Midbrain-Hindbrain Boundary Morphogenesis: At the Intersection of Wnt and Fgf Signaling

Abstract

A constriction in the neural tube at the junction of the midbrain and hindbrain is a conserved feature of vertebrate embryos. The constriction is a defining feature of the midbrain-hindbrain boundary (MHB), a signaling center that patterns the adjacent midbrain and rostral hindbrain and forms at the junction of two gene expression domains in the early neural plate: an anterior otx2/wnt1 positive domain and a posterior gbx/fgf8 positive domain. otx2 and gbx genes encode mutually repressive transcription factors that create a lineage restriction boundary at their expression interface. Wnt and Fgf genes form a mutually dependent feedback system that maintains their expression domains on the otx2 or gbx side of the boundary, respectively. Constriction morphogenesis occurs after these conserved gene expression domains are established and while their mutual interactions maintain their expression pattern; consequently, mutant studies in zebrafish have led to the suggestion that constriction morphogenesis should be considered a unique phase of MHB development. We analyzed MHB morphogenesis in fgf8 loss of function zebrafish embryos using a reporter driven by the conserved wnt1 enhancer to visualize anterior boundary cells. We found that fgf8 loss of function results in a re-activation of wnt1 reporter expression posterior to the boundary simultaneous with an inactivation of the wnt1 reporter in the anterior boundary cells, and that these events correlate with relaxation of the boundary constriction. In consideration of other results that correlate the boundary constriction with Wnt and Fgf expression, we propose that the maintenance of an active Wnt-Fgf feedback loop is a key factor in driving the morphogenesis of the MHB constriction.

Keywords: Fgf; MHB; Wnt; constriction morphogenesis; image analysis; mes/r1; two-photon fluorescence; zebrafish.

Figure 1

Morphogenetic and molecular ontogeny of the midbrain hindbrain boundary (MHB) in zebrafish embryos. Left column: schematic diagrams of zebrafish embryos, lateral view, at stages indicated on left. Gray shading highlights the brain primordium. Middle column: diagrams of pertinent morphogenetic movements. Orientation is indicated on the left side of each diagram. Examples of each morphogenetic property can be found in the corresponding references in the right column. fb, forebrain; mb, midbrain; hb, hindbrain.

Figure 2

Region dependent wnt1 reporter response in ace(fgf8a) background. (A) 3-D reconstructions generated using maximum intensity projection from tissue autofluorescence (with sections from the roof plate removed) reveal the formation of an isthmic constriction (yellow arrowheads) that fails to mature without fgf8a. (B) wnt1 lineage is present but improperly polarized in ace embryos. During normal development, the wnt1 lineage increases expression of wnt1 that can be visualized by increased eGFP reporter signal at the MHB boundary. (C) The wnt1 lineage in the dorsal neuroepithelium normally turns off expression of wnt1 in the anterior hindbrain as reflected by a decrease in reporter intensity (measured from the profiles marked by green lines over time). Red arrows point to a midbrain cell just anterior to the MHB constriction and yellow arrowheads point to a neighboring cell posterior to the constriction. The blue arrow shows the presumptive peripheral midbrain layer (PML). In ace(fgf8a) embryos, the constriction relaxes and neighboring cells in r1 begin re-expressing wnt1 as shown by increasing reporter intensity, reflecting a cerebellar-to-tectal transformation. Basal constriction of the boundary cells with highest wnt1 reporter intensity occurs in both cases, however, the presumptive PML fails to form in ace(fgf8a) embryos. (D) The wnt1 lineage in the ventral neuroepithelium normally undergoes cell shortening and compresses to a narrow ring of cells anterior to the physical MHB constriction (black markers) while in ace(fgf8a) embryos, this reorganization fails to occur and wnt1 is no longer expressed. Scale bar = 100 μm.

Figure 3

Adhesion failure in ace(fgf8a) embryos and regionally dependent wnt1 lineage response. (Left) Initially broad expression of wnt1 is normally refined to the dorsal midbrain and anterior midbrain hindbrain domain (MH), helping to maintain fgf8a dependent adhesion in the dorsal MH. (Right) wnt1 expression in the posterior midbrain (except for a dorsal stripe) is lost in ace(fgf8a) while wnt1 expression in a dorsal stripe of r1 is reactivated during a cerebellar-to-tectal transformation. Midline adhesion in the MH is not maintained during brain ventricle morphogenesis.

Figure 4

Modulation of Wnt/Fgf signaling and effect on MH morphology. (Left) Summary of wnt/fgf expression domains, MH morphology, and morphogenetic cell behaviors in ace mutants. (Middle, Right) The same model shown for wild-type embryos and otx/gbx loss of function embryos. Figure drawn based on data found in Su et al. (2014). Deficient cell behaviors are indicated with a minus sign (−), while overactive cell behaviors are indicated with a plus sign (+).

Figure 5

Potential avenues of Wnt/Fgf signaling contributing to MH formation.

Figure 6

Computer Aided Feature Extraction (CAFE) reconstruction of zebrafish brain embryo images. (A) Single slice 2-photon microscope images of zebrafish brain embryo at 20 hpf displaying autofluorescence (left) and GFP (right) channels. GFP channel marks wnt1 expression. (B) Zebrafish embryo structure representation of autofluorescence (wires) and GFP (green volume). Visualizing images 1–45 in the 79-image stack in dorsal (left) and ventral (right) views. Autofluorescence (wires) reconstructed using boundary-based detection function to recognize local pixel gradients and define ball-and-stick model along the edges of the image. GFP (green volume) reconstructed using an area-based detection function to place balls in areas of high pixel intensity. (C) Detailed ball-and-stick model of boxed region in (B) showing CAFE-defined morphological surface (light blue). (D) Local surface normals for surfaces highlighted in (C). (E) Dorsal view of overlay of autofluorescence and GFP reconstructions with surface normals.