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First published online May 30, 2007
doi: 10.1242/10.1242/dev.000620

Development 134, 2325-2335 (2007)
Published by The Company of Biologists 2007
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Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins

Sema K. Sgaier1,2,*, Zhimin Lao1,{dagger}, Melissa P. Villanueva1, Frada Berenshteyn1,{dagger}, Daniel Stephen1,{dagger}, Rowena K. Turnbull1 and Alexandra L. Joyner1,2,3,{dagger},{ddagger}

1 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
2 Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
3 Department of Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.


Figure 1
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  		Fig. 1. Dynamic expression of En1lacZ and En2tau-lacZ in the mes/r1 of mouse embryos. En1 (A) and En2 (G) mRNA expression detected by whole-mount RNA in situ hybridization. (B-F) En1-lacZ and (H-L) En2-tau-lacZ expression at the indicated stages detected by ß-gal analysis. En1 is induced earlier and becomes more restricted than En2 by E10.5. Staining in the fourth ventricle in I and D is a result of probe trapping. CbP, cerebellar primordium; mes, mesencephalon; Mb, midbrain; r1, rhombomere 1.

 

Figure 2
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  		Fig. 2. En1 is required prior to ~E9.0 to pattern the anterior cerebellum and after E9.0 to form the posterior midbrain. (A) Schematic illustrating the expression profile of En alleles in the mes/r1 region of En1flox/Cre mice. Note that only one functional En1 allele is present because the En1-Cre allele is an En1-null. (B,C) Dorsal view of posterior adult brains of (B) En1Cre/+ and (C) En1flox/Cre mice. (D-I) Cresyl Violet-stained sagittal sections of the brains were taken at the level of (D-F) the vermis and (G-I) hemispheres as indicated in B,C by the red and black dashed lines, respectively. E,H and F,I are sections taken from brains in B and C, respectively. Arrow in F indicates the predominant phenotypes of fusion of vermis folia I-II and III and the red asterisk indicates a slight truncation of the inferior colliculus. In some En1flox/Cre mice, a normally foliated Cb was also seen (D,G). H, hemisphere; V, vermis; Pv, paravermis; Cb, cerebellum; IC and SC are the inferior and superior colliculus, respectively outlined by red and green dashed lines. Vermis and hemisphere folia are indicated by roman numerals (I-X) and letters (A, S, CI, CII, Pm, P), respectively.

 

Figure 3
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  		Fig. 3. Differential requirement of En genes for mouse midbrain and cerebellar development. (A) Schematic illustrating the detailed expression profile of each En allele (En1, En2 and En1flox/Cre) within the mes/r1 region. (B-K) Cresyl Violet-stained mid-sagittal sections of E18.5 brains with the corresponding genotypes and all the functional En allele profiles indicated at the top. Asterisks indicate forming fissures. Black arrows indicate truncated inferior colliculus. The inset in F shows the lateral Cb tissue that forms in the En1-/- mutants.

 

Figure 4
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  		Fig. 4. Viable En1 and En2 double-mutant adult mice have midbrain and cerebellar defects. (A-D) Dorsal posterior views of adult brains of (A) wild-type, (B) En2-/-, (C) En1-/+;En2-/+ and (D) En1-/+;En2-/- mutant mice. (E-L) Cresyl Violet-stained sagittal sections of the cerebella shown in A-D at the level of the vermis (E-H) and hemispheres (I-L), as indicated in A by the red and black dashed lines, respectively. Black and red arrowheads indicate the anterior and posterior foliation defects, respectively. Blue and red asterisks indicate hemisphere foliation defects and truncation in the IC, respectively. See Fig. 2 legend for midbrain and cerebellum annotation.

 

Figure 5
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  		Fig. 5. In the absence of En2, expression of mouse En2 but not Drosophila en in place of En1 can rescue mes/r1 defects. Stained sagittal sections of wild-type (wt) (A,G), En12ki/2ki;En2-/+ (B,H), En12ki/2ki;En2-/- (C,I), En12ki/-;En2-/- (D,J), En1Denki/Denki;En2-/+ (E,K), and En1Denki/+;En2-/- (F,L) at the level of the vermis (A-F) and hemispheres (G-L). A-C,E,G-I,K are stained with Cresyl Violet; D,F,J,L with Hematoxylin and Eosin. For comparison, the En2-/- phenotype is shown in Fig. 4. See Fig. 2 legend for midbrain and cerebellum annotation, and Fig. 4 legend for key to asterisks and arrows.

 

Figure 6
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  		Fig. 6. mes and r1 cells behave differently in En1-/+;En2-/- mutants. (A-J) Tamoxifen was administered to mouse embryos carrying the indicated alleles at 18.00 h on E10.5. (A,B) Coronal sections of E12.5 brains and (C,D) horizontal and (E,F) midline sagittal sections of E16.5 brains stained for ß-gal activity. (G,H) Dorsal views of ß-gal-stained adult brains. (I,J) Sagittal sections of adult brains taken at the level of the vermis and stained for ß-gal. The domain of marked cells was comparable in the En1CreERT1/+;En2-/- and En1CreERT1/+;R26R/+ embryos at E12.5, but decreased in the tectum and increased in the Cb of En1CreERT1/-;En2-/-;R26R/+ embryos by E16.5. CbP, cerebellar primordium; EGL and associated bar, external granule layer. Ist, isthmus; Mb, midbrain; VZ, ventricular zone; A, anterior; P, posterior; M, medial; L, lateral. Red asterisks indicate truncation of the isthmus/inferior colliculus. CbP is outlined by a purple dashed line in C,D; in I,J, the IC and SC are outlined by dashed red and green lines, respectively. The red bar in A,B indicates the size of the CbP domain with marked cells.

 

Figure 7
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  		Fig. 7. Fgf8, Fgf17 and En1 are expressed normally in En2-/- and En1-/+;En2-/- mutant E11.5 mouse embryos. RNA in situ hybridization of Fgf8 (A-C'), Fgf17 (D-F') and En1 (G-I') on sagittal sections of En1-/+ (A,D,G), En2-/- (B,E,H) and En1-/+;En2-/- (C,F,I) E11.5 mutant embryos. Upper (A-I) and lower (A'-I') panels show medial and lateral sections, respectively. The Fgf8/17 expression domains are positioned normally but are slightly reduced in size in mutants (C,F). En1 is expressed normally in mutants (H,I). The isthmus corresponds to the Fgf8 expression domain and is outlined in red. Red asterisks indicate truncation of the isthmus/inferior colliculus. r1, rhombomere 1; Ist, isthmus, mes, mesencephalon.

 

Figure 8
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  		Fig. 8. An `En code' can account for genetic partitioning of the midbrain and cerebellum vermis into functionally related domains. (A) Schematic illustrating the domains (color coded) within the mouse early neural tube (left) along the A-P axis that express either or both of the En genes, and the regions of the adult cerebellum and midbrain (right) that they give rise to owing to a rotation of the neural tube. The red-outlined region delineates rhombomere 1. (B) Schematic illustrating the normal gene dosage requirement for En1 and En2 in sustaining development of distinct domains of the tectum and Cb. Note that based on a sensitive knock-in assay, En2 protein appears to be more active throughout the vermis than En1. Thus, the gene dosage effects are likely to reflect differences in En1 and En2 gene expression. Cb, cerebellum; Mes, mesencephalon; SC, superior colliculus; IC, inferior colliculus; A, anterior; P, posterior; M, medial; L, lateral.

 




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