β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

Abstract

The canonical Wnt/β-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into β-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of β-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for β-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by β-catenin should be distinguished and carefully re-examined.

Fig. 1.

The vegetal contraction controls animal-vegetal axis polarity in Saccoglossus kowalevskii. (A) Fate maps. Anterior ectoderm (orange), posterior ectoderm (purple) and endomesoderm (blue) fates are segregated by cleavage planes at the 16-cell stage. (B) When fertilized eggs are vitally stained with Nile Blue 25 minutes post-fertilization (i), the staining concentrates towards the vegetal pole at 50 minutes (ii) and is inherited at the 16-cell stage by the veg2 blastomeres (iii) that will eventually give rise to the entire endomesoderm. Snapshots of a time-lapse movie (see Movie 3 in the supplementary material) at the onset (iv) and at the end (v) of vegetal contraction. (C) Treatment with the actin polymerization inhibitor cytochalasin B during vegetal contraction leads to an expansion of the anterior ectoderm, as evidenced by the marker sfrp1/5 (i-iv) and to a reduction in endoderm formation revealed by foxA expression (v-viii) at day 4 of development. Control embryos (i and v, lateral views, dorsal towards the right, anterior towards the top) and embryos treated with 20 μg/ml of cytochalasin B during vegetal contraction (ii and vi, partial animalization; iii and vii, strong animalization; iv and viii, complete animalization).

Fig. 2.

The canonical Wnt pathway is active at the vegetal pole of blastulae. (A-G) Embryos injected with an mRNA encoding a fusion between the fluorescent protein Venus and β-catenin. (A,C,E,G) Increasing cytoplasmic fluorescent signals before both cytoplasmic and nuclear staining occurs in cells of the vegetal hemisphere at early blastula stages. (B,D,F) When GSK3β, the upstream negative regulator of β-catenin stability, is inhibited using 1-azakenpaullone, strong cytoplasmic and nuclear fluorescent signals are detected in all cells of the embryo.

Fig. 3.

β-Catenin is required for endomesoderm and posterior ectoderm formation. Embryos were injected with a siRNA targeting β-catenin and analyzed by in situ hybridization at (A) day 2 of development and (B) gastrula stages. The expression of the endomesoderm marker foxA and of the posterior ectoderm marker hox9/10 is completely abolished (A, parts ii, vi; B, parts ii, vi), whereas the anterior ectoderm markers sfrp1/5 (A, part iv) and six3 (B, part iv) are expressed throughout the embryo. Control embryos (A, parts i, iii, v; B, parts i, iii, v) are shown in a lateral view with anterior towards the top. β-Catenin siRNA-injected embryos (A, parts ii, iv, vi; B, parts ii, iv, vi). Schematic cross-section of control (B, part vii) or β-catenin siRNA-injected embryo (B, part viii) at gastrula stages.

Fig. 4.

Activation of the canonical Wnt pathway leads to ectopic endomesoderm formation. (A,B) Blocking the negative regulator of β-catenin GSK3β leads to ectopic endomesoderm formation in a dose-dependent manner examined at day 3 of development (A) and gastrula stages (B). (A) At day 3, the embryos treated with 5 μM of 1-azakenpaullone are composed of endoderm stained by foxA (A, part ii) and posterior ectoderm (A, parts v, viii and xi), whereas the embryos treated with 10 μM are only made of endoderm (A, parts iii, vi, ix and xii). (B) Treatments with sub-optimal doses of 1-azakenpaullone lead to a progressive shift of the endomesodermal markers otx and foxA at gastrula stages towards the animal pole as the dose increases (B, parts x-xii and xiv-xvi). The posterior ectodermal marker hox9/10 follows the new endomesoderm/ectoderm boundary (B, parts vi-viii), whereas the anterior ectodermal expression of sfrp1/5 is lost (B, parts ii-iv). Control embryos at day 3 of development are oriented with anterior towards the top and dorsal towards the right (A, parts i, iv, vii and x). Gastrulae have been bisected after in situ hybridization; animal side towards the top (B). Control embryos at gastrula stages are shown in lateral view with anterior towards the top (B, parts i, v, ix and xiii).

Fig. 5.

Activation of the canonical Wnt pathway is sufficient to induce endomesoderm at cleavage stages. (A) Activating the Wnt pathway is sufficient to convert isolated ectoderm precursors into endomesoderm. Animal blastomeres isolated at the eight-cell stage do not form endomesoderm (A, parts iii, iv) and treatment with 10 μM of 1-azakenpaullone converts them into endomesoderm expressing foxA and otx (A, parts v, vi). Control embryos at gastrula stages, animal towards the top (A, parts i, ii). Numbers indicate positively stained explants over analyzed explants. (B,C) The Wnt pathway must be activated before blastula stages to induce endomesoderm. (B) Experimental design. (C) In situ results at gastrula stages (parts i-xxi) and day 4 of development (parts xxii-xxxv). Complete vegetalization is observed for treatments starting at early cleavage stages (4 and 6 hpf), as revealed by the expression of foxA in all cells of the treated embryos (C, parts ii, iii, xxiii and xxiv) and the loss of expression of all ectodermal markers examined (C, parts ix, x, xvi, xvii, xxx and xxxi). Partial vegetalization occurs for treatments during late cleavage stages (C, parts iv, v, xxv and xxvi), posterior ectoderm forms at the new endomesoderm/ectoderm boundary (C, parts xi, xii), but anterior ectoderm expressing six3 does not form (C, parts xviii, xix, xxxii and xxxiii). Treatments at early blastula stages do not induce ectopic expression of foxA, marking the endomesoderm at gastrula stages (C, parts vi, vii) but lead to a reduction of endoderm at day 4 (C, parts xxvi, xxvii). Posterior ectoderm expression of hox9/10 is unaffected (C, parts xiii, xiv), but expression of six3 in anterior ectoderm is lost (C, parts xx, xxi, xxxiv and xxxv). Gastrulae have been bisected after in situ hybridization, animal pole towards the top (C, parts i-xxi). Embryos at 4 days of development are shown with anterior towards the top and dorsal towards the right (C, parts xxii-xxxv). hpf, hours post-fertilization.

Fig. 6.

Endomesoderm posteriorizes ectoderm that would otherwise develop anterior character. (A) Ectoderm precursors develop into anterior ectoderm upon isolation. Vegetal explants derived from blastomeres isolated at the eight-cell stage comprise endomesoderm (A, part vii) and posterior ectoderm (A, parts viii, ix), whereas all cells of animal explants express the anterior ectodermal marker sfrp1/5 (A, parts iv-vi). When the veg1 tier, which is fated to form posterior ectoderm, is isolated at the 16-cell stage, it gives rise to anterior-most ectoderm (A, parts xii, xiii) like the an tier explant (A, parts x, xi). Veg2 tier explants comprise endomesoderm and a small portion of posterior ectoderm expressing hox9/10 (A, parts xiv and xv). (B) Endomesoderm precursors send posteriorizing signals between the 16-cell and blastula stages. When endomesoderm is removed at early cleavage stages, the ectodermal explants are entirely stained by the anterior marker foxQ2-1 (B, parts iv-vi). However, the ectodermal explants obtained from removal of endomesoderm precursors at mid-blastula stages display an anteroposterior pattern (B, parts vii-ix). Embryos are shown at day 2 of development with anterior towards the top. Numbers indicate the proportion of explants that exhibited the expression depicted.

Fig. 7.

Schematic representation of the function of β-catenin in Saccoglossus kowalevskii. During cleavage and early blastula stages, β-catenin accumulates in the nuclei of vegetal blastomeres where it is necessary and sufficient for endomesoderm specification. Endomesoderm precursors are then involved in the definition of the posterior ectoderm by sending posteriorizing signals to the overlying ectoderm that would otherwise be of anterior character.