Sea anemone Frizzled receptors play partially redundant roles in oral-aboral axis patterning

Abstract

Canonical Wnt (cWnt) signalling is involved in a plethora of basic developmental processes such as endomesoderm specification, gastrulation and patterning the main body axis. To activate the signal, Wnt ligands form complexes with LRP5/6 and Frizzled receptors, which leads to nuclear translocation of β-catenin and a transcriptional response. In Bilateria, the expression of different Frizzled genes is often partially overlapping, and their functions are known to be redundant in several developmental contexts. Here, we demonstrate that all four Frizzled receptors take part in the cWnt-mediated oral-aboral axis patterning in the cnidarian Nematostella vectensis but show partially redundant functions. However, we do not see evidence for their involvement in the specification of the endoderm - an earlier event likely relying on maternal intracellular β-catenin signalling components. Finally, we demonstrate that the main Wnt ligands crucial for the early oral-aboral patterning are Wnt1, Wnt3 and Wnt4. Comparison of our data with knowledge from other models suggests that distinct but overlapping expression domains and partial functional redundancy of cnidarian and bilaterian Frizzled genes may represent a shared ancestral trait.

Keywords: Nematostella; Axial patterning; Cnidaria; Gastrulation.

Fig. 1.

Normal developmental expression of the Fz genes and LRP5/6. From 10 hpf onwards, the animal/oral pole of the embryo is pointing downwards. In situ hybridization with digoxigenin-labelled RNA probes followed by anti-Dig-AP staining and NBT/BCIP detection. Scale bar: 100 µm.

Fig. 2.

Effects of RNAi-mediated KD of LRP5/6 and Fz5, as well as triple and quadruple Fz gene KD combinations on the expression of the β-catenin-dependent markers of different axial domains in the 30 hpf late gastrula. To keep the row labels readable, simultaneous RNAi of, for example, Fz1, Fz5 and Fz10 is marked as shFz4+Fz5+Fz10 rather than shFz4+shFz5+shFz10. The same labelling convention applies to all the other figures showing simultaneous KDs. Orange arrows indicate the direction of the drastic expression shifts. There is curious asymmetric expression of Bra and Wnt2 upon Fz4+Fz5+Fz10 RNAi, indicating possible abnormal feedback from the directive axis patterning mechanism. Black arrows indicate the ring of stronger Bra expression in the midbody and the aboral expansion of Wnt2 domain, suggesting an ectopic enhancement of the β-catenin signalling. The numbers in the top right corners show the fraction of embryos demonstrating this phenotype. Scale bar: 100 µm. For each gene, lateral views (oral end down) are on the left and oral (or aboral in the case of Six3/6) views are on the right. In situ hybridization with digoxigenin-labelled RNA probes followed by anti-Dig-AP staining and NBT/BCIP detection.

Fig. 3.

Effects of the RNAi-mediated KD of LRP5/6, Fz genes and the Wnt3/Wnt4 combination on the later development of the embryo. (A-D) Effects of the KD of LRP5/6 (A), of individual Fz genes (B), of triple and quadruple Fz gene KDs (C), and of the double KD of Wnt3 and Wnt4 (D). 4 dpf embryos are stained using phalloidin-AlexaFluor488 to visualize actin filaments. Scale bars: 100 µm. In the lateral views, the oral end points downwards.

Fig. 4.

Endoderm specification is an early event that does not seem to depend on Fz and LRP5/6. (A) Identification of the time of endoderm specification. Lateral views of 30 hpf embryos, oral end downwards. (B) Endodermal marker expression is not affected by the KD of LRP5/6 or by knockdown of individual Fz genes. The numbers in the top right corners show the fraction of embryos showing this phenotype. For each gene, lateral views (oral end down) are at the top and oral (or aboral in the case of Six3/6) views are at the bottom. All embryos are late gastrulae at 30 hpf. Scale bars: 100 µm. In situ hybridization with digoxigenin-labelled RNA probes followed by anti-Dig-AP staining and NBT/BCIP detection.

Fig. 5.

Effect of the morpholino-mediated KD of LRP5/6 and orally expressed Fz genes on the early development of Nematostella. (A) Effect of the knockdowns on the expression of the markers of the distinct axial domains in the ectoderm and on the expression of the endodermal marker SnailA. In situ hybridization with digoxigenin-labelled RNA probes followed by anti-Dig-AP staining and NBT/BCIP detection. All embryos are fixed at 30 hpf. The numbers in the top right corners show the fractions of embryos demonstrating this phenotype. Scale bar: 100 µm. Lateral views, oral end downwards. The inset image of an oral view of the LRP5/6 morphant stained for Six3/6 shows that the pre-endodermal plate does not express Six3/6. (B) Effects of the LRP5/6 and Fz10 morpholino KDs on the later development of the embryos. Phalloidin staining of the 2 dpf and 4 dpf planulae. Scale bars: 100 µm.

Fig. 6.

Effect of the KD of Wnt genes on the expression of the oral, midbody and aboral ectoderm markers in the 30 hpf gastrulae. (A) KDs of individual Wnt genes. (B) Simultaneous KDs of Wnt3 or Wnt4 with the individual Fz receptor genes. (C) Simultaneous KDs of Wnt3 and Wnt4. Orange arrows indicate the direction of the particularly drastic expression shifts. The numbers in the top right corners show the fractions of embryos demonstrating this phenotype. Scale bar: 100 µm. For each gene, lateral views (oral end downwards) are at the top and oral (or aboral in the case of Six3/6) views are at the bottom. In situ hybridization with digoxigenin-labelled RNA probes followed by anti-Dig-AP staining and NBT/BCIP detection.