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. 2011 Sep;138(18):3915-20.
doi: 10.1242/dev.068833. Epub 2011 Aug 10.

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry

Yoshihiro Komatsu  1 , Vesa Kaartinen, Yuji Mishina
Affiliations

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry

Yoshihiro Komatsu et al. Development. 2011 Sep.

Erratum in

  • Development. 2011 Oct;138(19):4334

Abstract

Cilia at the node generate a leftward fluid flow that breaks left-right symmetry. However, the molecular mechanisms that regulate ciliogenesis at the node are largely unknown. Here, we show that the epiblast-specific deletion of the gene encoding the BMP type 1 receptor (Acvr1) compromised development of nodal cilia, which results in defects in leftward fluid flow and, thus, abnormalities in left-right patterning. Acvr1 deficiency in mouse embryonic fibroblasts (MEFs) resulted in severe defects in their quiescence-induced primary cilia. Although the induction of quiescence in wild-type MEFs leads to an increase in the level of the cyclin-dependent kinase inhibitor p27(Kip1) and to rapid p27(Kip1) phosphorylation on Ser(10), MEFs deficient in Acvr1 show a reduction in both p27(Kip1) protein levels and in p27(Kip1) Ser(10) phosphorylation. The observed defects in cilium development were rescued by the introduction of p27(Kip1) into Acvr1-deficient MEFs, implying that BMP signaling positively controls p27(Kip1) stability in the G0 phase via p27(Kip1) Ser(10) phosphorylation, which is a prerequisite for induction of primary cilia. Importantly, in control embryos, p27(Kip1) protein is clearly present and strongly phosphorylated on Ser(10) in cells on the quiescent ventral surface of the node. By contrast, the corresponding cells in the node of Acvr1 mutant embryos were proliferative and showed a dramatic attenuation in both p27(Kip1) protein levels and phosphorylation on Ser(10). Our data suggest that cell quiescence controlled by BMP signaling via ACVR1 is required for transient formation of nodal cilia, and provide insight into the fundamental question of how the node represents the mechanistic `node' that regulates the development of left-right symmetry in vertebrates.

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Figures

Fig. 1.
Fig. 1.
BMP signaling through ACVR1 regulates left-right patterning. (A) Whole-mount view of wild-type and Acvr1 cKO embryos at E10.5. Acvr1 cKO embryos showed abnormalities in head (white asterisks, top panel), embryonic turning (middle panel) and cardiac looping (bottom panel). (B) Expression patterns of Nodal, Lefty2, Pitx2, Shh, Foxa2 and Lefty1 are shown. Ventral view for Nodal. Frontal view for Lefty2, Pitx2, Foxa2 and Lefty1. Lateral view for Shh. Red arrows show aberrant right-sided expression of Nodal, Lefty2 and Pitx2 (second panel from the top), and absent midline expression of Lefty1 (bottom panel) in Acvr1 cKO mutants. A, anterior; P, posterior; L, left; R, right.
Fig. 2.
Fig. 2.
Structural abnormalities at node and alteration of the nodal flow. (A) Scanning electron microscopic analysis of the node at E7.75 and E8.0. Low- and high-magnification images are shown. (B) Nodal cilia were significantly shorter in Acvr1 cKO embryos at E7.75 and E8.0. Four embryos for each stage were analyzed and more than 50 nodal cilia per embryo were measured. Data are mean+s.e.m. (C) Examples of the movement of beads in the node. The movements of four individual beads represented by different colors were traced on videograms. Bead migration from the right edge to the left edge of the node within 10 seconds was regarded as a consistent leftward flow. Movements of at least four beads per embryo were monitored to judge the presence of the leftward flow in a given embryo. A, anterior; P, posterior; L, left; R, right.
Fig. 3.
Fig. 3.
BMP signaling through ACVR1 is required for the primary cilia formation via the regulation of p27Kip1. (A) A significant reduction in the number of primary cilia (green, acetylated tubulin) and increased levels of a cell proliferation marker (red, Ki-67) were observed in Acvr1-deficient MEFs (upper panels), whereas γ-tubulin (red) was seen in both control and mutant MEFs (lower panels). (B) Differences in the number of cilia and in cell proliferation rates between control MEFs (Ad-lacZ transduced cells, gray) and Acvr1-deficient MEFs (Ad-Cre transduced cells, black). More than 100 cells were randomly analyzed in three independent experiments. Data are mean+s.e.m. (C) Cell lysates were prepared at days 1, 3 and 5 during cilia formation and cell cycle regulators were analyzed by western blotting. (D) Defective formation of the primary cilia (green, acetylated tubulin) in Acvr1-deficient MEFs (middle) coincided with the increased levels of a cell proliferation marker (red, Ki-67) and attenuated phosphorylation of p27Kip1 on Ser10 (blue, phospho-Ser10 in p27Kip1). Cilia formation was restored with adenoviral transduction of p27Kip1 (Ad-p27) into Acvr1-deficient MEFs (right). (E) Differences in a number of cilia from D in three independent experiments. Data are mean+s.e.m.
Fig. 4.
Fig. 4.
Cell cycle arrest regulated by BMP signaling is crucial for the transient nodal cilia formation. (A) Levels of both p27Kip1 and p27Kip1 phosphorylation on Ser10 (P-p27Kip1) (green staining) were significantly lower on the ventral surface of the node in Acvr1 cKO embryos at E7.75. In wild-type embryos, cells on ventral surface of the node were quiescent (no detectable Ki-67 staining), whereas corresponding cells in Acvr1 cKO embryos were proliferative (stained positive for Ki-67). A, anterior; P, posterior. (B) A mechanism by which BMP signaling through ACVR1 regulates development of nodal cilia in mouse embryo. The presence of ACVR1 leads to a stabilization of p27Kip1 via phosphorylation on Ser10, thus inducing quiescence. Subsequently, nodal cilia develop and nodal flow is generated, which triggers the establishment of left-right asymmetry. If Acvr1 is deleted, phosphorylation on Ser10 is attenuated, leading to a destabilization of p27Kip1 at the node. As a result, nodal cilia are not developed appropriately, which leads to a failure of nodal flow and to a failure to break left-right symmetry.
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