Determining zebrafish dorsal organizer size by a negative feedback loop between canonical/non-canonical Wnts and Tlr4/NFκB
概要
Title
Determining zebrafish dorsal organizer size by a
negative feedback loop between canonical/noncanonical Wnts and Tlr4/NFκB
Author(s)
Zou, Juqi; Anai, Satoshi; Ota, Satoshi et al.
Citation
Nature Communications. 2023, 14, p. 7194
Version Type VoR
URL
rights
https://hdl.handle.net/11094/93372
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
Article
https://doi.org/10.1038/s41467-023-42963-3
Determining zebrafish dorsal organizer size
by a negative feedback loop between
canonical/non-canonical Wnts and
Tlr4/NFκB
Received: 25 January 2023
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Accepted: 26 October 2023
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Juqi Zou 1, Satoshi Anai 2, Satoshi Ota 3, Shizuka Ishitani
Masayuki Oginuma 1 & Tohru Ishitani 1,4
1
,
In vertebrate embryos, the canonical Wnt ligand primes the formation of
dorsal organizers that govern dorsal-ventral patterns by secreting BMP
antagonists. In contrast, in Drosophila embryos, Toll-like receptor (Tlr)-mediated NFκB activation initiates dorsal-ventral patterning, wherein Wntmediated negative feedback regulation of Tlr/NFκB generates a BMP
antagonist-secreting signalling centre to control the dorsal-ventral pattern.
Although both Wnt and BMP antagonist are conserved among species, the
involvement of Tlr/NFκB and feedback regulation in vertebrate organizer
formation remains unclear. By imaging and genetic modification, we reveal
that a negative feedback loop between canonical and non-canonical Wnts and
Tlr4/NFκB determines the size of zebrafish organizer, and that Tlr/NFκB and
Wnts switch initial cue and feedback mediator roles between Drosophila and
zebrafish. Here, we show that canonical Wnt signalling stimulates the expression of the non-canonical Wnt5b ligand, activating the Tlr4 receptor to stimulate NFκB-mediated transcription of the Wnt antagonist frzb, restricting
Wnt-dependent dorsal organizer formation.
Animals display various body plans comprising various anatomical
axes such as the dorsal-ventral (DV) and anterior-posterior (AP) axes.
The establishment of body axes is one of the most fundamental events
in the development of multicellular organisms. Since its discovery, the
Spemann-Mangold organizer1, a group of cells that initiate DV axis
formation in the amphibian embryo, has been intensively studied in a
variety of model animals. It was found that organizer formation is
induced by Wnt/β-catenin signaling. Canonical Wnt ligands, such as
zebrafish Wnt8a and Xenopus Wnt11, activate β-catenin signaling,
specifically in the dorsal embryonic region, thereby stimulating the
formation of the dorsal organizer2–6. The dorsal organizer secretes the
BMP antagonist Chordin into the ventral region; then, Chordin inhibits
BMP-dependent ventral specification7–9. Thus, Wnt/β-catenin signaling
initiates DV axis formation through Chordin/BMP in vertebrates. On
the other hand, in Drosophila, the DV axis formation is initiated by Tolllike receptor (Tlr)/NFκB signaling10–13. Spätzle (Spz) ligands are proteolytically cleaved, specifically in the ventral-most region, which then
activates the Tlr homolog (Toll). Activated Tlr stimulates the degradation of the IκB homolog, allowing nuclear translocation of the NFκB
family of transcription factors and consequent transcriptional activation of genes for ventral specification14,15. Concurrently, NFκB also
induces the expression of the Wnt family of extracellular protein
1
Department of Homeostatic Regulation, Division of Cellular and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka
565-0871, Japan. 2Yuuai Medical Center, Tomigusuku, Okinawa 901-0224, Japan. 3Genome Science Division, Research Center for Advanced Science and
Technology, The University of Tokyo, Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan. 4Center for Infectious Disease Education and Research (CiDER),
e-mail: ishitani@biken.osaka-u.ac.jp
Osaka University, Suita, Osaka 565-0871, Japan.
Nature Communications | (2023)14:7194
1
Article
WntD, functioning as an antagonist to attenuate Tlr/NFκB signaling16,17.
This Wnt-mediated negative feedback regulation of Tlr/NFκB signaling
is responsible for the precise size of the ventral embryonic region.
Moreover, Tlr/NFκB signaling represses the BMP homolog (Dpp)mediated dorsal specification by inducing the expression of the
Chordin homolog (Sog)18,19. Thus, Tlr/NFκB signaling initiates Drosophila DV axis formation through the regulation of Wnt, Chordin, and
BMP homologs. Taken together, Wnt, Chordin, and BMP are conserved
mediators of DV axis formation in vertebrates and Drosophila.
Since the Wnt/Chordin/BMP system is evolutionarily conserved
and Drosophila Tlr/NFκB signaling initiates DV axis formation, it is
expected that vertebrate Tlr/NFκB signaling might also be involved in
DV axis formation. Overexpression of NFκB family genes reportedly
inhibits dorsal formation in Xenopus laevis20,21. Other studies have
shown that injection of Drosophila Spätzle and Tlr homolog into
Xenopus embryos induced a secondary axis22, and that overexpression
of IκB (inhibition of NFκB) blocked Xenopus dorsal formation23. While
these findings indicate that Tlr/NFκB signaling may respectively function as a negative or positive regulator of dorsal formation, these
overexpression studies remain controversial. Furthermore, although
large-scale screening for isolating zebrafish mutants with dorsoventral
patterning defects has been performed24, Tlr/NFκB signaling-related
factors have not been isolated. Thus, the function and regulation of
endogenous Tlr/NFκB signaling during vertebrate DV axis formation
remain unclear.
The negative feedback loop plays an important role in axis formation and size control. For example, a Sizzled-mediated BMP-Chordin feedback loop is required for correct DV patterning and embryonic
size control25. Feedback regulation between Wnt and its secreted
inhibitor Dkk1 contributes to size control of sensory organs26. The Wnt
antagonist Sfrp1-mediated negative feedback regulation of Wnt/βcatenin signaling is essential for the development of a normal-sized
heart muscle27. Because the dorsal organizer is the signaling center
priming axis formation, organizer size should be properly controlled,
raising the possibility that Wnt/β-catenin signaling, the organizerinducer, may be restricted by negative feedback regulation. However,
this mechanism is poorly understood.
In this study, we examined the function and regulation of
endogenous Tlr and NFκB during zebrafish DV axis formation, using a
combination of in vivo reporter analysis, CRISPR/Cas9-mediated
knockout, and morpholino knockdown. We show that during the
initiation of dorsal organizer formation, Wnt/β-catenin signaling
stimulates the activation of the NFκB homolog Rel through Toll-like
receptor 4 (Tlr4), specifically in the dorsal embryonic tissue. Surprisingly, the non-canonical Wnt5 ligand mediates β-catenindependent Tlr4/Rel activation. Activated Rel then stimulates the
transcription of a Wnt antagonist, frizzled-related protein (frzb),
thereby restricting the Wnt/β-catenin-active area and dorsal organizer size. Thus, Wnt5-Tlr4/NFκB-mediated indirect negative feedback
regulation of Wnt/β-catenin signaling determines the precise size of
zebrafish dorsal organizer.
Results
NFκB activation in the dorsal region of zebrafish embryos
To clarify the spatiotemporal pattern of NFκB activity, we generated a
new NFκB reporter, NFκB-tkP:dGFP (Fig. 1a). We confirmed that activation of NFκB stimulated NFκB-tkP:dGFP activity in human HEK293
cells (Supplementary Fig. 1a) and then generated stable transgenic
zebrafish lines carrying a single copy of NFκB-tkP:dGFP (Supplementary Fig. 1b). NFκB-tkP:dGFP activity in transgenic fish was detected at
3.7 hours-post-fertilization (hpf) (Fig. 1b), indicating that the reporter
gene was zygotically activated. The reporter expression gradually
accumulated to the dorsal margin of the blastoderm, which corresponds to the future dorsal organizer, from the dome stage (4.3 hpf),
and completely localized in the dorsal region at the 50% epiboly stage
Nature Communications | (2023)14:7194
https://doi.org/10.1038/s41467-023-42963-3
(5.3 hpf) (Fig. 1b, c). These results suggest that NFκB functions in dorsal
organization.
Rel/NFκB negatively regulates dorsal organizer formation
To test whether NFκB is involved in dorsal organizer formation, we
overexpressed the zebrafish IκB homolog iκbab to block NFκB activity
in early zebrafish embryos (Fig. 1d). Overexpression of iκbab induced
expansion of the organizer area and dorsal tissue, marked by the
expression of dharma and chordin, respectively7,9,28–30, in early
embryos (Fig. 1e), resulting in class 2–3 (C2–3) dorsalizations31,32, with a
significant loss of ventral tail fin in larvae (Fig. 1f). These results suggest
that NFκB negatively regulates dorsal specification.
Next, we investigated which NFκB regulates dorsal cell fate. In
zebrafish, the NFκB family comprises five members: Rel (mammalian
c-Rel homolog), Rela, Relb, NFκB1, and NFκB2 (Supplementary Fig. 2a).
We focused on Rel because it is the most homologous to Drosophila
Dorsal, with high levels of rel transcripts being detected in early
embryos (Supplementary Fig. 2a, b). rel overexpression dramatically
activated the NFκB reporter, narrowed the size of organizer and dorsal
tissue, and reduced expression levels of the organizer marker dharma
and the dorsal tissue marker chordin in early embryos (Fig. 1g, h and
Supplementary Fig. 2c). This induced the ventralized V1–4 phenotype
in most larvae, characterized by the loss of dorsoanterior structures
and expanded ventral tissues32 (Fig. 1i), which indicates that Rel may
possibly inhibit the formation of dorsal organizer and dorsal tissue. ...