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Science 338 (6104): 226-231

Copyright © 2012 by the American Association for the Advancement of Science

Cilia at the Node of Mouse Embryos Sense Fluid Flow for Left-Right Determination via Pkd2

Satoko Yoshiba1, Hidetaka Shiratori1, Ivana Y. Kuo2, Aiko Kawasumi1,*, Kyosuke Shinohara1, Shigenori Nonaka3, Yasuko Asai1, Genta Sasaki1, Jose Antonio Belo4, Hiroshi Sasaki5, Junichi Nakai6, Bernd Dworniczak7, Barbara E. Ehrlich2, Petra Pennekamp7,8,{dagger}, and Hiroshi Hamada1,{dagger}

1 Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (CREST), Japan Science and Technology Corporation (JST), 1-3 Yamada-oka, Suita, 565-0871 Osaka, Japan.
2 Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
3 Laboratory for Spatiotemporal Regulations, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, 444-8585 Aichi, Japan.
4 Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, and IBB-Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-135 Faro, Portugal.
5 Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
6 Saitama University Brain Science Institute, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
7 Universitätsklinikum Münster, Institut für Humangenetik, Vesaliusweg 12-14, 48149 Münster, Germany.
8 Universitaetsklinikum Muenster, Klinik für Kinder- und Jugendmedizin, Allgemeine Pädiatrie, Albert-Schweitzer-Campus 1, 48149 Muenster, Germany.


Figure 1
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Fig. 1. Specific requirement for Pkd2 in crown cells of the node for correct L-R patterning. (A and B) Schematic representation of two types of node-specific Pkd2 transgene are shown on the top. Their expression patterns were examined by staining of transgenic embryos at embryonic day 8.0 (E8.0) with X-gal. Schematic (left) and actual (middle) views of whole embryos, and transverse sections of the node (right) are shown. Middle, inset of (B) shows the node from ventral view at higher magnification. Expression of the NDE-driven transgene is highly specific to crown cells of the node (B). A, anterior; P, posterior; L, left; R, right. Scale bars, 50 μm. (C) Whole-mount in situ hybridization analysis of the expression of Nodal and Pitx2 in E8.0 embryos of the indicated genotypes. Left-sided expression of Nodal and Pitx2 in LPM is lost in Pkd2–/– embryos but is restored (red arrowheads) in Pkd2–/–;Foxa2-Pkd2 and Pkd2–/–;NDE-Pkd2 embryos. Scale bar, 500 μm.

 

Figure 2
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Fig. 2. Pkd2 is necessary for sensing of nodal flow. (A) Nodal flows in wild-type and Pkd2–/– embryos were examined by means of PIV analysis at E8.0. Flow is maintained in the Pkd2–/– embryo. Each arrowhead denotes the direction and speed of the flow at the indicated position. The color scale indicates the direction and magnitude of the flow velocity (leftward in yellow and red, rightward in blue). White lines indicate the outlines of the node. Scale bar, 10 μm. (B) E8.0 Pkd2+/+ and Pkd2–/– embryos harboring the ANE-LacZ transgene were stained with X-gal. Scale bar, 50 μm. (C) The L-R patterns of ANE activities in embryos of the indicated genotypes either in situ or under conditions of artificial rightward flow in vitro are summarized. The numbers of embryos showing each pattern are indicated. (D) Pkd2+/+ embryos with ANE-LacZ were cultured under the influence of a rightward artificial flow from early headfold to six-somite stages. Artificial rightward flow reversed the pattern of Pitx2 expression in LPM (left, red arrowhead) and that of ANE activity in crown cells, as detected by means of in situ hybridization of LacZ mRNA (middle) and X-gal staining (right). Scale bars, 100 μm.

 

Figure 3
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Fig. 3. Calcium signal is essential for L-R symmetry breaking in the node crown cells. (A) Activity of ANE is disrupted when Ca2+ signal blockers are treated. Two pathways that lead to Ca2+ release from endoplasmic reticulum were examined (B). L > R asymmetric ANE activity is maintained in control and Ruthenium Red–treated embryos, whereas it is disrupted with Gd3+, 2-aminoethoxydiphenyl borate (2-APB), or Thapsigargin (TG). Scale bar, 50 μm. (C) The effects of each reagent on L-R asymmetric ANE activity are summarized. The numbers of embryos showing each pattern are indicated.

 

Figure 4
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Fig. 4. Ciliary localization of Pkd2 is required for correct L-R determination. (A) Schematic representation of NDE-driven Pkd2::Venus transgene is shown on the top. Crown cell–specific expression of the transgene in an E8.0 embryo was confirmed by means of whole-mount in situ hybridization with a Venus probe (top right). Scale bar, 50 μm. Left-sided expression of Nodal was restored in Pkd2–/–;NDE-Pkd2::Venus embryo (bottom right), suggesting that Pkd2::Venus is functional. Scale bar, 500 μm. Pkd2::Venus protein is preferentially localized to cilia (left, red arrowheads). Scale bar, 10 μm. (B) Transgenic embryos expressing Pkd2(E442G)::Venus or Pkd2(D509)::Venus in crown cells. Both proteins are unable to localize to cilia. Scale bar, 10 μm. (C) Localization of Pkd2(E442G)::Venus was confirmed by means of immunofluorescence staining with antibodies to acetylated tubulin (red), to Venus;green fluorescent protein (GFP) (green), and to phalloidin (blue). Scale bar, 10 μm. (D) Open probability of Pkd2(wt), Pkd2(E442G), and Pkd2(R6G-G819X) channels in the presence of increasing cytosolic Ca2+ concentration. Pkd2(E442G) retains normal channel activity, whereas Pkd2(R6G-G819X) loses it. Error bars represent ±SEM.

 

Figure 5
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Fig. 5. Nodal flow is sensed by the cilia of crown cells. (A) An E8.0 transgenic embryo harboring NDE-Kif3a-IRES-LacZ was stained with X-gal, revealing crown cell–specific expression of the transgene (left). Scale bar, 50 μm. In SEM of the node of an E8.0 Kif3a–/– embryo with NDE-Kif3a, cilia are apparent at the edge (red box) but not at the center (blue box) of the node. Pale blue lines indicate the border between the endoderm and crown cells, with the dotted circle enclosing pit cells. The boxed regions on the left are shown at a higher magnification on the right. Scale bars, 5 μm. (B) Expression of L-R marker genes in embryos of the indicated genotypes that were examined at E8.0 (in vivo) or cultured under the influence of a rightward artificial flow before analysis. Pitx2 expression pattern of the Kif3a–/–;NDE-Kif3a embryo responded to the flow and is right-sided (red arrowhead). Scale bar, 500 μm. (C) The numbers of embryos showing each pattern of gene expression are summarized.

 


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