Dkk1 Regulates Ventral Midbrain Dopaminergic Differentiation and Morphogenesis

Diogo Ribeiro,Kristina Ellwanger,Christof Niehrs,Ana Martin-Villalba,Ernest Arenas,Spyridon Theofilopoulos,Désirée Glagow,Nina S. Corsini,Mike Karl

doi:10.1371/journal.pone.0015786

Abstract

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1+/− embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1−/− embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.

Highlights

Dickkopf 1 (Dkk1) is a secreted glycoprotein belonging to the Dkk family, which consists of four members (Dkk-1, -2, -3, -4)
Dkk1 was detected at significant levels in the ventral midbrain (VM) at E9.5 and E10.5, but it was undetectable at E11.5–13.5
At E9.5, Dkk1 was highly expressed in the ventral diencephalon and was expressed in a salt and pepper pattern in the VM, where it followed a rostrocaudal gradient with higher expression levels rostrally than caudally (Figure 1A)

Summary

Introduction

Dickkopf 1 (Dkk1) is a secreted glycoprotein belonging to the Dkk family, which consists of four members (Dkk-1, -2, -3, -4). Dkk is a known Wnt/b-catenin pathway inhibitor: it binds to Lrp5/6, preventing its interaction with the Wnt protein and disrupting the Wnt-induced Frizzled-Lrp complex formation necessary for signal transduction [3,4,5]. Dkk can block the Wnt/b-catenin pathway by inducing Lrp endocytosis in the presence of Kremen proteins [6]. Dkk has been shown to have a major role in inducing head formation: injection of dkk mRNA in Xenopus embryos leads to anteriorized embryos with big heads and enlarged cement glands; together with a dominant-negative mutant of the BMP2/4 receptor, dkk mRNA is able to induce secondary axes with complete heads [1]. Loss-of-function studies further confirm that Dkk is essential for head induction: Xenopus embryos injected with an anti-Dkk antibody [1] and Dkk knockout mice [7] lack anterior head structures. The headinducing activity of Dkk is mediated through the inhibition of the

Methods

Results

Conclusion