treated embryos as compared to C-MO treated embryos. All groups were normalized to β-actin and expression in uninjected wildtype embryos was set at 100%. Asterisks (*) denote statistically significant changes as determines with Students t-test (p<0.05).