Positioning of the midbrain-hindbrain boundary organizer through global posteriorization of the neuroectoderm mediated by Wnt8 signaling

Muriel Rhinn,Michael Brand,Marta Luz,Klaus Lun,Michaela Werner

doi:10.1242/dev.01685

Abstract

The organizing center located at the midbrain-hindbrain boundary (MHB) patterns the midbrain and hindbrain primordia of the neural plate. Studies in several vertebrates showed that the interface between cells expressing Otx and Gbx transcription factors marks the location in the neural plate where the organizer forms, but it is unclear how this location is set up. Using mutant analyses and shield ablation experiments in zebrafish, we find that axial mesendoderm, as a candidate tissue, has only a minor role in positioning the MHB. Instead, the blastoderm margin of the gastrula embryo acts as a source of signal(s) involved in this process. We demonstrate that positioning of the MHB organizer is tightly linked to overall neuroectodermal posteriorization, and specifically depends on Wnt8 signaling emanating from lateral mesendodermal precursors. Wnt8 is required for the initial subdivision of the neuroectoderm, including onset of posterior gbx1 expression and establishment of the posterior border of otx2 expression. Cell transplantation experiments further show that Wnt8 signaling acts directly and non-cell-autonomously. Consistent with these findings, a GFP-Wnt8 fusion protein travels from donor cells through early neural plate tissue. Our findings argue that graded Wnt8 activity mediates overall neuroectodermal posteriorization and thus determines the location of the MHB organizer.

Highlights

The initial subdivision of the neural plate is the first step towards generating cellular diversity in the vertebrate brain
To address whether axial mesendoderm is required for correct AP positioning of the midbrain-hindbrain boundary (MHB) primordium in the neural plate, we analyzed the expression of otx2, gbx1 and the MHB markers eng2 and fgf8 in mutants for two transcription factors – no tail and floating head – that lack notochord differentiation (Halpern et al, 1993; Schulte-Merker et al, 1994; Talbot et al, 1995). otx2, gbx1, eng2 and fgf8 are expressed at their correct AP position in ntl mutant embryos (Fig. 1E-H) and in flh mutant embryos (Fig. 1I-L) when compared with wild-type embryos (Fig. 1A-D)
We find that the AP position of the MHB markers otx2 (Fig. 1A,M; n=5/5), gbx1 (Fig. 1B,N; n=3/3), eng2 (Fig. 1C,O; n=3/3), and fgf8 (Fig. 1D,P; n=4/4) is normal

Summary

Introduction

The initial subdivision of the neural plate is the first step towards generating cellular diversity in the vertebrate brain. A key question is how this initial subdivision is achieved along the anteroposterior (AP) axis. In the two-step ‘activationtransformation’ model, an activating signal induces the ectoderm to become anterior neural tissue. Inhibition of bone morphogenic protein (Bmp) activity by factors released from the organizer like chordin, noggin or follistatin is thought to be crucial for this step (Wilson and Hemmati-Brivanlou, 1997). A second, transforming signal is thought to convert part of the neuroectoderm into a more posterior identity (transformation, or ‘posteriorization’). Candidate molecules for posteriorizing signals are fibroblast growth factors (Fgfs), Wnt proteins and retinoic acid, but which signaling protein(s) are acting directly on target cells has been difficult to resolve (Gamse and Sive, 2000)

Methods

Results

Discussion

Conclusion