Central roles of the roof plate in telencephalic development and holoprosencephaly

Abstract

The roof plate is a well known signaling center in CNS development, but its roles in the developing telencephalon and the common holoprosencephaly (HPE) malformation have been uncertain. Using cellular ablations in mice, we show that roof plate cell loss causes failed midline induction and HPE in the dorsal telencephalon. This morphologic phenotype is accompanied by selective deficits in midline gene expression and a reduced activity gradient for bone morphogenetic proteins (Bmps), the major signals produced by the roof plate. In dissociated cells and mutant explants, exogenous Bmp4 is sufficient to mimic roof plate selectivity in midline gene regulation and to rescue roof plate-dependent midline patterning. Previously unrecognized neuroanatomical defects predicted by the mouse model are then confirmed in human HPE patients. These findings establish selective roles for roof plate-dependent Bmp signaling in dorsal telencephalic patterning and HPE and define novel candidate genes for the human disorder.

Figure 1.

RP ablation induces MIH HPE. A, E12.5 fixed embryo (top row) and freshly dissected CNS tissue (bottom row, except for the far right, which was fixed and stained with dilute ink for improved contrast); forebrain close-ups are dorsal views (top is anterior). RP ablated embryos display poor midline development and incomplete separation in the middle interhemispheric region (between arrows), whereas anterior and posterior regions remain separated bilaterally. An open neural tube is present caudally. The eyes and midline craniofacial region lack obvious defects. Scale bars, 0.5 mm. B, E12.5 coronal hematoxylin and eosin (H&E) stains and corresponding cortical outlines. Cortical primordia fail to separate in mutants but only at middle interhemispheric levels. The ventricles also collapse after ablation, a feature seen in some human MIH and classical HPE cases. Scale bar, 0.4 mm. C, D, Midline ventricular zone and mantle layer. Foxg1 and Lhx2 are normally absent from the E11.5 midline, but their expression becomes continuous across the midline in mutants. E12.5 mutants also possess telencephalic neurons (TuJ1⁺/Tbr1⁺) that are normally absent from the midline mantle layer. Scale bars, 0.1 mm. Dashed lines demarcate the ventricular surface in mutants; arrowheads designate the cortex–hem boundary. Top is dorsal for all section images. c, Cortex; cp, choroid plexus; d or di, diencephalon; ge, ganglionic eminence; h, hem; s, septum.

Figure 2.

Selective defects in midline gene expression after RP ablation. ISH and qRT-PCR analyses. All images are coronal (top is dorsal), except for Fgf8 (horizontal/axial; top is anterior). A, B, Midline signals. Wnt2b and Wnt3a, which are selectively expressed in the normal E12.5 cortical hem, are markedly reduced in mutants. Ventral Shh and rostral Fgf8 are well preserved. The Fgf8-expressing rostral midline is everted as a result of marked telencephalic hypoplasia. Scale bar, 0.2 mm. cl, Control; mt, mutant. C, D, Midline LIM homeobox genes. Lmx1a is selectively expressed in the tCPe and cortical hem at E12.5 and is markedly reduced in mutants. In contrast, Lhx5 expression is maintained and its domain expanded in E10.5 mutants (C), and Lhx5 transcripts are significantly increased in E12.5 mutants relative to controls (D). Scale bars: low power Lmx1a, 0.5 mm; all others, 0.1 mm. E, F, HPE genes. Tgif is preferentially expressed in cortical neuroepithelia, whereas Zic2 expression is highest in the RP. After RP ablation, Tgif levels are significantly reduced, whereas Zic2 expression is maintained or slightly increased. Like that of Lhx5, the expression domain of Zic2 appears expanded in mutants. Dashed lines demarcate the ventricular surface. Scale bars: low magnifications, 0.5 mm; high magnifications, 0.2 mm. Error bars indicate SEM; ∗∗p < 0.01.

Figure 3.

Selective defects in dorsal cortical patterning after RP ablation. A, E11.5–E12.5 ISH and Lhx2 immunostaining (red–orange signal; yellow represents autofluorescent red blood cells). Lhx2 and Emx2 levels in mutants are reduced in the dorsal cortical (hippocampal) primordium, resulting in flattened DV expression gradients. In contrast, overall levels and VD gradients of Pax6, Foxg1, and Ngn2 appear unaffected. Dashed lines demarcate the ventricular surface; top is dorsal for all images. Scale bars, 0.2 mm. B, ISH signal intensity scatter plots and linear curve fits of images in A, plotted on x- and y-axes of identical scale. C, qRT-PCR studies confirm the overall reduction in Lhx2 and Emx2 levels in E12.5 mutants (mt) relative to control (cl) littermates, whereas Foxg1 and Ngn2 expression are maintained. Error bars indicate SEM; ∗p < 0.05.

Figure 4.

Reduced and flattened Bmp activity gradient after RP ablation. E10.5 pSmad1/5/8 immunohistochemistry with Hoechst nuclear counterstaining. A, Normal embryos; boxed regions magnified to the right (M phase images are from a different focal plane). Immunoreactivity for nuclear pSmad displays a DV gradient within interphase regions of the E10.5 dorsal telencephalic ventricular zone and is particularly intense in M phase cells at the ventricular surface. Scale bars: dorsal telencephalon, 0.2 mm; all others, 20 μm. B, RP ablated embryos and controls; black and gray arrowheads designate medial and lateral thirds of the dorsal telencephalon, respectively, which were used in D. Overall pSmad immunoreactivity in interphase and M phase cells is reduced in the mutant dorsal telencephalon. Scale bar, 0.2 mm. C, Interphase cell intensity; scatter plots of average pSmad signal with second-order polynomial curve fits. Overall levels of pSmad are reduced and its DV gradient is flattened after RP ablation. Note the correlations with cortical DV gradient profiles in Figure 3. D, M phase cell intensity. Signal intensity is markedly reduced in mutants (mt) relative to controls (cl). Unlike interphase cells, no spatial gradient is detected in M phase cell intensity. Error bars indicate SD; ∗∗∗p < 0.0001.

Figure 5.

Sufficiency of exogenous Bmp4 to mimic midline patterning and gene regulation selectivity by the RP. A, E10.5 mutant explants treated with 50 ng/ml Bmp4; qRT-PCR analysis. Bmp4 significantly rescues Lmx1a expression. Msx1 and Ttr also demonstrate positive trends, but Tgif does not. Lhx5 and Zic2 levels are not significantly affected by exogenous Bmp4. Error bars indicate SEM; ∗∗p < 0.01. B, E10.5 mutant explants treated with 50 ng/ml Bmp4; whole-mount ISH and post hoc sections (ventricular surface up). Bmp4 treatment rescues Ttr expression in most mutant explants (6 of 9), whereas BSA does not (0 of 4). In all six rescued explants, Ttr was expressed in a thin midline epithelium at the ventricular surface. In two explants, Ttr rescue occurred in an extended simple epithelium that morphologically resembles normal CPe (inset). Scale bar, 0.1 mm. C, CYPA-normalized qRT-PCR data from dissociated E12.5 CD1 dorsal telencephalic cultures. Among the four genes reduced after RP ablation, Msx1 and Tgif show significant upregulation by Bmp4, whereas the Ttr and Lmx1a dose–response curves have consistent upward trends that occasionally reach statistical significance (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). In contrast, the two genes maintained or elevated in RP ablated mutants (Lhx5 and Zic2) consistently show flat dose–response curves that fail to reach statistical significance in any of the four datasets (CD1 and B6 cells, CYPA and 18S normalizations) (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Error bars indicate SEM; ∗p < 0.05; ∗∗p < 0.01.

Figure 6.

MIH HPE patient defects predicted by the mouse RP ablation phenotype. A, T1-weighted sagittal and T2-weighted axial MRI scans. The middle interhemispheric phenotype is demonstrated on the sagittal image by apparent absence of the midbody of the corpus callosum (arrow), whereas the rostrum and genu are better preserved (arrowheads). The axial scan demonstrates abnormal midline gray matter in central hemispheric regions (arrows) and the characteristic anterior displacement of Sylvian fissures (arrowheads). B, Choroid plexus and hippocampal defects; T1-weighted axial and T2-weighted coronal MRI images of patients shown in A. Choroid plexus, which enhances with contrast (arrows in top left panel), is undetectable in the contrast-enhanced scan of the MIH HPE patient. The coronal image displays hippocampi that are small and dysplastic compared with control (arrows in right panels). All 23 cases reviewed demonstrated marked tCPe deficiency, and 7 of 11 cases with adequate images exhibited reduced hippocampal size.

Figure 7.

The “central” RP–Bmp pathway in dorsal telencephalic patterning and MIH HPE. A, Normal development. Zic2 induces the RP, which produces Bmps that generate a Bmp activity gradient in the dorsal telencephalon. This activity gradient then directs dorsal midline induction (tCPe and cortical hem in orange and brown, respectively) and dorsal cortical patterning. LHX5 also acts upstream of the RP, and the RP exerts negative feedback that restricts Zic2 and Lhx5 expression to the midline. B, Pathways to MIH HPE. Two independent pathways give rise to RP deficits that ultimately lead to MIH HPE. In the first pathway, the RP deficit results from failed induction as a result of reduced Zic2 function. In the second (Zic2-independent) pathway, RP dysfunction arises from excessive RP cell death, which leads to reduced Bmp signaling in the dorsal telencephalon. In addition to ZIC2 mutations, candidate inducers of MIH HPE include LHX5 mutations, factors that exacerbate RP cell death (including increased Bmp signaling) and mutations that decrease Bmp signaling. For additional details, see Discussion.