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First published online September 12, 2006
doi: 10.1242/10.1242/dev.02557

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Megane/Heslike is required for normal GABAergic differentiation in the mouse superior colliculus

¹ GSF-National Research Center for Environment and Health, Institute of Developmental Genetics, 35/8006, Ingolstädter Landstrasse, 1, 85764-Neuherberg, Germany.
² Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804-Munich, Germany.

View larger version (38K): [in a new window]   Fig. 1. Schematic representation of the MgntZ targeting vector and recombination at the Mgn locus. (A) Restriction map of the wild-type Mgn locus. Broken lines indicate regions of homology in the targeting vector. (B) Restriction map of the MgntZ targeting vector. (C) The predicted structure of a mutated Mgn allele following homologous recombination. The horizontal bars (5' and 3' external probe) indicate the DNA fragment used for Southern blot analysis. (D) Targeted locus after removing neomycin cassette (neo) by Cre-mediated excision at the loxP sites. (E) Southern blot analysis of restriction enzyme-digested DNA from targeted MgntZ (+/tZ) and wild-type (+/+) animals with 5' and 3' external probes (the same strategy was used to detect littermates from heterozygous intercrosses). wt and mt indicate the position of the wild-type and the mutated allele, respectively. (F) Southern blot containing genomic DNA upon SacII digestion and probed with an internal probe, indicating proper excision of the neomycin gene (neo). (G) Heteroduplex PCR (primers for the mutant allele, MM107 and tlacZ; primers for the wild-type allele, MM108 and MM109) used to identify mutated embryos/mice. (H) RT-PCR with specific primers for the bHLH domain of Mgn (MM111D and MM112R). The 250 bp band is missing in the MgntZ/tZ mice.

View larger version (93K): [in a new window]   Fig. 2. Colocalization analysis of Mgn expression with GABAergic markers in the developing mesencephalon. (A-H) Expression of Mgn (C,D) and Gad67 (E,F) in adjacent coronal (A,C,E,G) and sagittal (B,D,F,H) sections of E12.5 mouse embryos. (A,B) Bright-field images. (G,H) Superimposed images of the two adjacent sections hybridized in parallel with Mgn (in yellow) and Gad67 (in blue) indicate that Mgn and Gad67 are expressed in close vicinity but do not colocalize. At E12.5, Mgn is expressed in the ventricular zone underlying the mantle layer in which Gad65/67 expression arises. However, no overlapping of the two expression domains can be observed. This spatial relationship between the two expression domains is especially intriguing in the developing mesencephalon (G), but also detectable in other expression domains such as the ZLi (H). SC, superior colliculus; IC, inferior colliculus; vMb, ventral midbrain.

View larger version (38K): [in a new window]   Fig. 3. Cramping, growth characteristics and lethal phenotype. (A-C) Fore/hindlimb cramping phenotype in postnatal homozygous MgntZ/tZ mutant at postnatal day 20 (P20). Wild-type (A), homozygous Mgn knockout (B) and heterozygous Mgn knockout (C) mice. When lifted by their tail, MgntZ/tZ mice displayed an obvious fore-/hindlimb clenching phenotype. Neither wild-type nor heterozygous Mgn+/tZ mice show this phenotype. (D) Phenotypic comparison of Mgn+/+ and MgntZ/tZ mice at P32. Both animals were on C57BL/6 genetic background. (E) Survival curve of MgntZ/tZ mice. Days of survival of MgntZ/tZ (red line) compared with Mgn+/+ (green line) and Mgn+/tZ (blue line) mice. (F) Representative growth curve comparison between littermates. Mean body weight at age P0-P45 is represented by a green line (Mgn+/+), a blue line (Mgn+/tZ) and a red line (MgntZ/tZ) with error bars displaying the standard errors of the day-specific means. Arrows indicate the day of death for each of the MgntZ/tZ mice. The mean difference in weight between heterozygous and wild-type mice was 0.227 g (P=0.9475).

View larger version (88K): [in a new window]   Fig. 4. Complete depletion of GABAergic neurons in the SC of postnatal mice. (A-D) Gad65 expression in the postnatal superior colliculus (SC) of wild-type (A,C) and homozygous mutant mice (B,D). There is complete depletion of Gad65-expressing cells in the SC, whereas the ventral midbrain (vMb) and cortex are unaffected. Gad65-expressing cells in the inferior colliculus of homozygous mouse (D) are reduced compared with in the wild-type mouse (C). (E-H) Calbindin immunohistochemical staining of the SC of wild-type and homozygous mutant mice. The layers and patches characteristic of the cytoarchitecture of the superior colliculus are unaffected (E,F). (G,H) Higher magnification of the SC cytoarchitecture showing no obvious difference in the wild-type (G) and the MgntZ/tZ (H) mice. Abbreviations: Zo, zonal layer of the SC; SuG, superficial grey layer of the SC; Op, optic layer of the SC; InG, intermediate grey layer of the SC; InWh, intermediate white layer of the SC.

View larger version (101K): [in a new window]   Fig. 5. GABAergic neurons in the dorsal mesencephalon are not induced. In situ hybridization with the Gad67 riboprobe of coronal (A-I) and sagittal (J-O) sections, showing the mesencephalon of E10.5 (A-C), the anterior of E11.5 (D-F) and the caudal regions of E11.5 (G-I), 13.5 (J-L) and E18.5 (M-O) of wild-type (B,E,H,K,N) and MgntZ/tZ (C,F,I,L,O) embryos. (A,D,G,J,M) Bright-field images of wild type. (B,E) Gad67 expression is prominent in the mantle zone of the developing ventral midbrain of wild-type embryo. (C,F) Gad67 expression is reduced in the developing ventral midbrain of homozygous mutant mice, and dorsal expression is not detectable. Arrows in E,F indicate the presence (E) and absence (F) of dorsal Gad67-expressing cells. (H,I) Gad67 expression is also reduced in the caudal region of the mutant SC at E11.5. (K) Expression of Gad67 is present in the dorsal mesencephalic vesicle of wild-type (arrows), while the Gad67 expression in the mutant (L) is completely absent in the dorsal mesencephalic vesicle (arrows); it is also reduced in the pretectal area (arrowheads) and slightly reduced in the ventral midbrain. Gad67 expression in other areas of the brain (spinal cord, ventral hindbrain, cerebellum, thalamus, hypothalamus and the ventral telencephalon) is not affected. Arrows in K,L indicate the presence (K) and absence (L) of Gad67-expressing cells in the dorsal midbrain at E13.5 of the indicated genotype. (N-O) The mid/hindbrain region. Strong Gad67 expression can be observed in the SC of the wild-type mouse (N). (O) In the mutant mouse, Gad67 expression is completely absent in the dorsal midbrain, weaker in the ventral midbrain and not affected in the cerebellum and ventral hindbrain. Abbreviations: Cb, cerebellum; IC, inferior colliculus; SC, superior colliculus; vMB, ventral midbrain.

View larger version (63K): [in a new window]   Fig. 6. Cells that failed to be GABAergic neurons are present in the midbrain of MgntZ/tZ embryos. (A,B,D,E) Immunohistochemical staining of activated caspase 3 in the mesencephalic vesicle at E12.5. In the mesencephalic vesicle, no increase in apoptosis can be observed in the mutant. Single cells could be found labelled in the wild-type (Mgn+/+) (A,B) as well as in the mutant (MgntZ/tZ) in this region (D,E). (C,F) Immunohistochemical staining of NeuN in the developing SC at E13.5. There are no alterations in the thickness of either the ventricular zone or the mantle zone in the mutant mice (F). (G-J) No differences are observed in the PH3-positive cells in the wild-type (G, magnified in H) when compared with the homozygous mutant (I, magnified in J). (K-P) lacZ expression of E13.5 mouse Mgn+/tZ (K-M) and MgntZ/tZ (N-P) embryos. Whole-mount lacZ expression is more intense in the MgntZ/tZ mice (N), whereas localization is identical to that in Mgn+/tZ mice (K). lacZ expression in a coronal section of the SC from Mgn+/tZ (L) and MgntZ/tZ (O) embryos at E13.5. (M,P) Horizontal sections of the SC are magnified, showing single cells migrating out from the SVZ into the differentiation zone (arrows).

View larger version (57K): [in a new window]   Fig. 7. Other GABAergic and non-GABAergic markers are present in the SC of MgntZ/tZ embryos. In situ hybridization with Mgn, Pitx2 and Ebf2 probes in adjacent coronal (A-H) at E12.5, or with Gat1, Brn3a and Gad67 in sagittal (I-P) sections at E13.5 in wild-type and in MgntZ/tZ embryos. (A,B,I,J) Bright-field images. No changes for Pitx2 (E,F) and Ebf2 (G,H) could be observed when comparing wild-type and mutant mice. Mgn was absent in the mutant (compare C with D). At E13.5, no changes in the expression of Gat1 (K,L) or Brn3a (M,N) were detectable in the SC of the mutant when compared with the wild type, whereas the expression of Gad67 is totally abolished in the SC of the homozygous mutant mice (P, compare with O; see arrows).

View larger version (32K): [in a new window]   Fig. 8. Summary model of the Mgn expression and function regarding the specification of GABAergic neurons in the mouse SC. Mgn expression in the VZ/SVZ has an instructive role in the acquisition of GABAergic identity, as determined later on in the mantle zone by the appearance of two specific independent markers for GABAergic cells (Gad65 and Gad67) in the postmitotic neurons. Given the adjacent and non-overlapping expression domains between Mgn and Gad genes, it is likely that the Mgn transcription factor activates downstream gene(s) that, in turn, transactivate the Gad65 and Gad67 promoters.