Development of the aboral domain in Nematostella requires β-catenin and the opposing activities of Six3/6 and Frizzled5/8

Abstract

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvβ-catenin regulates both oral and aboral development. In the oral hemisphere, Nvβ-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvβ-catenin knockdown. In addition, Nvβ-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvβ-catenin in the patterning of the entire oral-aboral axis of Nematostella.

Keywords: Anterior posterior axis; Apical organ; Axis formation; Cnidaria; Wnt signaling.

Fig. 1.

NvSix3/6 controls but does not initiate aboral domain development. (A-C) Nuclear staining of NvSix3/6-Venus fusion protein (green) and DAPI (blue) at blastula stage. (D-U) Lateral views (aboral pole to the left) of in situ hybridizations showing expression patterns of NvFoxQ2a, NvFGFa1 and NvWnt2 at mid-gastrula (26 hpf; D-O) or mid-blastula stage (14 hpf; P-U) embryos. Injected morpholinos, mRNA or AZ treatment (2 µM, 2-26 hpf) are indicated above the panels, probes on the left. Scale bars: 50 µm.

Fig. 2.

Nvβ-catenin is necessary for gastrulation and for transcription of both oral and aboral marker genes. (A-E) Early blastulae labeled with anti-mouse β-catenin antibody (white in A-C,E; yellow in D). In D, DAPI is blue and colocalization is white. Treatments are indicated on the left side. Dashed red line in A,B delimits the area of weak nuclear β-catenin antibody staining. The two images in B represent maximum projections of the two halves of the same embryo. Nvβ-cat MO (500 µM) reduces and AZ treatment enhances labeling. Note that the embryo in A is 6 hpf whereas the others are 8 hpf. MO injection causes a developmental delay (C,E) compared with wild-type (WT) embryos (A,B). (F-H) Phalloidin (green) staining of control MO (F) and Nvβ-cat MO (G, 500 μM, H, 100 μM) injected embryos at 26 hpf. (I) RT-qPCR of Nvβ-cat MO-injected embryos (28 hpf) at a concentration of 500 µM (dark gray) and 100 µM (light gray) compared with the control (control MO, 500 µM). Fold changes of the relative expression levels are shown; values between −1 and +1 mean no change (highlighted in light gray). Error bars represent the s.d. of three (500 µM) or two (100 µM) biological replicates in two technical replicates each. Scale bars: 50 µm.

Fig. 3.

Nvβ-cat is required for the establishment of oral and aboral patterning systems. (A-X) In situ hybridizations at mid-blastula stage (14 hpf). Probes are indicated on the left, injections indicated above. Lateral views, the future aboral pole to the left, assuming continuity of the expression of marker genes at gastrula stage, when the blastopore becomes visible. The presence of oral and aboral expression domains for NvFGFa1 makes the orientation in Q unreliable. Note the shift of NvFkh (U) and NvFoxB (V) away from the oral pole in low dose Nvβ-cat MO-injected blastulae. Expression patterns shown in U,V were observed in ∼50% of the embryos; the remaining 50% showed no staining (NvFkh: 16/34; NvFoxB: 13/25). Scale bars: 50 μm.

Fig. 4.

Loss of polarity upon Nvβ-cat knockdown is partially rescued by AZ treatment. (A-VV) Lateral views (aboral pole to the left) of in situ hybridizations at mid-gastrula stage (26 hpf). Probes are indicated on the left, treatments/injections indicated above. Nvβ-cat MO high: 500 µM; Nvβ-cat MO low: 100 µM; AZ: 5 µM, 4-26 hpf. Animals injected with high Nvβ-cat MO dose and treated with AZ (Y-FF) resemble those injected with low Nvβ-cat MO dose (Q-X); animals injected with low Nvβ-cat MO dose and treated with AZ (GG-NN) resemble those injected with control MO (A-H), except for NvWnt2 (F,LL). Scale bars: 50 μm.

Fig. 5.

Aboral domain expression of NvFz5/8 requires oral suppression by Nvβ-cat and maintenance by NvSix3/6. (A-S) NvFz5/8 in situ hybridizations of uninjected animals (A-H) and animals treated/injected as indicated (I-S). All images are lateral views, except H which is an aboral view. Stages are indicated above. Nvβ-cat MO high: 500 µM; Nvβ-cat MO low: 100 µM; AZ: 5 µM, 4-26 hpf. Scale bars: 50 µm.

Fig. 6.

NvFz5/8 limits the size of the aboral domain during gastrulation. (A-BB) Lateral views (aboral pole to the left) of in situ hybridizations at the stages indicated above. Injections are also indicated above, probes on the left. The expansion of aboral marker gene expression and the reduction of oral markers are first visible at early gastrula stage and become more pronounced at mid-gastrula stage; see main text for more details. Asterisk in N indicates non-specific staining. Scale bars: 50 µm.

Fig. 7.

NvFz5/8 regulates the size of the apical organ by controlling FGF activity in the aboral-most domain. (A) RT-qPCR of NvFz5/8 MO-injected embryos at mid-gastrula (26 hpf). Fold changes of the relative expression levels of the indicated genes are shown; values between −1 and +1 mean no change (highlighted in light gray). Error bars represent the s.d. of three biological replicates. (B-I) Lateral views (aboral pole to the left; B,D,F,H) and aboral views (C,E,G,I) of the apical ciliary tuft visualized by anti-acetylated tubulin antibody staining of mid-planulae. The injected MOs are indicated above. The red brackets highlight the size of the apical tuft in the different conditions. (J-T) Lateral views (aboral pole to the left) of NvFGFa1 and NvFz5/8 in situ expression patterns in mid-planula (J-Q) and mid-gastrula (R-T) MO-injected embryos. The injected MOs and probes used are indicated above and on the left, respectively. Scale bars: 50 µm.

Fig. 8.

Summary of the changes in gene expression in Nvβ-cat, NvFz5/8 and NvSix3/6 knockdown animals. (A,B) Schematics of blastula (A) and gastrula (B) stage embryos, aboral pole to the left. NvFoxQ2-, NvWnt2- and NvFkh-expressing domains are indicated in dark blue, orange and red, respectively. Nvβ-cat morphants (low concentration) show at blastula stage a reduced aboral NvSix3/6/NvFoxQ2a-expressing territory and an expanded oral NvWntA/NvSnailA-expressing territory, and at gastrula stage an expanded endoderm-like NvSnailA-expressing territory. In Nvβ-cat morphants (high concentration), no polarized gene expression can be detected, but both oral (NvWntA, NvSnailA) and aboral (NvFz5/8) markers are expressed throughout the body. NvFz5/8 and NvSix3/6 morphants do not display alterations in gene expression at blastula stage. At gastrula stage, aboral markers are expanded towards the oral pole and oral markers are reduced in NvFz5/8 morphants. Knockdown of NvSix3/6 results in reduction of aboral and expansion of oral markers at gastrula stage.