Research Article

Maternal Huluwa dictates the embryonic body axis through β-catenin in vertebrates

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Science  23 Nov 2018:
Vol. 362, Issue 6417, eaat1045
DOI: 10.1126/science.aat1045

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Maternal factor sets axis

The vertebrate body form changes from the round shape of the fertilized egg to a cylindrical shape when the body plan is established. However, it is unknown whether a maternal factor controls this body axis formation. Yan et al. identified such a maternal factor and named it Huluwa. Loss of maternal Huluwa, a transmembrane protein, in zebrafish or frog eggs resulted in embryos that lacked the body axis and were missing the head and dorsoanterior tissues. Huluwa promoted Axin degradation, likely independent of Wnt ligand–receptor signaling, to protect β-catenin from degradation and induce body axis development during embryogenesis.

Science, this issue p. eaat1045

Structured Abstract

INTRODUCTION

The formation of the body axis in vertebrate embryos relies on the formation of the dorsal organizer at the onset of gastrulation, which is called the Spemann-Mangold organizer in frogs and the embryonic shield in zebrafish. Previous studies have indicated that nuclear β-catenin signaling is essential for induction of the dorsal organizer.

RATIONALE

Maternal Wnt ligand/receptor signaling has been proposed for stabilization and nuclear transportation of cytoplasmic β-catenin during early blastulation for the dorsal organizer formation. It is unknown whether β-catenin signaling is activated for organizer formation by Wnt-independent mechanisms.

RESULTS

In a spontaneous maternal-effect mutant line of zebrafish, none of the maternal mutant embryos formed the dorsal organizer at the shield stage, nor did they have a head or other dorsoanterior tissues at later stages of development. Through positional cloning and candidate gene testing, we found the mutant gene to be a previously uncharacterized locus that we designate huluwa (hwa). The defects of Mhwa mutants could be fully rescued by overexpression of wild-type hwa mRNA. Using antisense oligodeoxynucleotides or morpholino, we found that maternal depletion of Xenopus hwa transcript in oocytes also causes loss of the body axis and dorsal tissues in the derived embryos. Zebrafish Hwa protein consists of 294 amino acids comprising a 23–amino acid extracellular domain, a 23–amino acid transmembrane domain, and a 248–amino acid intracellular domain. Immunostaining revealed that Hwa protein is located on the plasma membrane of blastomeres only in a region during zebrafish early blastulation in which β-catenin is translocated into nuclei. By performing rescue experiments in hwa mutant embryos using different hwa mutant mRNAs, we found that the transmembrane domain and the intracellular 164VPPNSP169 and 184SLRRSST190 motifs are essential for Hwa activity. Overexpression of β-catenin efficiently rescued the defects in zebrafish Mhwa mutants. Ectopic expression of hwa mRNA efficiently induced a secondary axis in zebrafish and Xenopus embryos, which absolutely required β-catenin. Although mammalian homologs of zebrafish Hwa have not yet been identified, we found that transfection of zebrafish Hwa in human HEK293T cells enhances β-catenin signaling. Zebrafish Hwa could directly bind to human Axin protein, and transfection of zebrafish hwa into mammalian cells promoted Axin degradation with participation of tankyrases. Consistent with in vitro data, levels of Axin1 and Axin2 proteins in Mhwa mutant embryos were up-regulated, and overexpression of the dominant negative form Axin1ΔRGS or Axin1ΔDIX of Axin1 in Mhwa mutant embryos could rescue the dorsal organizer and the body axis. Therefore, we conclude that maternal Hwa is absolutely required for the formation of the dorsal organizer and the body axis by protecting β-catenin from Axin-mediated degradation in vertebrate embryos.

Overexpression of the Wnt antagonist gene DKK1, the dominant negative Wnt8a form dnwnt8, or the dominant negative LRP5 form LRP5ΔC in zebrafish embryos neither disrupted the dorsal organizer nor blocked the organizer- and body axis–inducing activity of ectopic hwa; treatment with the Wnt inhibitor Wnt-C59 also had no such effects. Xenopus embryos derived from oocytes depleted of lrp6 or wnt11b transcript could form the full body axis after being injected with hwa mRNA at the four-cell stage.

CONCLUSION

Maternal Hwa protein in vertebrate embryos is essential for the dorsal organizer and body axis formation, which activates β-catenin signaling during early blastulation in a Wnt ligand/receptor-independent fashion.

Huluwa (Hwa) is essential for the organizer and body axis formation.

Hwa protein is located on the plasma membrane of the prospective dorsal blastomeres in the zebrafish blastula. Hwa binds to and promotes the degradation of Axin in a way independent of Wnt ligand/receptor signaling, resulting in stabilization and nuclear translocation of β-catenin for activating organizer-specific target gene expression. The notochord (red) is an organizer-derived tissue.

Abstract

The vertebrate body is formed by cell movements and shape change during embryogenesis. It remains undetermined which maternal signals govern the formation of the dorsal organizer and the body axis. We found that maternal depletion of huluwa, a previously unnamed gene, causes loss of the dorsal organizer, the head, and the body axis in zebrafish and Xenopus embryos. Huluwa protein is found on the plasma membrane of blastomeres in the future dorsal region in early zebrafish blastulas. Huluwa has strong dorsalizing and secondary axis–inducing activities, which require β-catenin but can function independent of Wnt ligand/receptor signaling. Mechanistically, Huluwa binds to and promotes the tankyrase-mediated degradation of Axin. Therefore, maternal Huluwa is an essential determinant of the dorsal organizer and body axis in vertebrate embryos.

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