Two oppositely localised frizzled RNAs as axis determinants in a cnidarian embryo

Abstract

In phylogenetically diverse animals, including the basally diverging cnidarians, "determinants" localised within the egg are responsible for directing development of the embryonic body plan. Many such determinants are known to regulate the Wnt signalling pathway, leading to regionalised stabilisation of the transcriptional coregulator beta-catenin; however, the only strong molecular candidate for a Wnt-activating determinant identified to date is the ligand Wnt11 in Xenopus. We have identified embryonic "oral-aboral" axis determinants in the cnidarian Clytia hemisphaerica in the form of RNAs encoding two Frizzled family Wnt receptors, localised at opposite poles of the egg. Morpholino-mediated inhibition of translation showed that CheFz1, localised at the animal pole, activates the canonical Wnt pathway, promotes oral fates including gastrulation, and may also mediate global polarity in the ectoderm. CheFz3, whose RNA is localised at the egg vegetal cortex, was found to oppose CheFz1 function and to define an aboral territory. Active downregulation mechanisms maintained the reciprocal localisation domains of the two RNAs during early development. Importantly, ectopic expression of either CheFz1 or CheFz3 was able to redirect axis development. These findings identify Frizzled RNAs as axis determinants in Clytia, and have implications for the evolution of embryonic patterning mechanisms, notably that diverse Wnt pathway regulators have been adopted to initiate asymmetric Wnt pathway activation.

Figure 1. Two Clytia Frizzled Genes Belonging to Distinct Families

(A) Domain structure of CheFz1 and CheFz3 proteins compared to the Drosophila Frizzled protein. Blue boxes: cysteine-rich domains; Pink/red boxes: seven-pass transmembrane domains (transmembrane regions in red); green boxes: KTXXXW motif. (B) Maximum likelihood analysis of relationships of Frizzled sequences from mouse (m), Drosophila melanogaster (Dm), and the known cnidarian Frizzleds: Hydra vulgaris, Hydractinia echinata, and N. vectensis (Nv). The tree was rooted using the Drosophila Smoothened sequence. Scale indicates number of inferred substitutions per site.

Figure 2. Opposing Localisation of CheFz1 and CheFz3 RNAs during Early Development

In situ hybridisation of C. hemisphaerica eggs, embryos, and planula larvae (fixed at 1 and 3 d after fertilisation), showing the concentration of CheFz1 RNA (top row) in the animal cytoplasm of the egg and around the nuclei during first cleavage, and its graded oral–aboral distribution in blastula and early gastrula stages. CheFz1RNA levels declined during gastrulation and subsequently remained low throughout all regions, with some increase in the oral endoderm during planula development. CheFz3 RNA (middle row) was localised strongly to the vegetal egg cortex, becoming concentrated in the vegetal part of the cleavage furrow during cytokinesis, and then to the presumptive aboral pole throughout embryonic and larval development. Asterisks mark nuclei in eggs. In medusa and polyp stages (bottom row), low levels of CheFz1 RNA were detected in all regions, including the tentacle bulb (tb), while CheFz3 RNA was detected strongly in specific regions of the endoderm: the circular canal (cc) and a juxta-oral band of the manubrium (arrows). All embryos and polyps are oriented with the oral pole pointing up. Bars = 40 μm.

Figure 3. CheFz1 and CheFz3 Regulate the Canonical Wnt Pathway and Direct Oral and Aboral-Specific Gene Expression

(A) Activation of the canonical Wnt pathway in mid-blastula–stage control embryos (top row) and embryos derived from CheFz1-Mo (middle row)– or CheFz3-Mo (bottom row)–injected eggs, visualised by injecting eggs prior to fertilisation with RNA coding for a β-catenin–Venus fusion protein. Coinjected rhodamine dextran was distributed uniformly (not shown). β-catenin–Venus was stabilised specifically in the oral half of control embryos. CheFz1-Mo reduced β-catenin–Venus to barely detectable levels, while CheFz3-Mo caused it to accumulate in all cells. (B) Characteristic phenotypes of embryos derived from CheFz1-Mo– or CheFz3-Mo–injected eggs compared with uninjected controls. Differential interference contrast images of early gastrula-stage embryos: arrows indicate ingressing cells. CheFz1-Mo severely reduced the extent of cell ingression, although did not prevent oral thickening of the epidermal layer, while CheFz3-Mo caused expansion of the zone of cell ingression across most of the embryo. (C) Confocal images of similar early gastrula-stage embryos, with cell contours visualised using fluorescent phalloidin (green) and nuclei with ToPro3 (red). (D) Equivalent images of planula-stage embryos (1 d after fertilisation). There is a clear deficit in endoderm formation in embryos of CheFz1-Mo–injected but not in control CheFz1-5mp-Mo–injected embryos. CheFz3-Mo embryos show both endoderm and ectoderm, but oral–aboral polarity is disrupted. (E) Representative in situ hybridisation images of early gastrula embryos showing abolition of the oral CheBra expression territory following CheFz1-Mo injection, and expansion following CheFz3-Mo injection. (F) Aboral CheFoxQ2a expression at the same stage was abolished following CheFz3-Mo injection but expanded following CheFz3-Mo injection. In all panels, embryos are oriented with the oral pole up. Bars = 40 μm.

Figure 4. Specific Inhibition of Translation by CheFz1-Mo

X-gal staining of embryos derived from eggs injected with a β-galactosidase reporter RNA bearing the CheFz1-Mo 5′ UTR target sequence. Co-injection of CheFz1-Mo but not CheFz1-5mp-Mo completely abolished β-galactosidase expression.

Figure 5. Reciprocal Regulation of CheFz1 and CheFz3

Representative in situ hybridisation images obtained with CheFz1 and CheFz3 probes on control and morpholino-injected embryos. The arrow points to the weak oral CheFz1 expression domain in controls at this stage. Each morpholino causes massive up-regulation of the other RNA, albeit with vestiges of an oral–aboral gradient still discernable. A mixture of both morpholinos caused simultaneous up-regulation of both RNAs.

Figure 6. Global β-Catenin Stabilisation Uncouples Oralisation from CheFz RNA Levels

(A) Confocal images of phalloidin/ToPro3-stained early gastrula embryos (see Figure 2) showing characteristic phenotypes obtained following Wnt pathway activation by treatment with 50 mM LiCl from the two-cell stage (bottom row) compared with untreated controls (top row). (B) Representative in situ hybridisation images of control and LiCl-treated embryos fixed at the early gastrula stage, with probes as indicated (weak oral CheFz1 expression indicated by arrows in controls).

Figure 7. Embryo Polarity Redirected by Ectopic CheFz1 or CheFz3

(A) Differential interference contrast images of embryos following injection of 0.5 mg/ml RNA coding for CheFz1 or CheFz3 into eggs before fertilisation compared to uninjected controls. Stages as indicated. (B) Confocal images of early gastrula embryos stained with phalloidin/ToPro3 following morpholino injection into the egg (left column) and with polarity restored by injection of 0.5 mg/ml RNA (lacking the morpholino target site) into one blastomere at the two-cell stage (right two columns). Top row: CheFz1 misexpression; Bottom row: CheFz3 misexpression. Blue indicates the progeny of the RNA-injected cell revealed by fluorescent dextran. Arrows indicate ectopic pointed oral poles. The original position of the egg animal pole is deduced to lie on the injected–uninjected boundary (asterisk) since the first cleavage passes through the animal pole, and can be further pinpointed by residual endogenous polarity in CheFz1-injected embryos. Bars = 40 μm.

Figure 8. Model for Cnidarian Embryonic Patterning Initiated by Localised Maternal Frizzled RNAs

CheFz1 (red) transcribed from animally localised RNA activates the canonical Wnt pathway to stabilise β-catenin (green) and so promotes development of oral fates (including gastrulation and endoderm formation). It may also play a role in the establishment of ectodermal polarity via PCP. CheFz3 (blue) transcribed from vegetally localised RNA spatially restricts canonical Wnt pathway activation by CheFz1, defining an aboral gene expression territory. The oral and aboral domains are reinforced by mutual downregulation of CheFz1 and CheFz3 (dotted lines), by mechanisms probably involving secondary signalling molecules.