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First published online 21 June 2006
doi: 10.1242/dev.02441

Development 133, 2793-2804 (2006)
Published by The Company of Biologists 2006
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The roof plate regulates cerebellar cell-type specification and proliferation

Victor V. Chizhikov1, Anne G. Lindgren2, D. Spencer Currle3, Matthew F. Rose4, Edwin S. Monuki3 and Kathleen J. Millen1,2,*

1 Department of Human Genetics, University of Chicago, 920 E. 58th Street, CLSC 319 Chicago, IL 60637, USA.
2 Committee on Genetics, University of Chicago, 920 E. 58th Street, CLSC 319 Chicago, IL 60637, USA.
3 Departments of Pathology and Developmental and Cell Biology, University of California, Irvine, D440 Medical Sciences I, Irvine, CA 92697-4800, USA.
4 Program in Developmental Biology, Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Cellular populations in dorsal rhombomere 1 at E10.5. (A) Dorsal view schematic of r1. Anterior (a) and posterior (p) domains of r1 are indicated. 4v, 4th ventricle, IsO, isthmus, RRL, rostral rhombic lip. (B-D) Whole-mount in situ staining of E10.5 r1 demonstrating anterior (a) and posterior (p) roof plate domains. Arrowhead indicates reduced expression of Lmx1a and Gdf7 and no Wnt1 expression in the anterior domain. (E-I) In situ and immunostained paramedial sagittal sections through r1 in wild-type (E,G-I) and Gdf7-Cre;ROSA (F) E10.5 mice. Markers are indicated. Arrowheads in e'-e''' point to the medial single cell layer. Arrows point to dorsal neuroepithelium, which expresses Lmx1a, Gdf7 and Wnt1.(F) Arrowheads and arrow point to the medial single cell layer and dorsal neuroepithelium, respectively. (H) Arrows point to BrdU+/Lmx1a+ double-labeled cells. Arrowheads point to the medial single cell layer. (I) Arrowhead indicates Lmx1a+/Msx1/2+ region and the arrow indicates the Msx1/2+ region outside of this domain (J,K) Summary diagrams of gene expression domains, with provided key. Roof plate is defined as the Lmx1a+/Gdf7+ region in both anterior and posterior r1.

 

Figure 2
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  		Fig. 2. Cellular populations within the E12.5 cerebellar anlage. (A,B,D-F) Paramedial sagittal and (C) lateral sagittal immunostained sections through dorsal r1 at E12.5 in wild-type (A,D,E) and Math1lacZ/+ (B,C,F) embryos. Markers are indicated. Solid arrowhead in B points to LacZ+ cells in the rhombic lip many of which are Lhx2/9 negative. Arrow points to LacZ+/Lhx2/9+ cells engaged in the RLS. The open arrowheads in B and C indicate LacZ+ fibers on the dorsal surface, which belong to LacZ+/Lhx2/9+ cells. Arrowheads in D inset indicate small numbers of Pft1a+/Lhx1/5+ double-labeled nuclei. Laterally, the junction (broken line in C) between the pons and cerebellum (Cb) is difficult to distinguish, since the aqueduct (aq in D) no longer separates them. Arrowhead in merge panel E indicates Lmx1a-/Msx1/2+ cells. (G,H) Schematic of the molecular map of roof plate r1 populations in whole-mount (G) and paramedial sagittal section (H). c1 cells extend to the isthmus adjacent to the anterior roof plate. E12.5 cerebellar anlage populations are excluded from this region, defining a negative (neg) zone of gene expression.

 

Figure 3
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  		Fig. 3. Roof plate cells do not significantly contribute to the cerebellar anlage. (A) Schematic of the genetic strategy to permanently label progeny of roof plate cells. Black triangles indicate loxP sites. (B) Side view of X-Gal-stained E10.5 r.p.Lmx1a-cre;ROSA embryo. LacZ expression (arrow) recapitulates dorsal Lmx1a expression at E10.5. (C) Sagittal section of the E12.5 anlage of immunostained r.p.Lmx1a-Cre;ROSA embryo. Arrowhead points to LacZ+ cells. Arrows point to Math1+ c1 cells (D) Sagittal section of X-Gal-stained E18.5 r.p.Lmx1a-Cre;ROSA embryo. External granule cell layer (EGL) is marked by broken line and arrows. Arrowhead indicates X-Gal+ choroid plexus.

 

Figure 4
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  		Fig. 4. Cerebellar anlage abnormalities in mouse embryos with ectopic expression of Lmx1a. (A-F). Dorsal view of E12.5 whole embryos (A,B) and their dissected neural tubes (C-F). Arrows indicate dramatically expanded medial single cell layer in transgenics (B,D,F). (G-W) Paramedial sagittal sections of E12.5 cerebella with genotypes and markers indicated, together with quantification of each cell type. In all panels, insets show a higher magnification of the boxed regions. Roof plate (arrow in H showing Gdf7 and arrows in J showing Lmx1a+/Msx1/2+ cells) was expanded to the ventricular surface within the anlage. Arrowheads in I and J point to Lmx1a-/Msx1/2+ cells located adjacent to Lmx1a+/Msx1/2+ roof plate cells. (K,N,Q,T,W) Quantification of number of c1, RLS, pc2 and c2-c4 cells in Lmx1a transgenic embryos. * indicates P<0.01. Bars indicate s.e.m.

 

Figure 5
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  		Fig. 5. Bmps are required for roof plate development and induction of c1 and RLS cells in vitro. (A-E) Isolation and culturing of e8.5 mid-hindbrain dorsal (d) neural fold explants. Addition of noggin and follistatin (N+F) decreased induction of Lmx1a (B-D) and Gdf7, as measured by RT-PCR (E). (D) Quantification of numbers of Lmx1a+ cells in dorsal explants cultured with and without Bmp inhibitors. (F-N) Examples of wild-type E8.75-E9.0 intermediate neural tube explants (int) at the level of r1, cultured with and without chick r1 roof plate (r.p.). Addition of noggin and follistatin (N+F) reduces induction of c1 and RLS cells. Intermediate explants and roof plate are labeled and demarked by the broken line. (J,N) Quantification of numbers of Math1+ (J) and Lhx2/9+ (N) cells appeared in wild-type intermediate explants. Blue, intermediate explants cultured alone (int). Red, intermediate explants cultured with chick roof plate (int+rp). Green, intermediate explants cultured with chick roof plate together with noggin and follistatin (int+rp+N+F). * indicates P<0.01. Bars indicate s.e.m.

 

Figure 6
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  		Fig. 6. Roof plate and early cerebellar anlage defects in dreher mouse embryos. (A-F) Dorsal views of whole-mount in situ-stained E10.5 embryos with genotypes and markers as indicated. In dreher, the anterior roof plate (a) is expanded at the expense of the posterior roof plate domain (p). IsO is still maintained (C,D). (E,F) The anterior limit of Math1 expression is found at the isthmus (broken line) in wild-type embryos. It is positioned more posteriorly in dreher. Open arrowheads show Math1-negative domain in dreher embryos. (I-P) Whole-mount in situ analysis reveals the anterior Math1 gene expression limit is still abnormal in E12.5 dreher embryos. The anterior limits of other genes, however, are normal in dreher embryos. Despite morphological changes in the roof plate a normal negative (neg) zone of gene expression is maintained adjacent to the isthmus (broken line in I-P). Open arrow points to Math1-negative domain in anterior r1 of dreher embryo. Solid arrowheads show pc2, c2 and c3 cells. (G,H,Q,R) Schematic illustrations summarize gene expression changes.

 

Figure 7
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  		Fig. 7. Roof plate ablation causes loss of Wnt1 and c1 populations, and reduces cerebellar anlage proliferation at E10.5. (A) Schematic of the genetic strategy to ablate roof plate cells. (B-I) Whole-mount in situ staining of E10.5 embryos with markers and genotypes indicated. (B,C and H,I) Dorsal views with broken line in C demarking the dorsal edge of the open neural tube. (D-G) Side views. Arrows in B-E indicate position of r1 roof plate. Arrowheads `o' and `v' mark normal otic and ventral midbrain Lmx1a expression in panels B and C. Upper and lower insets in panel E show Wnt1 and Fgf8 isthmic expression. Arrows in panels F and G point to the cerebellar rhombic lip. Arrows in panel H show broad expression of cyclin D2 which is eliminated in roof plate-ablated embryos (I). Weak staining in I is ventral, visible because of the open neural tube. Dark lines on dorsal edges are artifact. (J,K) Schematic illustration of E10.5 wild-type and roof plate-ablated embryos demonstrating the consequences of ablation. (L) Hematoxylin and eosin-stained sections at the level of the horizontal line in panels J and K in time series. Arrows point to the most dorsal neuroepithelium of roof plate-ablated embryo (M-P) Immunostained transverse sections at E10.5 demonstrating that cell proliferation and cyclin D2 expression is reduced across the entire cerebellar anlage in ablated embryos. Arrowheads point to the developing cerebellar anlage.

 

Figure 8
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  		Fig. 8. Effect of roof plate ablation on specification of cerebellar cells at E12.5. (A-D) Whole-mount in situ staining showing c3 and pc2 populations in wild-type and ablated embryos at E12.5. Arrow in A indicates roof plate expression of Lmx1a which is eliminated in panel B. Arrowheads `o' and `v' mark normal otic and ventral midbrain Lmx1a expression in panels A and B. Arrows in C and D demonstrate pc2-specific expression of Ptf1a and the reorientation of the open edges of the dorsal neural tube, illustrated in panels E and F. (G,H,J,K,M,N) Transverse immunostained sections at the level of the horizontal line in panels E and F, with markers and genotypes. c1 and RLS markers are missing in ablated embryos, while pc2, c2 and c3 populations are still present, in reduced numbers and altered positions in the anlage. (I,L) Quantification of pc2, c2 and c3 cells found on transverse sections from wild-type and roof plate-ablated embryos. Bars indicate s.e.m. (O,P) Schematic illustrations summarize gene expression changes.

 

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© The Company of Biologists Ltd 2006