beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

Abstract

In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal-vegetal axis in sea urchin embryos are largely unknown. Nuclear beta-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that beta-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that beta-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal-vegetal axis. Our results also reveal similarities between the sea urchin animal-vegetal axis and the vertebrate dorsal-ventral axis, suggesting that these axes share a common evolutionary origin.

Figure 1

Vegetalization of embryos by β-catenin and lithium. (A) A vegetalized embryo developing from an egg injected with pt β-catenin (amino terminal serine/threonine mutated to alanine) RNA. The arrow points to the remaining ectoderm in this embryo. The increased number of secondary mesoderm-derived pigment cells are seen clearly in this embryo. (B) A vegetalized embryo resulting from incubation in 35 mM lithium chloride. The arrow points to the remaining ectoderm in this embryo. (C) A pluteus larva developing from an egg injected with an RNA encoding a truncated β-catenin (HT-6) protein (lacking the armadillo repeats 5–13).

Figure 2

Induction of endoderm in animal halves by β-catenin. (A) Protocol for introducing mRNA into animal halves. (B) An animal half made from an embryo that was injected at the one-cell stage with an RNA encoding a truncated β-catenin protein (HT-6). It develops as a polarized embryoid that does not form aboral ectoderm, endoderm, or mesoderm. (C) Induction of endoderm and gastrulation in an animal half made from an embryo injected at the one-cell stage with pt β-catenin RNA.

Figure 3

Patterning of ectoderm by low concentrations of β-catenin. (A) Induction of aboral ectoderm in isolated animal halves by β-catenin. RT-PCR was used to monitor the expression of aboral ectoderm and endoderm-specific markers. LvS1 is an aboral ectoderm-specific marker; LvEndo16 and LvN1.2 are endoderm-specific markers. Actin primers were used to monitor input cDNA for each sample. The autoradiograph shows that aboral ectoderm is induced after injection of a low concentration of pt β-catenin RNA into animal halves whereas expression of endodermal markers is not detected. (B) Animal half made from an HT-6 RNA-injected embryo. (C and D) Patterning of ectoderm by low concentrations of pt β-catenin. (C) Induction of a stomodeum in a pt β-catenin-injected animal half (arrow). (D) Induction of a ciliary band in a pt β-catenin-injected animal half (arrows).

Figure 4

Animalization of sea urchin embryos by overexpression of C-cadherin. (A and B) Morphology of control and C-cadherin RNA-injected embryos. (A) Uninjected embryo at the early prism stage. (B) An embryo animalized by C-cadherin. It is radialized and does not develop endoderm or mesoderm, and long cilia are seen in the cuboidal epithelium. (C) RT-PCR analysis of C-cadherin RNA-injected embryos. In addition to the loss of the endodermal markers LvEndo16 and LvN1.2, these embryos do not express the aboral ectoderm-specific marker LvS1. (D, E, F, and G) Expression of the oral ectoderm marker Ecto V in control embryos (D and E) and C-cadherin RNA-injected embryos (F and G). (D and F) Differential interference contrast images. (E and G) Corresponding indirect immunofluorescent images. In control embryos, the Ecto V antigen is localized to the oral ectoderm whereas, in the C-cadherin RNA-injected embryos, the Ecto V antigen is seen on the surface of all blastomeres. (H) RT-PCR analysis of C-cadherin RNA and pt β-catenin RNA co-injected embryos. Autoradiograph shows that, although marker genes for endoderm and aboral ectoderm are not expressed in C-cadherin-injected embryos, they are rescued with injection of pt β-catenin RNA.