Abstract
Pharyngeal arch artery (PAA) progenitors undergo proliferative expansion and angioblast differentiation to build vessels connecting the heart with the dorsal aortae. However, it remains unclear whether and how these two processes are orchestrated. Here we demonstrate that Tmem88 is required to fine-tune PAA progenitor proliferation and differentiation. Loss of zebrafish tmem88a/b leads to an excessive expansion and a failure of differentiation of PAA progenitors. Moreover, tmem88a/b deficiency enhances cyclin D1 expression in PAA progenitors via aberrant Wnt signal activation. Mechanistically, cyclin D1-CDK4/6 promotes progenitor proliferation through accelerating the G1/S transition while suppressing angioblast differentiation by phosphorylating Nkx2.5/Smad3. Ectodermal Wnt2bb signaling is confined by Tmem88 in PAA progenitors to ensure a balance between proliferation and differentiation. Therefore, the proliferation and angioblast differentiation of PAA progenitors manifest an inverse relationship and are delicately regulated by cell cycle machinery downstream of the Tmem88-Wnt pathway.
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Acknowledgements
We acknowledge the financial support of the National Key Research and Development Program of China (2018YFA0800200 (Q.W.) and 2020YFA0804000 (Q.W.)) and the National Natural Science Foundation of China (32025014 (Q.W.) and 81921006 (Q.W.)).
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Q.W. conceived the study, designed experiments and created the figures. M.M.Z., J.L., A.H.M., G.Z.N. and Y.C. performed experiments and collected data. M.M.Z., W.Q.Z. and Q.W. analyzed the data and discussed results and strategy. M.M.Z. and Q.W. wrote the manuscript, with input from all authors. Q.W. reviewed and edited the manuscript.
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Nature Cardiovascular Research thanks Todd Evans, Lluis Fajas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Expression patterns of tmem88a and tmem88b during embryo development.
a,b, Expression of tmem88a and tmem88b in wild-type embryos at the indicated developmental stages was analyzed using whole-mount in situ hybridization. Arrowheads indicate pharynx expression of tmem88a (a) and tmem88b (b). Lateral views, anterior to the left.
Extended Data Fig. 2 Generation of tmem88a and tmem88b mutants using the CRISPR/Cas9 system.
a, The tmem88a mutant had a 17-base deletion and 1-base substitution that resulted in a truncated protein lacking the TM domain and PDZ-binding motif. Red arrowhead indicates the position of the gRNA target site. b, The tmem88b mutant had a 166-base deletion, which was predicted to encode a truncated protein lacking the TM domain and PDZ-binding motif. c, The tmem88a mutant was identified using HpyCH4III digestion. d, The tmem88b homozygous mutant was identified using PCR-based genotyping analyses. M indicates the DNA marker. e, The expression of tmem88a and tmem88b in wild-type and the relevant mutant embryos was examined using in situ hybridization. f, Morphological observation of wild-type, tmem88a−/− and tmem88b−/− embryos at the indicated stages. Scale bars, 600 μm. g,h, tmem88a−/− and tmem88b−/− embryos have no apparent defects on the formation of the heart and blood cells. Wild-type, tmem88a−/− and tmem88b−/− embryos were harvested at 48 hpf for in situ hybridization (g) or O-dianisidine staining (h). Ventral views, anterior to the top.
Extended Data Fig. 3 tmem88a/b−/− mutants exhibit evident pericardial edema and obvious defects in heart and blood cell development.
a-c, The expression of tmem88a and tmem88b in wild-type embryos and the indicated mutants was examined using in situ hybridization (a) and qRT-PCR (b and c). The expression levels of β-actin were used as a reference to normalize the amount of mRNAs in each sample. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. NS, non-significant. d, The expression of Tmem88a and Tmem88b proteins in wild-type embryos and tmem88a−/−, tmem88b−/− and tmem88a/b−/− mutants at 28 hpf was examined using Western blot. e, Morphological defects in tmem88a/b−/− embryos at the indicated stages. Scale bars, 600 μm. f, Wild-type and tmem88a/b−/− embryos were harvested at 48 hpf for in situ hybridization with a cmlc2 probe. Ventral views, anterior to the top. g, O-dianisidine staining of erythrocytes in wild-type and tmem88a/b−/− embryos at 48 hpf. Ventral views, anterior to the top. h, Wild-type and tmem88a/b−/− embryos were harvested at 18 hpf and 20 hpf for in situ hybridization with a nkx2.5 probe. Dorsal views, anterior to the top.
Extended Data Fig. 4 Ectopic activation of Wnt/β-catenin signaling impairs the PAA progenitor to angioblast transition.
a,b, Tg(hsp70l:wnt8a-EGFP) embryos were heat shocked at 20 hpf for 30 min, and then harvested at 38 hpf for in situ hybridization with etv2 and scl probes (a). The average numbers of PAA angioblast clusters were quantified in (b). n ≥ 22 per group. Student’s t-test was used. Data are mean ± SD.
Extended Data Fig. 5 The expression of cyclin D1 is increased in tmem88a/b-depleted PAA progenitors.
a-c, qRT-PCR analysis of the expression of cyclin D1 and cdk4 in ZsYellow+ cells sorted from the pharynx region of Tg(nkx2.5:ZsYellow) and tmem88a/b-deficient Tg(nkx2.5:ZsYellow) embryos at 28 hpf (a), 48 hpf (b) and 60 hpf (c) using fluorescence activated cell sorting purification. The expression levels of β-actin were used as a reference to normalize the amount of mRNAs in each sample. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. NS, non-significant. d, tmem88a/b−/− embryos were treated with either 10 μM Ro-3306 or PHA-767491 from 20 hpf, and then harvested at 38 hpf for in situ hybridization with nkx2.5 and etv2 probes.
Extended Data Fig. 6 CDK4-mediated phosphorylation on threonine 11 of zNkx2.5 restrained its transcriptional activity.
a, HEK293T cells were co-transfected with Flag-zNkx2.5 and increasing amounts of Myc-CDK4, then harvested for Western blot. Tubulin was used as the loading control. b, HeLa cells transfected with the indicated plasmids were immunostained with anti-Flag and anti-Myc antibodies. The nuclei were counterstained with DAPI. Scale bar, 10 µm. c-e, HEK293T cells co-transfected with ANF-luciferase plasmid and the indicated plasmids were harvested 36 h after transfection for luciferase assays. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. NS, no significant. WT, wild-type; KD, kinase dead. f,g, HEK293T cells were co-transfected with ANF-luciferase plasmid and a construct expressing either Flag-zNkx2.5 or its T11A mutant. Cells were then harvested for luciferase assays (f). Wild-type embryos were injected with 100 pg of the ANF-luciferase reporter constructs and 200 pg Flag-nkx2.5 mRNA or Flag-nkx2.5-T11A mRNA at the one-cell stage, and then these embryos were subjected to luciferase assays at 24 hpf (g). Data represent the mean ± SD of three independent experiments. Student’s t-test was used.
Extended Data Fig. 7 CDK4 interacts with and phosphorylates Smad3a and Smad3b.
a, Conserved CDK substrate motifs in Smad3 proteins from different species. Red stars indicate the critical residues in CDK substrate motifs. b,c, Both Smad3a and Smad3b interact with CDK4. HEK293T cells transfected with the indicated plasmids were subjected to immunoprecipitation with an anti-Flag antibody. d, CDK4 phosphorylates Smad3a and Smad3b. HEK293T cells were transfected with the indicated plasmids, and then treated with or without 0.5 μM PD0332991 for 5 h prior to harvest for Western blot. CDK4 substrates were blotted with an antibody against phosphorylated human Smad3 (Thr178). Tubulin was used as the loading control. WT, wild-type; KD, kinase dead.
Extended Data Fig. 8 CDK4-dependent phosphorylation of Smad3 suppresses etv2 transcription.
a, HEK293T cells co-transfected with increasing amounts of Smad3a expression plasmids and etv2-Luc or scl-Luc reporter, then harvested for luciferase assays. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. b,c, CDK4-mediated phosphorylation of Smad3 represses etv2 transcription. Note that the treatment with CDK4/6 inhibitor PD0332991 (b) or mutation of the three CDK phosphorylation sites in Smad3a (Smad3a-3A) (c) relieved the CDK4-induced suppression of etv2-Luc reporter expression. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. d, The −2516 to −2270 bp region of the etv2 promoter is important to respond to Smad3. HEK293T cells were transfected with various constructs as indicated. The cells were then harvested for luciferase assays 36 h later. Data represent the mean ± SD of three independent experiments. Student’s t-test was used. e, The potential Smad-binding elements in the etv2 promoter. There are two potential Smad-binding elements (SBEs) in the −2516 to −2270 bp region. The SBE in probe A was mutated with G-2396 replaced by A to generate MT probe A, and the SBE in probe B was mutated with G-2312 replaced by A to generate MT probe B. f, Smad3 directly bound to probe A and probe B as shown by EMSA. Cold A, unlabeled probe A; Cold B, unlabeled probe B. MTPA and MTPB, mutants of probe A and B. g,h, MT1-etv2-Luc and MT2-etv2-Luc contained the same mutations in MTPA and MTPB, respectively, while MT-etv2-Luc contained both of the mutations. HEK293T cells were co-transfected with various constructs as indicated. Cells were then harvested for luciferase assays 36 h later (g). Wild-type embryos were injected with 100 pg of the indicated reporter constructs together with or without 150 pg Flag-smad3a mRNA at the one-cell stage, and then these embryos were subjected to luciferase assays at 24 hpf (h). Data represent the mean ± SD of three independent experiments. Student’s t-test was used.
Extended Data Fig. 9 The expression of p21 remains unchanged during PAA development.
Tg(nkx2.5:ZsYellow) embryos stained with anti-ZsYellow antibody were hybridized with a fluorescence-labeled p21 probe at 24, 32, and 42 hpf. PAA clusters are indicated by white dotted lines. This experiment was repeated independently three times. Dorsal views, anterior to the left. Scale bar, 50 μm.
Extended Data Fig. 10 Reducing the expression of wnt2bb rebalances the proliferation and differentiation of PAA progenitors in tmem88a/b−/− mutants.
a, Wild-type, wnt2bb+/− and wnt2bb−/− embryos in the Tg(nkx2.5:ZsYellow) background were coimmunostained with anti-ZsYellow and anti-β-catenin antibodies. Nuclei were counterstained with DAPI. PAA progenitor clusters are indicated by the white dotted lines. This experiment was repeated independently three times. Dorsal views, anterior to the left. Scale bar, 50 μm. b, The PAAs 3–6 morphology of wild-type, wnt2bb+/− and wnt2bb−/− embryos in the Tg(flk:EGFP;gata1:DsRed) background at 60 hpf. This experiment was repeated independently three times. Lateral views, anterior to the left. Scale bar, 50 µm. c,d, tmem88a/b-deficient Tg(nkx2.5:ZsYellow) embryos were injected with 3 ng cMO or wnt2bb MO at the one-cell stage, then harvested at 28 hpf. PAA progenitors were labeled with anti-ZsYellow antibody. Proliferating cells were visualized by BrdU immunofluorescence. This experiment was repeated independently three times. Representative pictures are shown in (c). Dorsal views, anterior to the left. Scale bar, 50 μm. The percentage of BrdU-positive cells in PAA progenitors is shown in (d). n ≥ 4 per group. Student’s t-test was used. Data are mean ± SD. e,f, The expression changes of nkx2.5 and etv2 in tmem88a/b−/− embryos injected with 3 ng cMO or wnt2bb MO (e). The average numbers of etv2+ PAA angioblast clusters were quantified in (f). n ≥ 19 per group. Student’s t-test was used. Data are mean ± SD.
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Zhang, M., Liu, J., Mao, A. et al. Tmem88 confines ectodermal Wnt2bb signaling in pharyngeal arch artery progenitors for balancing cell cycle progression and cell fate decision. Nat Cardiovasc Res 2, 234–250 (2023). https://doi.org/10.1038/s44161-023-00215-z
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DOI: https://doi.org/10.1038/s44161-023-00215-z