Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation

Abstract

Gastrulation is a fundamental process during embryogenesis that shapes proper body architecture and establishes three germ layers through coordinated cellular actions of proliferation, fate specification, and movement. Although many molecular pathways involved in the specification of cell fate and polarity during vertebrate gastrulation have been identified, little is known of the signaling that imparts cell motility. Here we show that prostaglandin E(2) (PGE(2)) production by microsomal PGE(2) synthase (Ptges) is essential for gastrulation movements in zebrafish. Furthermore, PGE(2) signaling regulates morphogenetic movements of convergence and extension as well as epiboly through the G-protein-coupled PGE(2) receptor (EP4) via phosphatidylinositol 3-kinase (PI3K)/Akt. EP4 signaling is not required for proper cell shape or persistence of migration, but rather it promotes optimal cell migration speed during gastrulation. This work demonstrates a critical requirement of PGE(2) signaling in promoting cell motility through the COX-1-Ptges-EP4 pathway, a previously unrecognized role for this biologically active lipid in early animal development.

Figure 1.

cox1, ptges, and ep4 transcripts are expressed ubiquitously during gastrulation. (A–D) Lateral view, dorsal to the right. Expression of cox1 (A), cox2 (B), ptges (C), and ep4 (D) transcripts visualized by whole-mount in situ hybridization. (ant) Anterior.

Figure 2.

Ptges-deficient embryos exhibit gastrulation defects that are suppressed by coincubation with PGE₂. (A–H) Lateral view, dorsal to the right. (I–K) Animal view, dorsal to the bottom. (L–Q) Dorsal view, anterior to the top. (R,S) Lateral view, anterior to the left. (I–K) Boundaries indicate the distance from the posterior end of the forebrain to the anterior end of the notochord. (L–Q) Boundaries indicate the width of the notochord. (A,I,L,O) Control embryos treated with DMSO. (B,F–H,J,K,M,N,S) Embryos injected with 2 ng of ptges-MO2. (E,P,Q) Embryos injected with 4 ng of ptges-MO2. (C,D) Embryos injected with 10 ng of cox1-MO. (D) Embryos coinjected with cox1-MO (10 ng) and cox1 RNA (50 pg). (F) Embryos injected with 2 ng of ptges-MO2 + PGI₂. (G) Embryos injected with 2 ng of ptges-MO2 + PGF_2α. (H,K,N) Embryos injected with 2 ng of ptges-MO2 + PGE₂. (Q) Embryos injected with 4 ng of ptges-MO2 + PGE₂. (H) Embryos coinjected with ptges-MO2 (2 ng) + mouse ptges RNA (100 pg). (I–K) Expression patterns of six3b, pax2.1. (L–Q) Expression pattern of no tail. (T) Quantitative analyses of the distance between the forebrain (fb) and notochord (no) in DMSO, ptges-MO2, and ptges-MO2 + PGE₂ (n = 10 per each group). (U) Quantitative analyses of width of ntl expression in DMSO, ptges-MO2, and ptges-MO2 + PGE₂ (n = 10 per each group). (fb) Forebrain; (mh) mid-hindbrain; (no) notochord. The white star marks the blastopore to illustrate delayed epiboly. For details see text.

Figure 3.

EP4-deficient embryos exhibit convergence and extension defect during gastrulation. (A,B,D,E) Lateral view, dorsal to the right. (C,F,G,H) Lateral view, anterior to the left. (I,K) Animal view, dorsal to the right. (J,L) Dorsal view, animal pole to the top. (M,O) Dorsal view, anterior to the top. (N,P) Anterior view, dorsal to the bottom. (M,N) Boundaries indicate the width of the notochord. (N,P) Boundaries indicate the distance from the posterior end of the forebrain to the anterior end of the notochord. (A–C,I,J,M,N) Uninjected control. (D–F,H,K,L,O,P) ep4-MO2 (2 ng) injected embryos. (G,H) ep4 RNA (80 pg) injected embryos. (H) Embryos injected with ep4-MO2 (2 ng) + ep4 RNA (80 pg). (I,K) bmp4 expressions. (J,L) gsc expressions. (M,O) no tail (ntl) expressions. (N,P) six3, pax2, and ntl expressions. (h) Head; (tb) tailbud; (D) dorsal; (fb) forebrain; (mh) mid-hindbrain; (no) notochord. The arrowhead points to the location of the prechordal plate at the tailbud stage. The white star highlights the blastopore to illustrate delayed epiboly. The black star indicates the animal pole.

Figure 4.

EP4-deficient embryos exhibit decreased cell motility in dorsal migration, while maintaining normal cell shape. (A) Domains of convergence and extension movements in zebrafish gastrulae (Myers et al. 2002b). Orange arrows indicate domains of slow convergence and extension. (B) A diagram to demonstrate the difference between total versus net speed. (C) A schematic representation of the method used to measure cell shape. (LWR) Length-to-width ratio. (D–F) Cell behavior of mesoderm cells during slow convergence toward the dorsal midline. (D) Total and net dorsal speed of lateral mesodermal cells at 80% epiboly. (E) Meandering index of lateral mesodermal cells. The meandering index was determined by dividing the total speed by the net speed. (F) LWR of lateral mesodermal cells. Lateral mesoderm measurements were made in wild-type (two embryos, 84 cells), control EP4-MO-injected (EP4-MOc; two embryos, 80 cells), and EP4-deficient (six embryos, 224 cells) embryos.

Figure 5.

EP4-deficient embryos exhibit reduced net path, but not persistence of directional movement, during dorsal migration. Representation of the paths traveled by lateral mesodermal cells at 80% epiboly in embryos injected with control EP4-MO (EP4-MOc) and EP4-MO. The path of each individual cell movement from the lateral mesoderm region was plotted over 40 min, starting at 80% epiboly. Colored arrows demonstrate the vector representation of the average direction and net length of cells from EP4-MOc (blue) and EP4-deficient (red) embryos.

Figure 6.

PI3K/Akt is activated downstream of EP4 signaling. (A–C) Lateral view, dorsal to the right. (B) Ten micromolar PI3K/Akt inhibitor, LY294002. (C) Thirty micromolar PI3K/Akt inhibitor, LY294002. (D) Uninjected, ep4-MO2 (2 ng), or ptges-MO2 (2 ng) injected. (E) Uninjected, ep4-MO2 (2 ng), or ep2-MO2 (2 ng) injected. (F) Uninjected or ep4-MO2-injected (2 ng) embryos treated with or without exogenous PGE₂ (5 μM). (G) Uninjected, ep4-MO2 (2 ng), or ep2-MO (2 ng) injected embryos. (H) Molecular mechanisms for PGE₂ signaling in vertebrate gastrulation. Lysates from 2.5 embryos were loaded into each well for Western blot analyses. (pAKT) Phosphorylated AKT; (AKT) total AKT; (pERK) phosphorylated ERK; (ERK) total ERK. Please see text and Materials and Methods for details.