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. 2018 Dec 15;444(2):83-92.
doi: 10.1016/j.ydbio.2018.10.003. Epub 2018 Oct 15.

Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos

Affiliations

Canonical and non-canonical Wnt signaling pathways define the expression domains of Frizzled 5/8 and Frizzled 1/2/7 along the early anterior-posterior axis in sea urchin embryos

Ryan C Range. Dev Biol. .

Abstract

The spatiotemporal expression of Frizzled receptors is critical for patterning along the early anterior-posterior axis during embryonic development in many animal species. However, the molecular mechanisms that regulate the expression of Frizzled receptors are incompletely understood in any species. In this study, I examine how the expression of two Frizzled receptors, Fzl1/2/7 and Fzl5/8, is controlled by the Wnt signaling network which directs specification and positioning of early regulatory states along the anterior-posterior (AP) axis of sea urchin embryos. I used a combination of morpholino- and dominant negative-mediated interference to knock down each Wnt signaling pathway involved in the AP Wnt signaling network. I found that the expression of zygotic fzl5/8 as well as that of the anterior neuroectoderm gene regulatory network (ANE GRN) is activated by an unknown broadly expressed regulatory state and that posterior Wnt/β-catenin signaling is necessary to down regulate fzl5/8's expression in posterior blastomeres. I show that zygotic expression of fzl1/2/7 in the equatorial ectodermal belt is dependent on an uncharacterized regulatory mechanism that works in the same cells receiving the TGF-β signals patterning this territory along the dorsal-ventral axis. In addition, my data indicate that Fzl1/2/7 signaling represses its own expression in a negative feedback mechanism. Finally, we discovered that a balance between the activities of posterior Wnt8 and anterior Dkk1 is necessary to establish the correct spatial expression of zygotic fzl12/7 expression in the equatorial ectodermal domain during blastula and gastrula stages. Together, these studies lead to a better understanding of the complex interactions among the three Wnt signaling pathway governing AP axis specification and patterning in sea urchin embryos.

Keywords: Anterior-posterior; Deuterostome evolution; Fzl1/2/7; Fzl5/8; Gene regulatory networks; Wnt signal transduction.

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Figures

Fig. 1.
Fig. 1.
The spatiotemporal expression of fzl1/2/7 and fzl5/8 during early AP specification and patterning in invertebrate deuterostomes. (A) In sea urchin early development, the Wnt/β-catenin, Wnt/JNK, and Fzl1/2/7-PKC pathways all converge on the same developmental process: ANE restriction. Step 1(16-to 32-cell stage) Wnt/β-catenin signaling activates the endomesoderm GRN and represses the ANE GRN in posterior blastomeres. Step 2 (60-cell stage to early-mid-blastula stage) Wnt/β-catenin signaling activates posterior-to-anterior gradients of Wnt1 and Wnt8 that activate the Fzl5/8-JNK signaling pathway resulting in the down regulation of the ANE GRN in the posterior equatorial ectoderm. Step 3 (mid-blastula to mesenchyme blastula stage) In the regressing ANE GRN Fzl5/8 signaling activates Dkk1 and sFRP-1 expression. These Wnt antagonists perturb the posterior-to-anterior repression of the ANE GRN by Fzl5/8 signaling via a negative feedback loop. Around the same time, FoxQ2 activates the expression of two Wnt modulators, sFRP1/5 and Dkk3, that potentiate Fzl5/8 signaling (data taken from Range et al., 2013; Range, 2014; Range and Wei, 2016; Khadka et al., 2018). (B) Expression of fzl12/7 and fzl5/8 in embryos from the same mating pairs in S. purpuratus. (Ba) fzl5/8 is detected in anterior blastomeres beginning around the 120-cell stage. (Bb, c) In early blastula stages, stage fzl5/8 expression is progressively down regulated from equatorial ectodermal cells. (Bd, e) fzl5/8 is expressed around the anterior pole as well as the posterior endomesoderm cells at the mesenchyme blastula and early gastrula stage, as previously shown (Range et al., 2013). (Bf) fzl1/2/7 is expressed ubiquitously in the cleavage stage. (Bg) In blastula staged embryos, fzl1/2/7 expression is down regulated in posterior cells. (Bh) fzl1/2/7 expression is down regulated around the anterior pole at the early mesenchyme blastula stage. (Bi, j) Between mesenchyme blastula stage and early gastrula, fzl1/2/7 expression is restricted to an equatorial ectodermal belt and activated in the posterior endomesoderm. (C) Diagram of fzl5/8 and fzl1/2/7 expression in invertebrate deuterostomes. Data taken from (Darras et al., 2018; McCauley et al., 2013; Qian et al., 2013; Robert et al., 2014).
Fig. 2.
Fig. 2.
Zygotic control of fzl5/8 expression. (A) Control embryo showing anterior expression of fzl5/8. (B) fzl5/8 expression expands throughout the anterior ectodermal territory in embryos without functional Fzl5/8-JNK signaling and (C) is severely down regulated in Fzl1/2/7 knock down embryos. (D) In the absence of functional Wnt/β-catenin (Axin mRNA), fzl5/8 is expressed throughout the embryo. MO, morpholino; ΔFzl5/8, dominant negative Fzl5/8; Scale bar = 20 μm.
Fig. 3.
Fig. 3.
Initial activation of fzl5/8 and foxq2, a cardinal regulatory of the ANE GRN. The percentage of embryos examined that show the representative phenotypes depicted is indicated in each panel. In Six3 knockdown embryos the cardinal ANE regulator foxq2 (A, B) and fzl5/8 (D, E) are down regulated. In contrast, foxq2 (C) and six3 (F) are expressed broadly in Six3 morphants in the absence of Wnt/β-catenin signaling. MO, morpholino; Scale bar = 20 μm.
Fig. 4.
Fig. 4.
Control of early zygotic fzl1/2/7 expression by the Wnt signaling network. (A) Compared to control embryos (Aa), the anterior domain of fzl1/2/7 down regulation expands and the belt of fzl1/2/7 expression shifts toward the posterior/vegetal pole in embryos injected with ΔFzl5/8 mRNA (Ab). (Ac) In the absence of Fzl1/2/7, the expression of fzl1/2/7 expands throughout the entire embryo. fzl1/2/7 expression is down regulated in embryos lacking Wnt/β-catenin signaling (Ad) and in embryos with up regulated Wnt/β-catenin signaling (Ae). (B) fzl12/7 expression is similar in control (Ba) and Nodal morphants (Bb). MO, morpholino; ΔFzl5/8, dominant negative Fzl5/8; Scale bar = 20 μm.
Fig. 5.
Fig. 5.
Wnt8 and Dkk1 regulate the spatial expression of fzl1/2/7 in the ectoderm. (A) Compared to control (a), ectodermal fzl1/2/7 expression shifts towards the posterior of mesenchyme blastula embryos in Wnt8 morphants (b). (B) fzl5/8 expression expands towards the vegetal/posterior pole in embryos injected with Dkk1 mRNA (a, c) coincident with the expanded downregulation of fzl1/2/7 around the anterior pole and shift of the fzl1/2/7 expression belt towards the posterior/vegetal pole (c, d). (C) Consistent with previous experiments (Range, 2013), fzl5/8 expression around the anterior pole requires Dkk1 (a, d). The expression of fzl1/2/7 is shifted towards the anterior pole (b, e) and expressed throughout the anterior territory (e, f). MO, morpholino; AV, anterior view; Scale bar = 20 μm.
Fig. 6.
Fig. 6.
A four-step model for the activation of the ANE GRN and spatial regulation of fzl1/2/7 and fzl5/8 along the AP axis during early development. (A) A diagram illustrating that maternal regulatory mechanisms are necessary for the expression of fzl1/2/7 and fzl5/8 and that an unknown broadly expressed regulatory mechanism is necessary for the zygotic activation of the ANE GRN, which includes fzl5/8. (B) From the early cleavage stage to the mesenchyme blastula stages in the sea urchin, a balance among the activities of the Wnt/β-catenin, Wnt/JNK, and Fzl1/2/7 signaling pathways determine the spatial expression of fzl1/2/7 and fzl5/8 along the AP axis. The model is detailed in the figure.

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