Molecular regulation of the developing commissural plate

Abstract

Coordinated transfer of information between the brain hemispheres is essential for function and occurs via three axonal commissures in the telencephalon: the corpus callosum (CC), hippocampal commissure (HC), and anterior commissure (AC). Commissural malformations occur in over 50 human congenital syndromes causing mild to severe cognitive impairment. Disruption of multiple commissures in some syndromes suggests that common mechanisms may underpin their development. Diffusion tensor magnetic resonance imaging revealed that forebrain commissures crossed the midline in a highly specific manner within an oblique plane of tissue, referred to as the commissural plate. This specific anatomical positioning suggests that correct patterning of the commissural plate may influence forebrain commissure formation. No analysis of the molecular specification of the commissural plate has been performed in any species; therefore, we utilized specific transcription factor markers to delineate the commissural plate and identify its various subdomains. We found that the mouse commissural plate consists of four domains and tested the hypothesis that disruption of these domains might affect commissure formation. Disruption of the dorsal domains occurred in strains with commissural defects such as Emx2 and Nfia knockout mice but commissural plate patterning was normal in other acallosal strains such as Satb2(-/-). Finally, we demonstrate an essential role for the morphogen Fgf8 in establishing the commissural plate at later developmental stages. The results demonstrate that correct patterning of the commissural plate is an important mechanism in forebrain commissure formation.

Figure 1

DTMRI analysis of the developing mouse commissural plate shows the midline crossing of the commissures occurs at an oblique coronal angle. A-D″, Color maps generated from fractional anisotropy values and primary eigenvectors. Diffusion directions: green, medial to lateral; blue, anterior to posterior; red, dorsal to ventral. The CC, HC and AC are indicated by the arrow, open arrowhead and closed arrowhead, respectively. In A, the difference in sectioning angle between the coronal plane (A′) and the oblique coronal plane (A″) can be appreciated by the dashed lines. Development of the commissures can be observed in the sagittal plane (A, B, C, D). The AC can be visualized by DTMRI proximal to the midline at E14 (A). B: By E15, the HC can also be visualized. At E16, the AC and HC have grown (C). D: By E17 all forebrain commissures have crossed the midline. The medial to lateral development of these commissures cannot be appreciated simultaneously in the traditional coronal plane alone (A′, B′, C′, D′). However, in an oblique coronal plane, determined from the sagittal images, all three commissures can be observed (A″, B″, C″, D″). For example, the CC (closed arrow) and HC (open arrowhead) are not present in the coronal plane at E17 (D′), but appear together in the oblique coronal plane (D″). In both C″ and D″ the descending columns of the fornix (DCF) can be seen in purple (open arrow), dorsal to the AC (closed arrowhead). Scale bar = 200 μm.

Figure 2

Four distinct fiber tracts emerge within the mouse commissural plate by E17. A: Representative photomicrograph of a mid-sagittal section of E17 C57Bl/6 brain stained with hematoxylin. B: A 2.5× enlargement of the commissural plate in A. Axon tracts of the corpus callosum (CC, green), dorsal hippocampal commissure (DHC, blue), ventral hippocampal commissure (VHC, red) and anterior commissure (AC, pale blue) are outlined. The putative commissural plate has been outlined in black (A). Dashed vertical lines indicate the oblique coronal angle used to obtain sections in C-E. C-E: Representative rostral to caudal sections of the commissural plate at E17, immunolabeled with anti-GAP43 (brown) for growing neuronal processes and counterstained with hematoxylin (blue). C′, D′, E ′: Enlargements of highlighted regions in C-E. C′: In a rostral plane of the commissural plate, the CC crosses the midline at the boundary between the cingulate cortex (CgCtx) and the septum. CC crossing at this boundary continues caudally. D′: In addition to the CC, ipsilateral descending columns of the fornix (arrows) and dorsal and ventral segments of the HC begin to emerge (arrowheads). E′: At the most caudal extent of the commissural plate all four forebrain commissures cross the midline within the same oblique plane. From dorsal to ventral these are the CC, DHC (arrowheads), VHC (arrowheads) and AC. Also seen in E′ is the caudal extent of the descending columns of the fornix (arrows) projecting ventrally between the VHC and the AC. Fibers of the AC are in direct contact with the ventricular zone of the third ventricle at the midline (open arrowhead). Scale bar in A = 1 mm, scale bars in C-E′ = 500 μm.

Figure 3

Molecular delineation of the mouse commissural plate at E17 reveals four subdomains. Fluorescence immunohistochemistry of the dorsal telencephalic markers NFIA (closed arrowhead, A) and EMX1 (open arrowhead, A) at E17 in the oblique coronal plane and sagittal plane, where two (5μm think) adjacent sections have been overlaid (A, C). The ventral telencephalic markers SIX3 (open arrowhead) and ZIC2 (closed arrowhead) are expressed in the same sections (B, D). GAP43 labeling is shown in yellow to delineate the commissural axons. DAPI counterstain is shown in blue. While both NFIA and EMX1 encircle the rostral to caudal extent of the CC and surround the DHC, only NFIA expression spans the commissural plate ventrally to end rostral and dorsal of the AC (arrow). From the VHC towards the AC, the ventral markers ZIC2 and SIX3 are expressed in opposing gradients. ZIC2 is predominantly expressed around the fornix, while SIX3 is predominantly expressed around the AC. Laterally, ZIC2 is expressed within the septum, bounded by the lateral ventricles. However, SIX3 is expressed along the mediolateral extent of the AC. Immunoreactivity is also present in the choroid plexus and the epithelium of the rostral wall of the third ventricle (asterisks in C and D). These features are not considered part of the commissural plate. From these expression patterns dorsal domains (MC) and ventral domains (SA) are characterized as follows: MC or MC₁, NFIA⁺ and EMX1⁺; MC₂ NFIA⁺ and ZIC2⁺; SA₁, NFIA⁺ and ZIC2⁺ and SIX3⁺; SA₂, SIX3⁺. Scale bars: A-B = 200 μm, C-D = 150 μm.

Figure 4

Molecular patterning and morphology of the developing mouse commissural plate demonstrates the emergence of the four subdomains. Fluorescence immunohistochemistry for the markers NFIA (white), EMX1 (red), SIX3 (magenta) and ZIC2 (green) throughout the developmental oblique coronal plane of the commissural plate in mouse embryos at E14 (A-E), E15 (F-J), E16 (K-O) and E17 (P-T). GAP43 staining is shown in yellow. DAPI counterstain is shown in blue. An overlay of this molecular patterning is shown with boundaries indicated by dashed lines, defining dorsal domains (MC) and ventral domains (SA), characterized as follows: MC or MC₁, NFIA⁺ and EMX1^+; MC₂ NFIA⁺ and ZIC2⁺; SA₁, NFIA⁺ and ZIC2⁺ and SIX3⁺; SA₂, SIX3⁺. Scale bar = 200 μm.

Figure 5

Commissural plate domains are disrupted in Emx2^{−/ −} and Nfia^−/−, but not Satb2^{−/ −}mice. Fluorescence immunohistochemistry in the sagittal plane for GAP43 (yellow), EMX1 (red), ZIC2 (green) and SIX3 (magenta) reveals the anatomical and molecular features of the commissural plate in wildtype (A), Nfia^{−/ −} (B), Emx2^−/− (C), and Satb2^{−/ −} mice (D). NFIA immunoreactivity is not shown here because measurements were taken from additional sequential, but not adjacent, sagittal sections that did not allow overlay. Loss of the CC is seen in Nfia^−/− mice (B), where DHC and VHC dysgenesis is also apparent. Agenesis of the CC and HC is apparent in Emx2^{−/ −} (C) and Satb2^{−/ −} mice (D). The AC is enlarged in Satb2^{−/ −} mice (D). E: Quantification of the commissural plate molecular boundaries as dorsoventral distances from the center of the AC. The MC, SA₁ and SA₂ domains are indicated to the left of the bars. Key to symbols: squares, dorsal and ventral surfaces of the brain; open upward triangles, EMX1; closed downward triangles, ZIC2; closed circles, SIX3. Wildtype littermates served as controls for the mutant mice. * p < 0.05 compared to the dorsal height of control. ^# p < 0.05 compared to the dorsal extent of EMX1 expression of control. ^† p < 0.05 compared to MC of control. F: Molecular domain lengths for each of the mouse strains. Domain length calculations were based on linear distance along the oblique angle of the commissural plate, as demonstrated in Supp. Fig. 2. ^## p < 0.05 compared to EMX1 of control. ** p < 0.05 compared to ZIC2 of control. Star = p < 0.05 compared to SIX3 of control. Scale bar = 150 μm.

Figure 6

Glial populations form normally in Satb2 knockout mice. Glial populations within the commissural plate at E17 include the indusium griseum glia (IGG) at the dorsal limit of the MC₁, the glial wedge (GW) at the ventral limit of the MC₁, and the midline zipper glia (MZG) also at the boundary of the MC₁ and MC₂. A small population of glia surrounds the AC at the boundary of the SA₁ and SA₂. These populations can be seen in the wildtype CoP in A. In Satb2^−/− mice all glial populations are present and appear normal (B). The indusium griseum glia and midline zipper glia are usually split by the formation of the corpus callosum but this does not occur due to the absence of the corpus callosum in Satb2^−/− mice. Scale bar = 400 μm.

Figure 7

Absence of Fgf8 at the commissural plate in the Emx1-cre;Fgf8^flox/flox mice. A: Immunohistochemistry shows EMX1 expression in the pallium and medial telencephalon extending ventrally to the lamina terminalis. B: Emx1-cre and ROSA26^flox/flox mice were crossed to obtain a green fluorescent protein (GFP) profile of the extent of Emx1-cre recombinase activity. Immunohistochemistry for GFP at E14 reveals recombinase activity in the pallium. In particular, recombinase activity occurs around the dorsal part of the lamina terminalis (arrow). C and D: In situ hybridization in Emx1^+/+;Fgf8^flox/flox (Ctl) mice demonstrates that Fgf8 is expressed in the commissural plate at E14 at the border of the MC and SA domains. By contrast, sense in situ hybridization in control mice (E) and antisense in situ hybridization in Emx1-cre;Fgf8^flox/flox (cKO) mice (F) reveal no Fgf8 expression. Due to the restricted overlay of expression domains, the telencephalon develops without major deficit until E14. Scale bar = 400 μm.

Figure 8

Agenesis of the commissural plate in Fgf8 hypomorphs. Fractional anisotropy images of the Emx1-cre;Fgf8^flox/flox (cKO) heads and littermate controls (Ctl) at E14 (A-D) and E18 (E-I) where the intensity of white features indicates tissue and white matter tracts, while black indicates fluid spaces such as ventricles. At E14, cKO mutants show absence of the commissural plate (arrowhead in D), failure of midline fusion (arrow in B) and hypoplasia of the olfactory bulbs (arrows in C and D). At E18, further dysmorphologies are seen, including agenesis of the corpus callosum and hippocampal commissure (arrowhead, I), displacement of the corticoseptal boundary in the anterior telencephalon (compare hatched lines in E and F) and communication of the lateral ventricles can be seen (asterisk, H). H and I correspond to hatched lines in F. J-K: Hematoxylin stain shows the architecture of the brain at the coronal plane approximate to I. Hypoplasia of the neocortex can be seen in J, and hypoplasia of the commissural plate is evident J′. K-K′: All but few glia are eliminated following conditional knockout of Fgf8 as shown by immunohistochemistry for GFAP at the coronal plane approximate to I. A few fimbrial glia are indicated by the arrow in K′. L-L′: While axons of the fimbria stained with anti-GAP43 and the AC are present in cKO mice, midline crossing only occurs for the AC. M-P: Expression of the commissural plate markers are disrupted, but not eliminated by conditional knockout of Fgf8, as shown by immunohistochemistry for commissural plate markers EMX1, NFIA, ZIC2 and SIX3 in the mid-sagittal plane of E17 cKO mice. Scale bars: A-D = 600 μm; E-I = 700 μm; J, L, N = 1 mm; K, M, O = 500 μm; P-S = 300 μm.

Figure 9

DTMRI tractography in E18 brains of Emx1-cre;Fgf8^flox/flox mice (cKO). From a region of interest (ROI) in the corpus callosum (A-F), tractography in mutants shows profound misprojections that fail to cross the midline (hatched line) compared to wildtype littermates (Ctl). Probst bundles are apparent from an ROI in the corpus callosum (E and F). Tractography was performed with DTI-Studio (tracking thresholds: angle <70 degrees, FA>0.3). Scale bar A, D = 0.7 mm; B, E = 0.8 mm; C, F = 1 mm.

Figure 10

Schematic of the commissural plate at an oblique coronal angle showing the commissures and their associated molecular domains. Abbreviations: 3^rd V, third ventricle; CgCtx, cingulate cortex; LV, lateral ventricle; NCtx, neocortex.