Central Roles of the Roof Plate in Telencephalic Development and Holoprosencephaly

RP ablation causes MIH HPE

For simplicity, we generally use dorsal (i.e., toward the RP) and ventral (away from the RP) to describe spatial position and refer to the tCPe and cortical hem as “midline” structures. Our previously reported ablation studies (Currle et al., 2005) are summarized in supplemental Figure 1 (available at www.jneurosci.org as supplemental material). In ACTBCre;Gdf7DTA embryos, excessive cell death occurs transiently in the RP before differentiation of the tCPe and cortical hem, which results in the reduction of Bmp mRNA levels (Bmp4, Bmp6, and Bmp7). Although RP cell lineages do not contribute to most of the tCPe (or cortical hem), RP cell ablation results in near-total tCPe absence, thus indicating a major cell-nonautonomous requirement for RP cells in tCPe induction (Currle et al., 2005).

Consistent with the absence of tCPe, RP ablation also resulted in failed separation of the cortical primordia at the dorsal midline (Fig. 1A,B). Interestingly, this failure was regionally restricted to middle interhemispheric regions (i.e., telencephalic vesicles were normally separated anteriorly and posteriorly) in all mutant embryos examined by serial section analysis (Fig. 1B) (n = 14). The lack of middle interhemispheric separation was evident at the histologic and molecular levels in both the precursor (ventricular zone) and mantle layers (Fig. 1C,D). Mutants often retained partial midline character (e.g., thinner epithelium, partial Foxg1 and Lhx2 suppression, and Tbr1 expression) but uniformly displayed total to near-total absence of tCPe markers and morphology (Currle et al., 2005) as well as hem markers (Fig. 2A–D). RP ablation therefore leads to an MIH HPE phenotype (Barkovich and Quint, 1993; Simon et al., 2002), which is consistent with the Gdf7 genetic fate map and the spatiotemporal pattern of ablation (Currle et al., 2005).

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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.

The MIH HPE phenotype was also associated with partial to total ventricular reduction (Fig. 1B) by E12.5, a feature seen in some MIH (Ming and Golden, 2004) and classical HPE cases in humans (Norman, 1995; Takahashi et al., 2003). Other defects included a small forebrain preferentially affecting the dorsal telencephalon and reduced dorsal diencephalon volume consistent with diencephalic RP ablation (Currle et al., 2005). Although distorted secondarily by the malformed dorsal telencephalon and ventricles, the ventral forebrain did not exhibit midline fusion or other definitive defects, particularly in earlier stage embryos without collapsed ventricles (Fig. 2) (see Fig. 4). The relative lack of craniofacial and ventral forebrain anomalies also distinguishes human MIH HPE from classical forms (Barkovich and Quint, 1993; Simon et al., 2002).

RP ablation causes selective expression deficits in midline genes

To evaluate the cortical hem, we examined the expression of hem-restricted Wnt2b and Wnt3a (Grove et al., 1998) after RP ablation. Both Wnts were undetectable in E12.5 mutant embryos by ISH (Fig. 2A) and were significantly reduced by qRT-PCR (Fig. 2B). Similar reductions were seen at E13.5 (data not shown), whereas ventral Shh and rostral Fgf8 expression were well preserved in sections and whole-mount embryos (Fig. 2A) (data not shown). The maintained Shh and Fgf8 expression was consistent with the absence of Gdf7 cell lineages (in Gdf7Cre;R26R embryos) or excessive cell death (in ACTBCre;Gdf7DTA embryos) in the ventral Shh and rostral Fgf8 expression domains (Currle et al., 2005).

To further evaluate the cortical hem, we examined the LIM homeobox genes Lmx1a (Millonig et al., 2000) and Lhx5 (Zhao et al., 1999). Lmx1a, which is selectively expressed in the normal hem and tCPe, was markedly reduced by ISH and qRT-PCR after RP ablation (Fig. 2C,D). In contrast, midline Lhx5 expression was not reduced after RP ablation but was instead spatially expanded at E10.5 (Fig. 2C) and increased in overall levels by E12.5 (Fig. 2D). These expression studies indicate that Wnt2b, Wnt3a and Lmx1a expression (and the induction of normal cortical hem fate) are dependent on an intact RP, whereas Lhx5 expression lacks this dependence.

RP ablation causes selective expression deficits in HPE-associated genes

To identify potential genetic pathways involved in RP-dependent HPE, we examined the expression of human HPE-associated genes. Tgif and Zic2 (Fig. 2E), but not Shh or Six3 (data not shown), demonstrated dorsal telencephalic expression in normal E10.5 and E12.5 embryos. Both genes were expressed in the midline and cortical primordia, but Zic2 levels were higher at the midline whereas Tgif was preferentially expressed in cortex. After ablation, Tgif expression was significantly reduced at E10.5 and E12.5, whereas Zic2 expression was maintained and perhaps slightly increased (Fig. 2E,F). Like Lhx5 (Fig. 2C), Zic2 expression was seen in a more expansive domain in RP ablated mutants (Fig. 2E), suggesting that the RP is necessary to restrict Lhx5 and Zic2 expression to the midline. Thus, unlike the other midline genes, Lhx5 and Zic2 do not depend on the RP for upregulated or maintained expression.

RP ablation causes selective disruption of cortical transcription factor gradients

In addition to Tgif, we examined essential cortical patterning genes that normally have graded expression relative to the midline. As seen previously in RP ablated mutants with open forebrain defects (Monuki et al., 2001), mutant embryos with closed forebrains exhibited reduced Lhx2 levels in dorsal cortex. This resulted in decreased Lhx2 levels overall (as seen by ISH, immunohistochemistry, and qRT-PCR), as well as flattening and subtle inversion of its normal high dorsal-to-low ventral (DV) gradient (Fig. 3A–C). A similar extent and pattern of reduction was seen for Emx2, another DV graded gene. In contrast, three transcription factors normally expressed in high ventral-to-low dorsal (VD) gradients, Pax6, Foxg1, and Ngn2, were relatively unaffected in mutants (Fig. 3A–C). Additional section and whole-mount studies indicated that mutant embryos invariably induce Lhx2 and Emx2 but fail to upregulate and establish their DV gradients between E9.5 and E11.5, the period when intracortical gradients become detectable (data not shown). Thus, RP cells regulate dorsal cortical patterning in a selective manner, which includes upregulation of Tgif, Lhx2, and Emx2 and establishment of their DV gradients. The presence of dorsal cortical patterning defects by E10.5 (Fig. 2E) (data not shown) indicates that initial RP-dependent cortical patterning does not depend on the tCPe, whose definitive differentiation ensues later (by E11.5) (Currle et al., 2005).

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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.

RP ablation causes reduction and flattening of the Bmp activity gradient

Although steady-state levels of Bmp and Msx1 (msh homeobox homolog 1) mRNAs are reduced after RP ablation (Currle et al., 2005), direct studies of Bmp activity have been lacking. Indeed, graded Bmp activity has been presumed in the developing telencephalon based on precedent in other experimental systems but has not been directly demonstrated. Like other morphogens, TGFβ family signals, which include Bmps, are transduced in a pathway that appears to be strictly linear (Ashe and Briscoe, 2006). Linear TGFβ signal transduction is accomplished via the phosphorylation of Smad proteins and their subsequent translocation to the nucleus (Feng and Derynck, 2005). Because of its function as a direct and continuous transducer of extracellular TGFβ concentration with a short timescale (Bourillot et al., 2002), nuclear pSmad (or pMad in Drosophila) is a widely accepted integrative readout of Bmp signaling. In vertebrates, Bmp signals are specifically transduced via phosphorylation of Smad1, Smad5, and Smad8 (Feng and Derynck, 2005). A commercially available pSmad1/5/8 antibody (Chemicon) demonstrated appropriate specificity in tissue sections (Fig. 4) and in cultured E12.5 CD1 cortical cells treated with and without Bmp4 (for additional details regarding specificity, see Materials and Methods).

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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.

Studies of the E10.5 telencephalon revealed two distinct patterns of pSmad1/5/8 immunoreactivity (Fig. 4A). First, signal was particularly bright in many, but not all, cells lining the ventricles (M phase cells). This pattern occurred throughout the telencephalon and lacked any obvious spatial gradient within the dorsal telencephalon (Fig. 4D). Second, within interphase cells of the dorsal telencephalon, pSmad signal was selectively nuclear and displayed a smooth DV gradient (Fig. 4A–C). Interestingly, pSmad immunoreactivity was also relatively high in ventricular lining and interphase cells of the ventral telencephalon (Fig. 4A,B). Similar findings were observed in multiple control embryos at E12.5 (data not shown).

Within the dorsal telencephalon of RP ablated embryos, pSmad interphase and M phase cell labeling were markedly diminished (Fig. 4B–D). Within interphase cells, overall signal intensity was reduced by >40% (42.5%), with a greater reduction medially (48.6% over the medial 0.2 mm) than laterally (34.8% over the lateral 0.2 mm). This resulted in a flattening of the DV gradient in mutants relative to controls (Fig. 4C) (linear curve fit slopes, −0.018 gray value units/μm in mutants, −0.048 units/μm in controls). Likewise, average M phase cell intensity was markedly reduced in mutants (47.4% reduction medially, 46.1% reduction laterally). Unlike the interphase cells, no spatial gradient in average M phase signal intensity was detected within the dorsal telencephalon at E10.5 (Fig. 4D) (p = 0.951, control medial vs lateral; p = 0.763, mutant medial vs lateral). Although expression and activity of Smads and other Bmp pathway components could be altered after RP ablation, the pSmad data are consistent with the reduced dorsal midline Bmp production (Bmp4, Bmp6, and Bmp7) seen in RP ablated mutants (Currle et al., 2005). Importantly, these data also provide primary evidence for a true Bmp activity gradient in the normal dorsal telencephalon and its dependence on an intact RP.

Exogenous Bmp4 selectively rescues RP-dependent midline patterning in explants

To determine whether Bmp signals alone could replace an intact RP, we bathed dorsal forebrain explants from E10.5 RP ablated embryos in exogenous Bmp4 or BSA and then quantified mRNA levels by qRT-PCR. The four downregulated midline/HPE genes (Msx1, Ttr, Lmx1a, and Tgif) and the two genes unaffected or increased after RP ablation (Lhx5 and Zic2) were assayed. Because of developmental, phenotypic, and experimental variability, baseline expression levels in control explants varied significantly (Fig. 5A). Nonetheless, statistically significant rescue was achieved for Lmx1a (Fig. 5A). In addition, trends toward upregulation in Bmp4-treated explants were seen for Msx1 and Ttr, whereas Tgif levels remained relatively unchanged. Exogenous Bmp4 had no apparent effect on Lhx5 or Zic2 levels.

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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.

In addition to the aforementioned variability, rescues at the midline were likely to be masked by qRT-PCR assays on whole explants. We therefore performed identical explant cultures and assayed for tCPe, the most dorsal and most distinctive midline cell fate, by whole-mount ISH. Although none of the four mutant explants treated with BSA demonstrated Ttr rescue, six of the nine explants treated with Bmp4 demonstrated significant midline Ttr expression (Fig. 5B). Post hoc sectioning revealed Ttr expression within a thin (one to two cell layers thick) epithelium at the ventricular surface of all six Ttr-expressing explants. Remarkably, in two explants, Ttr rescue occurred in an extended simple epithelium that resembled normal tCPe (Fig. 5B, inset). These findings suggest that Bmp4 was sufficient to rescue not only tCPe gene expression but also tCPe morphogenesis.

Exogenous Bmp4 selectively upregulates RP-dependent midline genes in dissociated cells

To further test how well Bmp4 could recapitulate RP-dependent selectivity in midline gene regulation, we quantified the same battery of genes in dissociated dorsal telencephalic cells cultured with Bmp4. By dissociating cells, and thereby homogenizing variations normally found within intact tissues, embryo and explant variation could be better controlled and the potential primacy of extracellular Bmp concentration tested more directly. This test of selectivity was particularly stringent, because cells from E12.5 embryos were used, a stage well past the midline inductive period in vivo.

Nonetheless, exogenous Bmp4 significantly upregulated two of the four RP-dependent genes (Msx1 and Tgif) in a consistent manner across three CD1 cultures and two B6 cultures in two different base media (Fig. 5C) (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Ttr and Lmx1a also displayed upregulatory trends in all dose–response curves, occasionally reaching statistical significance (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). In contrast, the Lhx5 and Zic2 dose–response curves were consistently flat and failed to reach statistical significance in any culture.

The embryonic mouse model accurately predicts the human MIH HPE phenotype

Our mouse studies led to three specific predictions about neuroanatomical defects in human MIH HPE patients. First, surviving patients should have markedly reduced to absent tCPe (Currle et al., 2005). Second, because Wnt3a, Lhx2, and Emx2 are positive regulators of dorsal cortical (hippocampal) precursor proliferation (Porter et al., 1997; Lee et al., 2000b; Tole et al., 2000), their reduced expression should lead to reduced hippocampal size. Third, because mouse RP ablation defects are more severe toward the dorsal midline, tCPe should be more significantly affected than the hippocampus. Although ACTBCre;Gdf7DTA embryos rarely survive past E14.5, human MIH HPE patients can survive postnatally. Hippocampal and CPe reductions have been noted in at least two and nine MIH HPE cases, respectively (Barkovich and Quint, 1993; Marcorelles et al., 2002), but their prevalence and association with the disorder remained uncertain.

We therefore examined the hippocampus and tCPe in magnetic resonance imaging (MRI) scans from a cohort of 23 MIH HPE patients (Fig. 6A). Despite the uneven quality of imaging studies, tCPe appeared to be either totally absent or significantly reduced in all 23 patients (Fig. 6B). Eleven patients had coronal images that were adequate to assess hippocampal size; among these, seven showed small, and often dysplastic, hippocampi (Fig. 6B). Thus, marked reduction in tCPe volume and small hippocampi, with the tCPe phenotype appearing more severe, are characteristic features of human MIH HPE, as predicted by the mouse model.

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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.