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GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2–4 cell stage in Lymnaea stagnalis

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Abstract

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3β, β-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3β and β-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3β inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of β-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3β in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

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Authors and Affiliations

  1. Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan

    Hiromi Takahashi & Reiko Kuroda

  2. Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-8510, Japan

    Masanori Abe & Reiko Kuroda

Authors
  1. Hiromi Takahashi
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  2. Masanori Abe
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  3. Reiko Kuroda
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Corresponding author

Correspondence to Reiko Kuroda.

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The care and use of animals were in accordance with the guidelines for animal experiments of Tokyo University of Science.

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Communicated by Mark Q. Martindale

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Supplementary Fig. 1

(a) A scheme showing point treatment with lithium chloride (LiCl). Dots indicate the drug-treated developmental stages. Synchronized embryos were treated with 50 mM LiCl and washed out at 4 °C. (b) The effects of LiCl treatment at various developmental stages on embryo development. The type of malformations was classified as by Morrill with some modifications (See Materials and Methods). (PNG 308 kb)

High resolution image (TIF 23480 kb)

Supplementary Fig. 2

Diagram of the L. stagnalis Wnt signaling proteins. Numbers denote the position of amino acid residues in the polypeptide chain counted from the N-terminus. Black bars indicate the WISH-probe designed regions. Lsβ-Catenin contains 12 highly conserved armadillo repeats, the α helix of the C-terminal domain (Helix C) and a GSK3β phosphorylation site in the sequence DSGXXS within the N-terminal domain. LsDsh has three highly conserved domains known as DIX, PDZ and DEP. The kinase domain of LsGSK3 is also highly conserved. LsDsh has been found to have at least two types of major splicing variants, LsDsh1 and LsDsh2, in the non-functional domains. However, they are not distinguished in this study. Some minor splicing variants are also shown in the non-functional domains of LsGSK3. (PNG 195 kb)

High resolution image (TIF 19007 kb)

Supplementary Fig. 3

mRNA localization for Wnt signaling genes, Lsβ-catenin, Lsdsh and Lsgsk3, from 25-cell to gastrula stages of the Lymnaea stagnalis embryo. Scale bar: 50 μm (PNG 2989 kb)

High resolution image (TIF 33208 kb)

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Takahashi, H., Abe, M. & Kuroda, R. GSK3β controls the timing and pattern of the fifth spiral cleavage at the 2–4 cell stage in Lymnaea stagnalis. Dev Genes Evol 229, 73–81 (2019). https://doi.org/10.1007/s00427-018-00625-1

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