Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

Abstract

Rationale: The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown.

Objective: Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development.

Methods and results: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell-derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development.

Conclusions: Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development.

Keywords: BMP2 protein; developmental biology; lymphatic vessels; microRNAs.

Figure 1. Bmp2b signaling may function as an antilymphangiogenic cue in zebrafish embryos

A, Transverse section taken from the trunk region of 4 days postfertilization (dpf) wild-type and Tg(hsp70l:bmp2b)^fr13 embryos in Tg(kdrl:EGFP)^s843;Tg(fli1a.ep:DsRedEx)^um13 background. Overexpression of Bmp2b abrogates thoracic duct (TD; arrowhead in wild-type) formation. B, Quantification of percent TD formation measured over 10 segments within the trunk region. Compared with wild-type embryos (n=50), the formation of TD is substantially reduced in embryos with an elevated level of Bmp2b activity by heatshock treatment at either 26 hours postfertilization (hpf; n=39) or 50 hpf (n=36). C, Lateral view of wild-type and Bmp2b-overexpressing embryos at 4 dpf. TD (arrow) is absent in Tg(hsp70l:bmp2b)^fr13 embryo. Embryos shown have TgBAC(prox1:KalT4-UAS:uncTagRFP)^nim5;Tg(kdrl:GFP)^s843 background. D, Quantification of percent TD formation in wild-type and Tg(hsp70l:bmp2b)^fr13 embryos measured over 10 segments (n=36 for wild-type embryos, and n=32 for Tg(hsp70l:bmp2b)^fr13 embryos). E, Number of ventral sprouts from the TD (*) increased in 1 μm DMH1-treated embryos compared with DMSO-treated control embryos. F, Quantification of the numbers of ventral sprouts from the TD in control and DMH1-treated embryos (n=23 for DMSO-treated control embryos and n=25 for DMH1-treated embryos). Compared with control group, DMH1-treated embryos had a significant increase in the number of ventral sprouts (P<0.001). G, Quantification of flow-sorted prox1a:RFP⁺, fli1:EGFP⁺ lymphatic endothelial cells relative to all fli1:EGFP⁺ ECs in DMSO-treated or DMH1-treated embryos (N=3). CV indicates cardinal vein; and DA, dorsal aorta. Scale bars: 25 μm (A), 50 μm (C), and 100 μm (E).

Figure 2. Antilymphangiogenic effects of Bmp2b signaling are mediated by SMAD proteins

A, Four days postfertilization Tg(hsp70l:bmp2b)^fr13;TgBAC(prox1:KalT4-UAS:uncTagRFP)^nim5 embryos treated with DMSO, 1 μm DMH1 (Smad1/5/8 inhibitor), or 10 μm U0126 (Erk1/2 inhibitor). Arrow points the TD in DMH1-treated embryos. Asterisks mark lack of TD. B, Quantification of percent TD formation in DMSO, DMH1, or U0126-treated Tg(hsp70l:bmp2b)^fr13 embryos measured over 10 segments (n=30 for DMSO treatment, 37 for DMH1 treatment, and 32 for U0126 treatment). The percentage of Tg(hsp70l:bmp2b)^fr13 embryos with lymphatic structure development increased significantly between DMSO- and DMH1-treated embryos (25% and 94%, respectively; P<001), whereas U0126-treated embryos were unaffected (21%). CV indicates cardinal vein; DA, dorsal aorta; and TD, thoracic duct. Scale bar, 50 μm.

Figure 3. BMP2 attenuates lymphatic endothelial cell (LEC) differentiation from cultured mouse embryoid bodies (EBs)

A, Mouse EBs were prepared by hanging drop and then grown in 2-dimensional culture to induce lymphatic differentiation. At EB day 8, the expression of Vegfr2 and Prox1 increased together (arrow), indicating that LECs emerge within the EBs. B, Differentiation of lymphatic endothelial cells (CD31⁺/LYVE1⁺) in mouse EBs was increased in the presence of VEGF-C. C, Differentiated lymphatic endothelial cells within mouse EBs express hallmark of lymphatic lineage–specific genes, including LYVE1 (red) and Podoplanin (blue). D, Representative micrographs of the periphery of EBs on treatment with VEGF-C, BMP2, VEGF-C and BMP2, or with VEGF-C, BMP2, and DMH1. E, Quantification of (D) measuring the ratio of lymphatic vessel area (LYVE1⁺/CD31⁺) to total vasculature (CD31⁺) at the EB periphery (N=3; 4–6 EBs analyzed per condition per experiment). Scale bars: 50 μm (B), 50 μm (C), and 100 μm (D).

Figure 4. Different levels of BMP signaling activity in blood endothelial cells (BECs) and lymphatic endothelial cells (LECs)

A, Emerging parachordal lymphangioblasts (red arrows), the lympahtic progenitors in zebrafish, lack BRE:mCherry expression at 60 hours postfertilization, whereas endothelial cells (ECs) within intersegmental vessels (yellow arrowheads) are strongly positive for BRE:mCherry. B, LECs are largely devoid of BMP signaling activity. Arrows point to mCherry⁻/EGFP⁺ LECs, and arrowheads point to mCherry⁺/EGFP⁺ BECs. C, Quantification of the numbers of EGFP⁺ and EGFP⁺/mCherry⁺ ECs within the dorsal aorta, cardinal vein, and thoracic duct (TD; N=3; 10 embryos per experiment). D, Transverse section of embryonic day (E) 11.5 mouse embryos stained with PROX1 and P-SMAD1/5/8 antibodies. Two sections adjacent to each other are shown. p-SMAD1/5/8 staining is largely absent in developing LS but is strongly shown (arrowheads) in cardinal vein (CV). E, Transverse section of E10.5 and E11.5 Tg(BRE:EGFP) mouse embryos through the jugular region. PROX1 (red) and PECAM (blue) are visualized by antibody staining. Areas containing the wall of CV are shown. Arrows point to LECs within the vein wall, and arrowheads point to delaminated LECs. F, Quantification of the numbers of EGFP⁺ and EGFP⁺/PROX1⁺ ECs in CV and thoracic duct (TD; N=4). DA indicates dorsal aorta; ISV, intersegmental vessel; LS, lymph sac; and PL, parachordal lymphangioblast. Scale bars: 50 μm (A), 50 μm (B), 20 μm (C), and 20 μm (E).

Figure 5. Bmp2b signaling selectively represses the expression of prox1a in zebrafish embryos

A, Expression of prox1a mRNA substantially decreased on Bmp2b stimulation. Quantitative reverse-transcription polymerase chain reaction on selected markers for lymphatic endothelial cells (LECs) and blood endothelial cells (ECs) from fli1a:EGFP⁺ ECs isolated from 72 hours postfertilization (hpf) wild-type and Tg(hsp70l:bmp2b)^fr13 embryos after heatshock at 50 hpf. B, Expression level of PROX1was substantially downregulated on BMP2 stimulation in hLECs starting at 60 minutes (N=4). C, Inhibition of SMAD4 relieves the suppression of PROX1 by BMP2 signaling.

Figure 6. BMP2/Bmp2b signaling induces expression of miR-31 and miR-181a in zebrafish embryos and human lymphatic endothelial cells (hLECs)

A, miRNA polymerase chain reaction array expression analysis of cultured HDMVEC-Ly (hLECs) cells after treatment with 50 ng/mL BMP2 for 1 hour. Several miRNAs with significant expression changes were found, including miR-31 and miR-181a (N=4). Only miRNAs whose expression level exhibited statistically significant changes (P<0.05) in response to BMP2 stimulation are shown. B, Time course of miR-31 and miR-181a expression from fli1a:EGFP⁺ ECs isolated at indicated times from wild-type and Tg(hsp70l:bmp2b)^fr13 embryos after heatshock at 26 hours postfertilization (N=4). C, siRNA knockdown of SMAD4 in LECs prevented BMP2-mediated induction of miR-31 and miR-181a. Additionally, miR-31 and miR-181a expression levels after BMP2 treatment in the SMAD4 knockdown were decreased compared with vehicle treatment, suggesting potential negative feedback regulation (N=4). D, Pretreatment of hLECs with 5 μg/ mL actinomycin D to block transcription completely inhibited BMP2-induced expression of miR-31 and miR-181a, suggesting that BMP2-induced upregulation of miR-31 and miR-181a may require transcriptional activation of these miRNAs (N=4).

Figure 7. BMP2 signaling represses the expression of PROX1 via miRNA-dependent mechanism

A, Confocal projection of Tg(hsp70l:bmp2b)^fr13 embryos injected with 5 ng control, miR-31, or miR-181a MOs, followed by heatshock at 26 hours postfertilization. Inhibition of miR-31 or miR-181a can partially rescue the lymphatic defects induced by Bmp2b overexpression. Arrows point rescued thoracic duct (TD) in MO-injected embryos. B, Quantification of percent TD formation in control, miR-31, or miR-181a MO-injected wild-type and Tg(hsp70l:bmp2b)^fr13 embryos measured over 10 segments (n>40 for all conditions). TD formation was largely unaffected by MO knockdown of miR-31 or miR-181a in wild-type embryos. However, MO-mediated knockdown of miR-31 or miR-181a in the Tg(hsp70l:bmp2b)^fr13 background resulted in 50% to 70% of embryos forming at least a partial TD and between 20% to 25% forming a complete TD compared with 25% and 0% in control and MO-injected, respectively (P<0.001 for both groups). Scale bar, 50 μm. C, During development, active BMP2 signaling promotes the expression of miR-31/miR-181a in blood endothelial cells (blue) and, therefore, aids BECs to maintain their fate as venous endothelial cells. However, in presumptive lymphatic endothelial cells (green), the activity of BMP2 signaling is attenuated by unknown mechanism, thereby releasing miRNA-mediated repression of PROX1. CV indicates cardinal vein; and DA, dorsal aorta.