Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis

Abstract

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation.

Keywords: LPA; embryonic stem cell; hemangioblast; hematopoiesis; zebrafish.

Figure 1. Pharmacological blockage of ATX-LPA signaling inhibits hematopoietic differentiation

A Representative flow cytometry data for CD41 staining in day 6 EBs, treated with DMSO or 30 μM Ki16425 from day 2 to day 6. B Dose effect of Ki16425 on mESC hematopoietic differentiation. The relative fold changes of CD41⁺ cell percentage were shown (n = 5). C qPCR analyses of hematopoietic marker expressions (n = 4). D, E Methylcellulose colony-forming cell assay (M3434). Primitive erythroid colonies were scored 6 days after culture in M3434 (D) (n = 4). Definitive colonies were scored 10 days after culture in M3434 (E) (n = 4). Insets show average distribution of hematopoietic colonies. Ery-P: primitive erythroid; cfu-E: definitive erythroid; cfu-G/M/GM: granulocytes/macrophages/granulocyte macrophages; cfu-GEMM: granulocyte/erythroid/myeloid megakaryocytes. F Dose effect of HA130 on CD41⁺ cell percentage (n = 3). G qPCR analyses of hematopoietic marker expressions (n = 4). H, I Methylcellulose colony-forming cell assay (M3434). Primitive (H) and definitive (I) colonies were cultured and scored as in (D) and (E) (n = 3 per group). Data information: Data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control.

Figure 2. Genetic blockage of ATX–LPA signaling inhibits hematopoietic differentiation

A qPCR analyses of lpar1 or lpar3 knockdown efficiency (n = 4 per group). B Flow cytometry analyses of CD41⁺ cell percentage. The relative fold changes of CD41⁺ cell percentage were shown (n = 4). C qPCR analyses of hematopoietic markers (n = 5). D, E Methylcellulose colony-forming cell assay (M3434). Primitive erythroid colonies were scored 6 days after culture in M3434 (D) (n = 4). Definitive colonies were scored 10 days after culture in M3434 (E) (n = 4). Insets show average distribution of hematopoietic colonies. F qPCR analyses of atx knockdown efficiency (n = 3). expressions in whole EBs of day 6 (n = 4). G Effect of atx knockdown on CD41⁺ cell percentage (n = 6). H qPCR analyses of hematopoietic marker expressions in day 6 whole EBs (n = 4). I, J Methylcellulose colony-forming cell assay (M3434). Primitive (I) and definitive (J) colonies were cultured and scored as in (D) and (E) (n = 4 per group). Data information: Data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control.

Figure 3. Pharmacological blockage of ATX-LPA signaling inhibits hemangioblast formation

1. Representative flow cytometry data for Flk1 staining in day 4 whole EBs. EBs were treated with DMSO or 30 μM Ki16425 from day 2 to day 4 and analyzed by flow cytometry.

2. Effect of Ki16425 treatment on Flk1⁺ cell percentage (n = 5).

3. qPCR analyses of hematopoietic and germ layer marker expressions (n = 4). Endoderm marker: lamb1; mesoderm marker: brachyury; ectoderm marker: beta-tub3.

4. BL-CFC assay. Day 4 EBs treated as in (A) were digested with trypsin and cultured in BL-CFC medium. Blast colonies were identified and scored by their distinctive morphology 4 days later (n = 4).

5. Effect of HA130 on Flk1⁺ cell percentage (n = 4).

6. qPCR analyses of hematopoietic and germ layer marker expressions (n = 3).

7. BL-CFC assay (n = 3). Blast colonies were cultured and scored as in (D).

Data information: Data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control.

Figure 4. Genetic blockage of ATX-LPA signaling inhibits hemangioblast formation

1. Effects of lpar1 and/or lpar3 knockdown on Flk1⁺ cell percentage in day 4 whole EBs (n = 4).

2. qPCR analyses of hematopoietic and germ layer marker expressions (n = 4).

3. BL-CFC assay. Day 4 EBs were digested with trypsin and cultured in BL-CFC medium. Blast colonies were identified and scored 4 days later (n = 3).

4. Effect of atx knockdown on Flk1⁺ cell percentage (n = 3).

5. qPCR analyses of hematopoietic and germ layer marker expressions (n = 4).

6. BL-CFC assay (n = 4). Blast colonies were cultured and scored as in (C).

Data information: Data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control.

Figure 5. ATX–LPA signaling regulates hematopoietic differentiation through PI3K/Akt-Smad pathway

A, B Flow cytometry analyses of CD41⁺ cell percentage. EBs were treated with 100 nM LPA alone, or together with ROCK inhibitor Y-27632 (10 μM), Rac1 inhibitor NSC23766 (100 μM), Gi inhibitor PTX (80 ng/ml), or PKC inhibitor GF109203X (1 μM) from EB day 2 to day 6 in serum-free culture (A) (n = 3). EBs were treated with 100 nM LPA alone, or together with PI3K inhibitor LY294002 (4 μM), wortmannin (3 μM) or AKT inhibitor AKT VIII (2 μM) from EB day 2 to day 6 in serum-free culture (B) (n = 3). C, D Western blot analyses. Day 4 EBs, stably expressing Lpar1 or Lpar3 siRNA, were digested with trypsin, and protein samples were prepared. Beta-actin was used as the loading control. The quantification results were plotted as ratio between phosphorylated Akt and total AKT after normalization to beta-actin levels (C) (n = 3). The quantification result of phosphorylated Smad1/5/8 was plotted as ratio between phosphorylated Smad1/5/8 and total Smad1 after normalization to beta-actin levels (D) (n = 4). E, F Epistatic analyses. EBs were treated with LPA alone, or together with PI3K inhibitor wortmannin (3 μM) or AKT inhibitor AKT VIII (2 μM) from EB day 2 to day 4 in serum-free culture. Day 4 EBs were digested with trypsin, and protein samples were prepared. Beta-actin was used as the loading control (E) (n = 3). mESCs stably expressing vector or CA-Akt were differentiated as EBs and treated with DMSO or 10 μM Ki6425 from day 2 to day 4 (F) (n = 3). Beta-actin was used as the loading control. Data information: Data shown are means ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding control.

Figure 6. ATX–LPA signaling is required for hemangioblast formation and primitive hematopoiesis in zebrafish

A–H WISH analyses for embryos treated with Ki16425. Zebrafish embryos treated with DMSO or 30 μM Ki16425 were hybridized with riboprobes to hemangioblast markers lmo2 (A and B) and scl (C and D) at 12 hpf, or with riboprobes to primitive hematopoietic markers gata1 (E and F) and scl (G and H) at 24 hpf. Embryos were dorsal views with anterior to the top (A–D), or lateral views with anterior to the left (E–H). I–T WISH analyses for embryos injected with Lpar1 or Lpar3 MO. Zebrafish embryos injected with 4 ng Ctrl MO, 6 ng Lpar1 MO, or 2.5 ng Lpar3 MO at one-cell stage were hybridized with riboprobes to hemangioblast markers lmo2 (I–K) and scl (L–N) at 12 hpf, or with riboprobes to primitive hematopoietic markers gata1 (O–Q) and scl (R–T) at 24 hpf. Embryos were dorsal views with anterior to the top (I–N) or lateral views with anterior to the left (O–T). U–Zii WISH analyses for embryos injected with Atx MO. Zebrafish embryos injected with 0.5 ng Ctrl MO or 0.5 ng Atx MO at one-cell stage were hybridized with riboprobes to hemangioblast markers lmo2 (U and V) and scl (W and X) at 12 hpf, or with riboprobes to primitive hematopoietic markers gata1 (Yi and Yii) and scl (Zi and Zii) at 24 hpf. Embryos were dorsal views with anterior to the top (U–X) or lateral views with anterior to the left (Yi–Zii).