Cnidarian-bilaterian comparison reveals the ancestral regulatory logic of the β-catenin dependent axial patterning

Abstract

In animals, body axis patterning is based on the concentration-dependent interpretation of graded morphogen signals, which enables correct positioning of the anatomical structures. The most ancient axis patterning system acting across animal phyla relies on β-catenin signaling, which directs gastrulation, and patterns the main body axis. However, within Bilateria, the patterning logic varies significantly between protostomes and deuterostomes. To deduce the ancestral principles of β-catenin-dependent axial patterning, we investigate the oral-aboral axis patterning in the sea anemone Nematostella-a member of the bilaterian sister group Cnidaria. Here we elucidate the regulatory logic by which more orally expressed β-catenin targets repress more aborally expressed β-catenin targets, and progressively restrict the initially global, maternally provided aboral identity. Similar regulatory logic of β-catenin-dependent patterning in Nematostella and deuterostomes suggests a common evolutionary origin of these processes and the equivalence of the cnidarian oral-aboral and the bilaterian posterior-anterior body axes.

Fig. 1. The “repressor X” concept and the search strategy.

a Scheme of the Wnt/β-catenin signaling pathway indicating the members manipulated in this study in order to artificially upregulate it. We use two types of treatments (red) to upregulate β-catenin signaling: pharmacological inhibition of GSK3β by AZK and mutation of APC. b Oral, midbody, and aboral domains of the 1 day post fertilization (1 dpf) gastrula visualized by molecular markers. Lateral views, oral to the left. Asterisk denotes the blastopore. Arrowheads demarcate corresponding positions. Scale bar 100 µm. c Hypothetical mechanism of the response of the “saturating” and “window” genes to different intensities of the β-catenin signaling and the putative role of the transcriptional repressor X in regulating the “window” expression behavior. Hypothetical oral-to-aboral gradient of β-catenin signaling is shown in light blue on the upper panels. Repressor X is a saturating gene expressed above a certain β-catenin signaling intensity indicated by the red dashed line, i.e., orally (pink expression domain on graphs and middle panels). The window gene (blue expression domain) is activated above the β-catenin signaling intensity indicated by the black dashed line, however, it becomes repressed in the area of repressor X expression. Upon AZK treatment, the β-catenin signaling intensity increases eventually reaching saturation (blue arrowhead on the Y-axis). In increasing AZK concentrations, the minimal β-catenin signaling intensity sufficient for repressor X activation shifts aborally, displacing the area available for the window gene expression until it becomes impossible for the window gene to be expressed anywhere in the embryo. Upon repressor X knockdown (bottom panel), the window gene starts to behave as a saturating gene. O and A on graphs indicate the oral and the aboral end. d Search strategy used to identify transcriptional repressor X. e Scheme of treatments. At 1 dpf, AZK treatments were stopped at 30 h post fertilization (hpf), and either RNA was extracted immediately, or the embryos were washed out and incubated in Nematostella medium until 3 dpf (72 hpf). Asterisks indicate time points of RNA extraction. f Venn diagram with the numbers of the putative transcription factor coding genes upregulated by different treatments. The color code corresponds to that on e.

Fig. 2. Double FISH analysis of the expression domains of the four main repressor X candidates, oral Wnt genes, midbody markers Wnt2 and Sp6-9, and aboral marker Six3/6.

a FISH analysis of the expression domains of the transcription factor genes Bra, FoxA, FoxB, Lmx, Sp6-9 and Six3/6 in relation to each other. b FISH analysis of the expression domains of the abovementioned transcription factor genes in relation to the expression domains of the ectodermally expressed Wnt genes. c FISH analysis of the expression domains of the ectodermally expressed Wnt genes in relation to each other. d Schematic representation of the expression boundaries of the transcription factors in the Nematostella gastrula. e Schematic representation of the expression boundaries of the Wnt genes in the Nematostella gastrula. On a–c, lateral views (oral to the left) and oral views (unless specified otherwise) of representative embryos from two independent experiments with n > 30 for each combination of in situ hybridization probes are shown. Scale bars 100 µm. Dashed lines on d and e represent the same molecular boundaries.

Fig. 3. The effect of the repressor X candidates knockdown on the expression of the “window” genes Wnt1 and Wnt2.

Bra and Lmx knockdowns convert Wnt2 into a “saturating” gene, while FoxA knockdown does the same with Wnt1. The effect of Lmx knockdown appears to be similar but weaker than that of Bra. FoxB knockdown results in a “weak AZK effect” on both Wnt1 and Wnt2 suggesting that FoxB mildly represses both. The effects of the knockdowns of Bra, Lmx, and FoxB on Wnt2 expression are non-redundant, but similar and additive (see Supplementary Figs. 6 and 7). Quadruple knockdown with shRNA against Bra, Lmx, FoxA and FoxB (=shBLAB) removes oral molecular identity of the embryo completely. Red arrow indicates the bottom of the pharynx expressing the midbody marker Wnt2. On lateral views, asterisk denotes the blastopore. The numbers in the top right corner show the ratio of embryos displaying the phenotype shown on the image to the total number of embryos treated and stained as indicated on the figure. Scale bar 100 µm.

Fig. 4. Midbody domain prevents oral expansion of the aboral domain.

a Scheme of the treatments and Venn diagram showing the number of putative transcription factors downregulated by various treatments. b Sp6-9 prevents oral expansion of the aboral marker Six3/6. In BraMO, Six3/6 expression is also expanded orally, likely due to the oral shift of the Sp6-9 expression upon Bra knockdown (see Supplementary Fig. 5). Oral expansion of Six3/6 is enhanced upon double knockdown of Sp6-9 and Bra. Lateral views, oral to the left; asterisk denotes the blastopore. c Sp6-9 is a “window” gene shifting orally upon simultaneous knockdown of Bra, Lmx, FoxA and FoxB (=shBLAB) and expanding globally upon shBLAB knockdown followed by AZK treatment. Sp6-9-free area disappears in shBLAB. Lateral views, oral to the left; asterisk denotes the blastopore. The numbers in the top right corner on b, c show the ratio of embryos displaying the phenotype shown on the image to the total number of embryos treated and stained as indicated on the figure. Scale bars 100 µm.

Fig. 5. Nematostella embryo initially has aboral identity, which later becomes restricted to the aboral domain.

a Six3/6 is detectable in the aboral portion of the embryo from 12 h post fertilization (hpf) on. Bra becomes detectable in a group of cells on the future oral side of the embryo as early as 8 hpf, and by 10 hpf it forms a ring around the future preendodermal plate. Fz5/8 is a maternally deposited transcript. Fz5/8 expression shifts to the future aboral side by 12 hpf. SoxB1 is also a maternally deposited transcript. The loss of SoxB1 staining in the future endodermal territory occurs simultaneously with the formation of the Bra ring, and is likely regulated by the same mechanism. By gastrula stage, SoxB1 is expressed in the blastopore lip and aborally. On all lateral views, on which the O–A axis is discernible, the oral end is marked with an asterisk. Inset images of 10, 12 and 14 hpf embryos stained for Bra and SoxB1 show the lack of expression in the putative preendodermal plate on embryos orientated with their oral ends facing the viewer. 6 hpf images of Fz5/8 and SoxB1 expression show the optical midsection (left) and the surface view (right) of the same embryos. b Fz5/8 and SoxB1 expression remains ubiquitous in the β-catenin morphants. Lateral views of the 30 hpf gastrulae, oral ends are marked with an asterisk. c SoxB1 expression upon Bra knockdown appears weaker in the oral domain and expanded in the aboral domain, which is likely due to the oral shift of the Sp6-9 expression. Sp6-9 knockdown significantly expands SoxB1 expression fusing the oral and aboral expression domains. Simultaneous knockdown of Bra and Sp6-9 makes this effect even more pronounced consistent with the general aboralization of the embryo. The numbers in the top right corner on b, c show the ratio of embryos displaying the phenotype shown on the image to the total number of embryos treated and stained as indicated on the figure. Scale bars 100 µm.

Fig. 6. Endoderm has no influence on O–A patterning of the ectoderm.

a Fluorescence in situ hybridization shows that Bra expression does not extend into the endoderm of the embryos (pink outline), which were placed in 5 µM AZK after the time of the specification of the endodermal domain. b 5 µM AZK incubation starting before the time of the specification of the endodermal domain prevents endoderm formation but still leads to the abolishment of Wnt2 expression in shControl and to the conversion of Wnt2 into a “saturating” gene upon shBLAB knockdown (compare with Fig. 3). The numbers in the bottom right corner show the ratio of embryos displaying the phenotype shown on the image to the total number of embryos treated and stained as indicated on the figure. Scale bars 100 µm.

Fig. 7. Oral–aboral patterning regulation in Nematostella and P-A patterning in sea urchin are comparable.

a–c Scenarios of the direct correspondence of the cnidarian and bilaterian body axes. pb – polar bodies, aHox – anterior Hox gene, naHox – non-anterior Hox gene, asterisk denotes the mouth. Triangles with a β denote the direction of the β-catenin signaling gradient. d Putative topology of the gene regulatory network of the β-catenin-dependent O–A patterning in Nematostella. The GRN explains why the midbody domain does not expand into the oral and into the aboral domains, and why the aboral domain does not expand into the midbody. It does not explain, however, why the oral domain does not expand aborally. e Comparison of the early β-catenin-dependent patterning in sea urchin and Nematostella shows clear similarities. Unfertilized egg with maternal Fz5/8 and SoxB1 mRNA (future anterior/aboral markers) and maternal Dsh protein localized at the gastrulation pole^,. Upon activation of β-catenin signaling in the embryo, first in the endomesodermal domain and then in the posterior/oral ectoderm the expression of Fz5/8 and SoxB1 is suppressed, and the anterior/aboral markers (including the zygotic genes Six3/6 and FoxQ2) become progressively confined to one side of the axis. The axis becomes patterned by mutually repressive transcription factors (T). Gray “T” in Nematostella indicate repressive interactions, for which candidate transcription factors are not known. Triangles with a β denote the direction of the β-catenin signaling gradient. β? indicates that in Nematostella, nuclear β-catenin could only be experimentally detected until midblastula stage, after which the presence of nuclear β-catenin gradient is deduced based on target gene response. After preendodermal plate is specified in Nematostella, β-catenin signaling becomes repressed there by an unknown mechanism, possibly involving ERG.