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. 2013;11(2):e1001488.
doi: 10.1371/journal.pbio.1001488. Epub 2013 Feb 19.

The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian

Chiara Sinigaglia  1 , Henriette Busengdal, Lucas Leclère, Ulrich Technau, Fabian Rentzsch
Affiliations

The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian

Chiara Sinigaglia et al. PLoS Biol. 2013.

Abstract

The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis. We show that orthologs of the bilaterian anterior developmental genes six3/6, foxQ2, and irx have dynamic expression patterns in the aboral region of Nematostella. Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella. NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bilaterian anterior marker genes are expressed at the aboral pole of Nematostella.
(A1–E7) In situ hybridizations, probes are indicated on the left; developmental stages above the images. (A–E2, A–E3, A–E5, and A–E6) are lateral views, from planula stage on with the aboral pole to the left. At the blastula stage, embryos are oriented with the assumption that the staining is continuous between the blastula and gastrula stage. Note that the future oral and aboral sides cannot be distinguished morphologically prior to gastrulation. (A–E4 and A–E7) are aboral views. Expression domains of NvSix3/6, NvFoxQ2a, NvIrx, and NvFGFa1 are highly similar until the gastrula stage and then segregate into mutually exclusive domains. NvFoxJ1 expression becomes detectable only after gastrulation. (F1 and F2) Double in situ hybridization with NvSix3/6 (red) and NvFGFa2 (blue) probes, showing the early restriction of the NvFGFa2 expression from the gastrula stage on. (G–I) Schematic representation of the three types of aboral expression patterns: (G) “ring genes,” (H) “spot genes,” and (I) “late genes.” Synexpression groups are indicated on the right side; genes that have been described previously are shown in gray. Note that NvSoxB(1), NvHoxF/Anthox1, and NvFGF8a have additional expression domains not indicated in the cartoon.
Figure 2
Figure 2. FGF signalling specifically controls gene expression in the apical organ territory.
(A–C) Schematic illustration of the phenotype caused by knockdown of NvFGFa1 (B) and NvFGFa2 (C); NvFGFa1 morphants lack the apical tuft (indicated in red) and the small indentation at the aboral pole, from which it develops. NvFGFa2 morphants have an expanded indentation and apical tuft. (D–U) In situ hybridizations at the midplanula stage (72 h postfertilization, hpf). Morpholinos are indicated above the images, with in situ probes on the left side. (D–I) The aboral gap in the expression of “ring genes” is lost in NvFGFa1 and expanded in NvFGFa2 morphants. (J–U) Expression of “spot” and “late genes” is abolished in NvFGFa1 morphants and expanded in NvFGFa2 morphants. (N and Q) Note that knockdown of NvFGFa1 specifically affects the aboral expression of NvSoxB(1) and NvHoxF/Anthox1. For each experimental condition and analysis, a lateral view with aboral pole to the left is shown next to an aboral view (lateral view, left; aboral view, right); all images at the planula stage. Scale bar, 100 µm.
Figure 3
Figure 3. NvSix3/6 controls the development of the aboral territory.
(A–C) Illustration of the morphological phenotype after knockdown of NvSix3/6 (B) and NvFoxQ2a (C). (B) NvSix3/6 morphants have a shortened oral-aboral body axis and lack the apical tuft. (C) NvFoxQ2a morphants have a smaller apical tuft. (D–AA) In situ hybridizations at the midplanula stage (72 hpf); morpholinos are indicated above the images, with in situ probes on the left side. (E, H, K, N, Q, T, W, and Z) In NvSix3/6 morphants, expression of all aboral pole markers except NvSix3/6 is reduced. (T) Pharyngeal expression of NvSoxB(1) is retained and visible in the centre of the aboral view. (W) Note that NvHoxF/Anthox1 expression in the apical organ and in scattered aboral cells is absent/reduced. (F, I, L, O, R, U, X, and AA) NvFoxQ2a morpholino injection leads to a modest reduction in the expression of “spot” and “late genes” and a corresponding reduction in the gap of “ring genes.” For each experimental condition and analysis, a lateral view with aboral pole to the left is shown next to an aboral view (lateral view, left; aboral view, right); all images at planula stage. Aboral view in (U) is tilted to allow discrimination of the aboral (dashed circle) from pharyngeal expression. Scale bar, 100 µm.
Figure 4
Figure 4. FGF receptor signalling suppresses NvSix3/6 expression after gastrulation and is sufficient to induce apical organ formation.
(A–D) Schematic representations of the morphological phenotype obtained after morpholino double injections. (D) Co-injection of NvSix3/6 and NvFGFa2 morpholinos reverses the loss of the apical tuft of NvSix3/6 morphants (C). (E–P) Aboral views of in situ hybridizations at the midplanula stage (72 hpf); probes are indicated on the left side and morpholinos on top. Co-injection of NvSix3/6 and NvFGFa2 morpholinos leads to a moderate expansion in the expression of “spot genes” (E–L) and expansion of the aboral gap of the NvSix3/6 expression (M–P). (Q–X) Effects of FGFR inhibitor on the double injected animals. Probes are indicated above the images, with morpholinos on the left side. Embryos were treated, from the late gastrula stage on, with DMSO (Q, R, U, V) or with the FGF receptor inhibitor SU5402 in DMSO (S, T, W, X). Inhibition of FGFR activity suppresses apical organ formation in double injected embryos. Scale bar, 100 µm.
Figure 5
Figure 5. NvHoxF/Anthox1 and NvSoxB(1) have distinct roles in the regulation of aboral domain and apical organ genes.
(A–C) Schematic drawings illustrating the morphology of NvHoxF/Anthox1 (B) and NvSoxB(1) MO-injected planulae. Both genes are required for apical tuft formation, but only NvSoxB(1) morphants show a slightly elongated body column (C). (D–O) In situ hybridizations, lateral views (left) with the aboral pole to the left are shown next to aboral views (right) at the midplanula stage (72 hpf). In situ probes are indicated on the left, with morpholinos on top. NvHoxF/Anthox1 and NvSoxB(1) are required for the expression of NvFGFa1 (D–F) but not NvFGFa2 (G–I). NvSoxB(1) morphants lack the gap in the expression of NvSix3/6 and NvFoxQ2a (L, O), while NvHoxF/Anthox1 morphants only lack the gap in NvSix3/6 expression (K, N). Scale bar, 100 µm.
Figure 6
Figure 6. NvSix3/6 and NvFGFRa are required for the initiation of a FGF signalling feedback loop at gastrulation.
(A–T) In situ hybridizations with probes indicated on top and injected morpholinos on the left. All animals are at the midgastrula stage (24 hpf); lateral views with aboral pole to the left are shown next to aboral views. In NvSix3/6 morphants, the expression of NvFGFa1 and NvFGFa2 is reduced (C, D, G, H). In contrast to the situation at the planula stage, the expression of NvFGFa2 but not NvFGFa1 is reduced in NvFGFa1 morphants (K, L). NvFGFRa is required for the expression of NvFGFa1 and NvFGFa2, but not for NvSix3/6 and NvFoxQ2a (Q–T). (U–W) Quantitative RT-PCR of (U) NvSix3/6 MO-, (V) NvFGFa1 MO-, and (W) NvFGFa2 MO-injected embryos at the midgastrula stage (24 hpf). Fold changes of the relative expression levels of the indicated genes are shown; values between [−1, +1] mean no change, and +2 corresponds to 100% increase. Error bars represent the standard deviation of three biological replicates.
Figure 7
Figure 7. NvSix3/6 and canonical Wnt signalling interact in aboral domain development.
(A–F) Lateral views with aboral pole to the left of in situ hybridizations with a NvWnt2 probe from gastrula stage to the mid-planula (stages indicated on top), injected with a control (A–C) or an NvSix3/6 (D–F) morpholino. The expression domain of NvWnt2 progressively expands toward the aboral pole upon knockdown of NvSix3/6. (G) Quantification of the number of DAPI stained nuclei in control MO- (blue) and NvSix3/6 MO- (red) injected embryos. Number of nuclei is indicated on the y-axis, with time point of analysis on the x-axis. (H–O) Lateral views at the gastrula stage, with aboral pole to the left. Probes are indicated on the top, with treatments on the left. At 2 µM Azakenpaullone, expression of aboral markers is suppressed, and central and oral markers are shifted and expanded aborally, respectively. Scale bar, 100 µm.
Figure 8
Figure 8. Reduction of aboral neural markers and loss of aboral morphology in NvSix3/6 morphants.
(A–D) Lateral views of in situ hybridizations at the gastrula stage, with aboral pole to the left. Probes are indicated on the left, with morpholinos on top. Injection of NvSix3/6 MO leads to a reduction of aboral NvDmrtB positive neurons, but not to a general loss of neurons. (E–H) Lateral views of midplanula (72 hpf) animals labelled with DAPI (blue), Phalloidin (green), and anti-acetylated tubulin antibody (red). Aboral pole is to the left, and injected morpholinos are indicated on top. Dashed boxes mark the apical organ region; NvFGFa1 morphants lack basally positioned nuclei (F); in NvSix3/6 morphants (G), no difference between aboral and central ectoderm is visible. In NvHoxF/Anthox1 morphants, the aboral indentation with basal migration of aboral pole nuclei occurs, but no apical tuft develops (H).
Figure 9
Figure 9. A conserved anterior patterning system in Nematostella planula and sea urchin larva.
(A and B) Model for gene regulatory network in place at gastrula and planula stages during aboral patterning of Nematostella larvae. At the gastrula stage (A), all the genes are co-expressed in the aboral area and NvSix3/6 positively regulates the expression of FGF ligands and NvFoxQ2a. The positive regulation of NvFGFa2 by NvFGFa1 is already in place, and it is likely responsible for the early restriction of FGFs' expression domains. At the planula stage (B), the aboral territory is divided in two domains, one surrounding the apical organ (in purple and blue/red) and one in the apical organ (orange and yellow). The first corresponds to the expression domain of NvSix3/6 and NvFoxQ2a (and NvFoxD1), the latter to the expression of FGF ligands (drawing). The regulatory interactions change at this stage and NvFGFa1 represses NvSix3/6; additionally, NvFGFa1 starts a positive autoregulation (direct or indirect), responsible for the maintenance of the FGF expressing area. (C) Segregation of expression domains in sea urchin larva. Similar to Nematostella gastrula, the genes are co-expressed at the contra-blastoporal (anterior) pole of sea urchin (data from Strongylocentrotus purpuratus and Paracentrotus lividus) at early blastula and then segregate in distinct domains from the mesenchymal blastula stage on, when the tuft of cilia appears. In this case, however, the genes segregate in a slightly different way: six3 forms a ring around the apical plate, while foxQ2 is restricted to the apical plate itself. The apical domain expression of the FGF receptor is restricted to the apical plate.
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Comment in

  • Which came first, the head or the brain?
    Mejia R. Mejia R. PLoS Biol. 2013;11(2):e1001484. doi: 10.1371/journal.pbio.1001484. Epub 2013 Feb 19. PLoS Biol. 2013. PMID: 23431265 Free PMC article. No abstract available.

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