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.2007 Jun;134(12):2325-35.
doi: 10.1242/dev.000620.

Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins

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Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins

Sema K Sgaier et al. Development.2007 Jun.

Abstract

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. Our previous studies indicated that the difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. Here, we uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele we demonstrate that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, we show that Drosophila en is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, our studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. Our study suggests that an ;engrailed code' is integral to partitioning the tectum and cerebellum into functional domains.

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Figures

Fig. 1
Fig. 1. Dynamic expression ofEn1lacZ andEn2tau-lacZ in the mes/r1 of mouse embryos
En1 (A) andEn2 (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 β-gal analysis.En1 is induced earlier and becomes more restricted thanEn2 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.
Fig. 2
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 ofEn1flox/Cre mice. Note that only one functionalEn1 allele is present because theEn1-Cre allele is anEn1-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 someEn1flox/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.
Fig. 3
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 andEn1flox/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 theEn1−/− mutants.
Fig. 4
Fig. 4. ViableEn1 andEn2 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.
Fig. 5
Fig. 5. In the absence ofEn2, expression of mouseEn2 but notDrosophila en in place ofEn1 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), andEn1Denki/+;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, theEn2−/− 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.
Fig. 6
Fig. 6. mes and r1 cells behave differently inEn1−/+;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 β-gal activity. (G,H) Dorsal views of β-gal-stained adult brains. (I,J) Sagittal sections of adult brains taken at the level of the vermis and stained for β-gal. The domain of marked cells was comparable in theEn1CreERT1/+;En2−/− andEn1CreERT1/+;R26R/+ embryos at E12.5, but decreased in the tectum and increased in the Cb ofEn1CreERT1/−;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.
Fig. 7
Fig. 7.Fgf8, Fgf17 andEn1 are expressed normally inEn2−/− andEn1−/+;En2−/− mutant E11.5 mouse embryos
RNA in situ hybridization ofFgf8 (A-C′),Fgf17 (D-F′) andEn1 (G-I′) on sagittal sections ofEn1−/+ (A,D,G),En2−/− (B,E,H) andEn1−/+;En2−/− (C,F,I) E11.5 mutant embryos. Upper (A-I) and lower (A′-I′) panels show medial and lateral sections, respectively. TheFgf8/17 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 theFgf8 expression domain and is outlined in red. Red asterisks indicate truncation of the isthmus/inferior colliculus. r1, rhombomere 1; Ist, isthmus, mes, mesencephalon.
Fig. 8
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 forEn1 andEn2 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 inEn1 andEn2 gene expression. Cb, cerebellum; Mes, mesencephalon; SC, superior colliculus; IC, inferior colliculus; A, anterior; P, posterior; M, medial; L, lateral.
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