Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis

Abstract

The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral-aboral (O-Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O-Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3β inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O-Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal-vegetal patterning at earlier stages of anthozoan development.

Keywords: Axial evolution; Cnidarian; Wnt.

Fig. 1

(A) Phylogenetic tree depicting the outgroup relationship of cnidarians (arrow) with relation to bilaterian taxa (based on Hejnol et al. (2009)). (B) Ciliated Nematostella vectensis planula stage. The mouth is marked with an asterisk and the apical tuft is demarcated with an arrow.

Fig. 2

Spatial expression of three signaling pathway components (Nvwnt4, Nvwnt2 and NvfgfRA) and eight transcription factors at three developmental time points (30 hpf, 48 hpf and 72 hpf). Nvhbn is shown only at 30 hpf and 48 hpf and NvpaxD is shown only at 72 hpf. Two views of each time point are shown for each embryo. Upper view is an optical cross section and lower view is a surface view of the larval epithelium. The oral pole (asterisk) is to the left and the apical tuft is located at the aboral tip on the right. Cells which lie outside the main band of expression of Nvotp and Nvrx are indicated with arrows.

Fig. 3

The N. vectensis apical organ forms within a territory devoid of Nvsix3/6 transcript expression. (A) Cnidarian planula (apical view looking down on the apical tuft). The apical tuft, marked by acetylated tubulin staining (magenta) is outlined with a dashed circle. Nuclei are labeled with DAPI (white). (B) Cilia of the apical organ are labeled with acetylated tubulin (green) and the apical tuft marker NvfgfRA is shown in red. (C) A monochromatic image of the acetylated tubulin staining of the apical tuft (apical view) shown in (B). Note that cilia surrounding the apical tuft are also labeled, but that the apical tuft cilia are considerably longer and are localized to a singe apical spot. (D)–(H) Expression domains of mRNAs expressed at the apicat tuft at 48 hpf. Views are DIC images of the aboral pole centered on the position of the apical tuft following in situ hybridization to mRNA transcripts. (D) Nvsix3/6 is expressed in the aboral ectoderm surrounding the apical tuft but is not present in the apical tuft domain. (E) NvfgfRA is expressed in the apical tuft and surrounding cells in a continuous domain. (F) NvNK3 labels a narrowly restricted patch of cells at the apical tuft, with the exception of a small domain directly in the center of the apical tuft domain. (G) Nvanthox1 is found expressed throughout the apical tuft and expands in scattered cells beyond the apical tuft domain (arrows). (H) Nvirx is localized primarily to the oral pole and to the small patch of Nvsix3/6 negative territory at the apical tuft.

Fig. 4

(A) Aboral pole of a control embryo at 72 hpf stained with acetylated tubulin antibody in which the prominent cilia of the apical tuft (red dashed circle) are visible. (B) An embryo treated from 30 hpf to 72 hpf with 0.5 μM alsterpaullone in which the apical tuft is absent. Note the absence of prominent apical tuft cilia. (C)–(E″) Nvwnt4, Nvsp5, and Nvsix3/6 in control DMSO-treated embryos ((C)–(E)) and those treated from 30 hpf to 48 hpf with alsterpaullone at 1 μM (C′–E′) and 5 μM (C″–E″). (C) Nvwnt4 is expressed around the future site of the mouth (the blastopore) in control embryos. (C′) In embryos treated with alsterpaullone, Nvwnt4 expands to include an aboral expression domain in 1 μM treatments, and covers the entire epithelium in 5 μM treatments (C″). (D) Nvsp5 is found in an oral and aboral domain in control embryos and expands considerably in the aboral pole of 1 μM treated embryos (D′) and covers the entire epithelium in 5 μM treated embryos (D″). E. Nvsix3/6 is localized to the aboral pole of wildtype embryos. Treatment of embryos with alsterpaullone results in a complete loss of Nvsix3/6 expressionatboth1 μM (E′) and 5 μM (E″). (F)–(G) Spatial relationships between Nvwnt2 and Nvwnt4 ligand expression and Nvsix3/6 and Nvirx expression. (H)–(J) The dashed vertical line is placed in the same position in each image to illustrate the interface between Nvsix3/6 and Nvirx staining. (H) Expression of Nvsix3/6 as assayed by in situ hybridization and imaged using fluorescent signal of FastRed substrate. (I) Expression of Nvirx as assayed by in situ hybridization and imaged using reflection microscopy. (J) Overlay of Nvsix3/6 and Nvirx fluorescent signal in an embryo subjected to double in situ hybridization. (C)–(E), (C′)–(E′), (C″)–(E″), and (F) and (G) are lateral views with the oral pole (asterisk) to the left and the apical tuft to the right. (D),(E), (H) and (I) are aboral views looking down on the apical tuft (arrow).

Fig. 5

Transformation of epithelial identity following activation of canonical wnt signaling with alsterpaullone. (A) and (B) Markers of oral ectoderm, Nvotp and Nvhbn,in DMSO treated embryos (upper panels) and in alsterpaullone treated embryos (lower panels). Graphs of N. vectensis embryos below photos indicate the average expression domain boundaries calculated from 10 measured embryos. (A′) and (B′). Nvotp and Nvhbn are greatly expanded toward aboral territories in alsterpaullone treated embryos. (C)–(G) Aboral territories marked by expression of Nvdlx, Nvrx, Nvanthox1, NvfgfrA and NvNK3 in DMSO treated control embryos. (D)–(G′). Markers of aboral epithelial molecular identity are lost following alsterpaullone treatment.

Fig. 6

Recovery of epithelial mRNA transcript expression in alsterpaullone-treated embryos following washout, as scored by in situ hybridization. All views are lateral, optical cross-sections with the oral pole to the left. (A)–(C′) Expression of Nvwnt4. (D)–(F′)– Expression of Nvwnt2. (G)–(I′–. Expression of Nvsp5. (J)–(L′– Expression of Nvotp. (M)–(O′– Expression of Nvdlx. (P)–(R′) Expression of Nvrx. (S)–(U′)– Expression of NvfgfRA. (V)–(X′) Expression of Nvanthox1. (A), (D), (G), (J), (M), (P), (S), and (V) DMSO-treated control embryos incubated in 0.5% DMSO from 30 hpf to 72 hpf. (A′), (D′), (G′), (J′), (M′), (P′), (S′) and (V′). Embryos treated from 30 hpf to 48 hpf in 0.5% DMSO and then washed into a new DMSO control treatment until 72 hpf (washout control). (B), (E), (H), (K), (N), (Q), (T) and (W). Embryos treated with 1 μM alsterpaullone from 30 hpf to 72 hpf. (B′), (E′), (H′),(K′), (N′),(Q′), (T′) and (W′a. Embryos treated from 30 hpf to 48 hpf in 1 μM alsterpaullone and then washed out into DMSO only until 72 hpf. (C), (F), (I), (L), (O), (R), (U), and (X) Embryos treated with 5 μM alsterpaullone from hpf 30 to 72 hpf. (C′) (F′). (I′) (L′) (O′) (R′), (U′), and (X′) Embryos treated from 30 hpf to 48 hpf in 5 μM alsterpaullone and then washed out into DMSO only until 72 hpf.

Fig. 7

Effects of canonical wnt activation via alsterpaullone treatment on epithelial patterning and later formation of neural cell types. (A)–(A′) Nv-op85309 expression restricts to the aboral pole after incubation with low alsterpaullone concentrations (A′) compared to DMSO-treated controls (A) and is absent in high alsterpaullone concentrations (A″). Light staining in the endoderm (en) of the DMSO control embryo is background staining. Darkly staining cells in the ectoderm (ec) is specific signal. (B) NvanthoRFamide is expressed in oral neurons and a few scattered aboral neurons. (B′e Following treatment with alsterpaullone, anthoRFamide neurons are expressed throughout the larval epithelium in an approximately even distribution. (B″) At high concentrations, NvanthoRFamide neurons are found primarily at the aboral pole. (C)–(C′)– Summary of wnt pathway components expressed in wildtype planula larvae (C) and those treated with alsterpaullone (C′). The ectodermally expressed wnt ligands, Nvwnt4 (orange) and Nvwnt2 (yellow), the transcription factors Nvsix3/6 and Nvirx are indicated. (D) Epithelial patterning from the oral pole to the apical tuft of 48 hpf embryos. Genes are indicated by colored bars and standard deviations of measured embryos are indicated with black lines. Regions indicating predicted unique identity based on novel combinatorial overlap of expression of molecular markers assayed by in situ hybridization are indicated by alternating light and dark gray bars. See methods for description of measurements. (E)–(E″)– A summary of the distribution of opsin-bearing (red) and anthoRFamide-bearing (black) cells in control embryos and (E) and embryos treated with 1 uM (E′) as well as 5 uM (E″) concentrtions of alsterpaullone at 72 hpf. Views of all embryos are lateral with the oral pole to the left.