QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

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

This Article Summary Figures Only Full Text (PDF) Supplementary Material Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guimera, J. Articles by Wurst, W. Search for Related Content PubMed PubMed Citation Articles by Guimera, J. Articles by Wurst, W.

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.

^* Authors for correspondence (e-mail: guimera{at}gsf.de; wurst{at}gsf.de)

Accepted 31 July 2006

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The mouse Mgn protein (Helt) is structurally related to the neurogenic Drosophila hairy and Enhancer of split [h/E(spl)] proteins, but its unique structural properties distinguish it from other members of the family. Mgn expression shows a spatiotemporal correlation with GABAergic markers in several brain regions. We report here that homozygous Mgn-null mice die between the second and the fifth postnatal week of age, and show a complete depletion of Gad65 and Gad67 expression in the superior colliculus and a reduction in the inferior colliculus. Other brain regions, as well as other neural systems, are not affected. The progenitor GABAergic cells appear to be generated in right numbers but fail to become GABAergic neurons. The phenotype of the mice is consistent with reduced GABAergic activity. Thus, our in vivo study provides evidence that Mgn is the key regulator of GABAergic neurons, controlling their specification in the dorsal midbrain. Another conclusion from our results is that the function of Mgn shows a previously unrecognized role for h/E(spl)-related transcription factors in the dorsal midbrain GABAergic cell differentiation. Vertebrate h/E(spl)-related genes can no longer be regarded solely as a factors that confer generic neurogenic properties, but as key components for the subtype-neuronal identity in the mammalian CNS.

Key words: bHLH transcription factor, GABAergic neurons, Gad65 (Gad2), Gad67 (Gad1), Megane (Heslike, Helt), Superior colliculus, Midbrain, Specification, Mouse

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The GABAergic neurons are the principal inhibitory neurotransmitter system in the mammalian central nervous system (CNS) and their impairment is involved in the aetiology of several neurological disorders. At the molecular level, much of our knowledge about their specification comes from cerebral cortical developmental studies. In the forebrain, these neurotransmitteridentified neurons arise from proneural bHLH Mash1 (Ascl1 - Mouse Genome Informatics)-expressing progenitors in the subventricular zone (SVZ) of the ganglionic eminence and migrate tangentially into the cerebral cortex (Corbin et al., 2001; Parnavelas, 2000). Loss-of-function studies suggest a key role for the Nkx2.1 and Dlx1/2 homeobox gene families: providing the positional information needed to commit the undifferentiated subpallial progenitor cells into a GABAergic interneuron precursor (Ross et al., 2003). The transcription factor Ptf1a is also associated with specification of GABAergic neurons in the cerebellum (Hoshino et al., 2005). By contrast, the molecular mechanisms underlying the development of midbrain GABAergic neurons remains elusive because, among other reasons, most of the players mentioned above are not expressed in the midbrain, which suggests different pathways towards this cell fate.

bHLH transcription factors comprise an evolutionarily ancient group of proteins conserved across many species that mainly function as key regulators of cell-fate decisions and neuronal differentiation in the developing CNS of invertebrates and vertebrates (Jan and Jan, 1994; Kageyama and Nakanishi, 1997; Lee, 1997; Guillemot, 1999; Bertrand et al., 2002). In Drosophila, a most prominent group of neurogenic bHLH factors are the members of the Drosophila hairy and Enhancer of split [E(spl)] family. These proteins function as transcriptional repressors to maintain neural progenitors in a proliferative state and generally antagonize the activity of proneural bHLH proteins (e.g. Mash1, neurogenin 1, neurogenin 2 and neurogenin 3), promoting or suppressing neuronal determination-differentiation programs (Heitzler and Simpson, 1991; Kageyama and Nakanishi, 1997; Fisher and Caudy, 1998). In mammals, accumulating evidence suggests that, as in Drosophila, neurogenesis also relies upon h/E(spl)-related factors and proneural factors, and is associated with comparable functions (Davis and Turner, 2001). Recent evidence suggests that vertebrate proneural bHLH genes not only confer generic neuronal properties, but also play a crucial role as determinants of neuronal identity, controlling the appearance of cell type-specific traits (Brunet and Ghysen, 1999; Parras et al., 2002; Nakada et al., 2004). However, it has not been demonstrated so far whether h/E(spl) genes can also promote neuronal subtype identities.

We isolated a new member of the h/E(spl)-related bHLH protein referred to as megane (Mgn; Helt - Mouse Genome Informatics) (Guimera et al., 2006; Miyoshi et al., 2004; Nakatani et al., 2004). Mgn shows an expression pattern restricted to the midbrain at mouse developmental day 9.5 (E9.5). Mgn mRNA displays spatiotemporal transcriptional correlation with that of 65 kDa and 67 kDa isoforms of glutamic acid decarboxylase (Gad65 and Gad67, respectively; Gad2 and Gad1 - Mouse Genome Informatics), which are two independent biosynthetic enzymes for the neurotransmitter GABA (Erlander et al., 1991; Erlander and Tobin, 1991). A neuron is GABAergic if it contains either isoform of GAD (the rate-limiting enzyme in GABA synthesis).

Gain-of-function experiments suggest a possible involvement of Mgn in GABAergic neurogenesis (Miyoshi et al., 2004); however, the processes requiring Mgn in vivo as well as the involvement of Mgn in any specific GABAergic lineage remained unknown. To address this issue, we have performed loss-of-function studies using a gene targeting approach. Homozygous Mgn mutant mice show normal brain histology and morphology. However, expression of Gad65 and Gad67 is completely abolished in the superior colliculus (SC), and homozygous mutant animals display a cramping phenotype and symptoms resembling tonic-clonic seizures preceding postnatal death. Thus, our results demonstrate a key role of Mgn in the proper development of GABAergic neurons of the SC and, in addition, suggest a vital role of Mgn in early postnatal survival. Hence, we designated this bHLH gene as Mgn (mesencephalic GABAergic neurons h/E(spl)-related gene).

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction of an Mgn targeting vector and production of Mgn mutant mice
An Mgn gene targeting vector (Fig. 1B) was designed to delete the bHLHO domain (part of exon 2 and exon 3). Mgn genomic fragments were cloned from a 129SV-derived BAC clone. The targeting vector contains a 1.8 kb fragment of 5' homologous genomic DNA and a 5.2 kb fragment of 3' homologous genomic DNA flanking the deletion. A tau-lacZ (tZ) fusion protein was inserted in frame with the 12th codon in exon 2, followed by a neomycin cassette flanked by two loxP sites for subsequent removal by Cre recombinase-mediated excision. The plasmid was linearized with SpeI, electroporated into embryonic stem cells (129SVderived) and selected with G418. Genomic DNA was isolated from surviving ES cells, digested with BamHI and screened by Southern blot with 5' and 3' external probes, in parallel experiments. Two correctly targeted ES cell clones were identified out of 1360 ES clones analysed, then injected individually into blastocysts derived from C57BL/6 mice to produce chimaeric mice. Chimaeric pups were identified by their agouti coat colour. Chimaera males were bred to C57BL/6 and 129SV/J females and germline transmission was determined by Southern blot analysis of tail genomic DNA (Fig. 1E,F) as described above. Later, a PCR-based strategy was designed to facilitate the genotyping of the progeny. Primers for the wild-type band were MM108 and MM109, whereas primers for the mutant allele were MM107 and tlacZ. The PCR assay generated a 334 bp fragment for the wild-type allele and a 395 bp fragment for the mutant allele (Fig. 1G). Null allele animals were confirmed by the absence of the PCR fragment when specific primers for the deletion were used (MM111D and MM112R) (Fig. 1H). The mutation is maintained on both a 129SV/J and C57BL/6 genetic background. The neo-selectable marker was removed by crossing mice heterozygous for the Mgn floxed neo allele with C57BL/6 inbred CMV-Cre transgenic mouse line (Schwenk et al., 1995). The resulting progeny were analysed for the excision of the neo cassette by Southern blot (Fig. 1F). The animal experiments were conducted under federal guidelines for the use and care of laboratory animals and were approved by the GSF Institutional Animal Care and Use Committee.

Whole mount in situ hybridization and lacZ staining
Embryos were obtained from natural matings of wild-type C3H mice. Noon on the day when the vaginal plug was detected was considered to be embryonic day 0.5 of gestation (E0.5). Embryos were collected and staged precisely according to Theiler (Theiler, 1989). Embryos and dissected brains from different stages (E9-E14) were fixed overnight at 4°C in 4% paraformaldehyde. Whole-mount in situ hybridization was performed as described by Sporle and Schughart (Sporle and Schughart, 1998). Antisense and sense digoxigenin (DIG)-labelled riboprobes transcribed from linearized plasmids containing a partial cDNA for Brn3a (Pou4f1 - Mouse Genome Informatics) (bp 614-938; XM_135366); Chat1 (Slc5a7 - Mouse Genome Informatics) (bp 27-1769; NM_022025); Dat (Slc6a3 - Mouse Genome Informatics) (bp 2332-2725; AF109072); Ebf2 (bp 653-1195; BC050922); Gad65 (Gad2 - Mouse Genome Informatics) (bp 753-1600; BC018380); Gad67 (Gad1 - Mouse Genome Informatics) (bp 934-1786; NM_008077); Gat1 (Slc6a1 - Mouse Genome Informatics) (bp 1381-2053; M92378); islet 1 (Isl1) (bp 1013-1322; NM_021459); Mgn (Helt - Mouse Genome Informatics) (bp 166-1000, DQ294234); Pitx2 (bp 1172-1863; NM_011098); Plp1 (bp 106-857; AK077564); Th (bp 23-788; NM_009377); and Vglut1 (Slc17a7 - Mouse Genome Informatics) (bp 1720-2332; NM_182993) were produced using a DIG-RNA labelling kit (Roche). lacZ expression was monitored by a colour reaction catalysed by the lacZ gene product ß-galactosidase as described (Gossler and Zachgo, 1993).

Immunohistochemistry
For immunohistochemistry, tissue sections were stained overnight with the following primary antibodies: rabbit anti-phospho-histone H3 (1:500; Upstate Biotechnology), which recognizes only M-phase nuclei as a mitosis marker (Mahadevan et al., 1991; Ajiro et al., 1996); anti-NeuN (specific marker for neuronal nuclei) (1:1000, Chemicon); anti-calbindin (1:2000; Swant); and anti-cleaved caspase 3 (1:200; Cell Signaling), which recognizes cleaved caspase 3 cell death protease activated during apoptosis (Krajewski et al., 1997). Subsequently sections were incubated with a biotinylated goat-anti-rabbit antibody (1:300, Dianova) followed by incubation with an HRP-coupled ABC complex according to the manufacturer's protocol (Vector Laboratories). Staining was visualized using diaminobenzidine as a chromogen.

Histological analysis
Brains and whole embryos were obtained from animals that were transcardially perfused with 4% paraformaldehyde, then fixed by immersion in 4% paraformaldehyde overnight at 4°C. Some of the postnatal brains were shock frozen on dry ice. Perfused brains were either cut on a cryostat at 30 µm or paraffin embedded and cut on a microtome at 4-8 µm. Frozen brains were cut on a cryostat at 18 µm and processed for in situ hybridization. In situ hybridization of frozen and paraffin sections was performed according to a modified version of the procedure described by Dagerlind et al. (Dagerlind et al., 1992). Following in situ hybridization, sections were counterstained with Cresyl Violet.

Oligonucleotides
MM107 (5'-cagagactggaaggagagtccttg-3'); MM108 (5'-gcggaatgcagagctctgag-3'); MM109 (5'-gaacgagctgggcaagacag-3'); MM111D (5'-cccggtttctcataaagtgatag-3'); MM112R (5'-gcactcgtggtaaccgtagtg-3'); and tlacZ (5'-agcatgatcttccatcacgtcg-3') oligonucleotides were used. Mgn cDNA was taken from GenBank (Accession Number DQ294234).

BrdU cell proliferation assay and TUNEL assay
Pregnant females were intraperitoneally injected with BrdU 15 minutes and 2 hours before they were sacrificed. Incorporation and detection of BrdU into cellular DNA were carried out with BrdU labelling and detection kit II (Roche), according to the manufacturer's instructions. For the TUNEL assay, paraffin sections of midgestational embryos were dewaxed and apoptosis was detected using In Situ Cell Death Detection Kit, Fluorescein (Roche) according to the manufacturer's instructions.

Statistical analysis
To compare the body weight between wild-type and heterozygous mice, the autocorrelation of the data from several mice for each mouse line was taken into account. A weighted linear model was fitted (weighted ANOVA with AR₁-autocorrelated errors) using the gls (generalized least squares) function in the nlme-package. The R statistical software was used to perform the statistical analysis (Pinheiro and Bates, 2000).

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Production of mutant mice
An Mgn gene targeting vector was designed to replace the entire bHLH and the nuclear localization signal domains with a tau-lacZ fusion protein by homologous recombination (Fig. 1A-D). Two targeted ES cell clones gave rise to virtually 100% chimaeric mice that transmitted the mutant allele into the germline (50% of descendents) by mating chimaeric mice with both C57BL/6 and 129SV/J mice. The resulting heterozygous mice (Mgn^+/tZ) generated from those two independently targeted ES clones were viable, fertile and not distinguishable from wild-type littermates. The Mgn mutant mouse was consecutively back crossed 11 generations to C57BL/6. Corresponding homozygous mutant mice were produced and analysed. RT-PCR analysis revealed that Mgn full-length mRNA is not present in the brains of Mgn^tZ/tZ mice (Fig. 1H). Next, Mgn expression was determined by in situ hybridization on mouse embryo sections. Mgn mRNA was detected in wild-type mice, but not in Mgn^tZ/tZ mice (Fig. 7C,D). The gene targeting strategy used in creating the Mgn^tZ allele deletes intron II. Thus, it is possible that the lack of this intronic sequence could disrupt a transcriptional regulatory element. To examine transcription from the Mgn locus, lacZ staining was performed at different midgestational times on Mgn mutant embryos and compared with the Mgn expression in wild-type embryos, as determined by in situ hybridization. The findings suggest that the tlacZ transcripts are essentially expressed in an Mgn-specific pattern in Mgn heterozygous and homozygous mutant embryos. Thus, this mouse provides a marker that reflects the endogenous expression of Mgn.

View larger version (38K): [in this window] [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 this window] [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.

After birth, during the first postnatal week, newborn Mgn^tZ/tZ mutant mice appeared healthy: the stomach and the intestine of homozygous mutants were filled with milk, pups were of similar size and weight to wild-type and heterozygous Mgn^+/tZ littermate controls, and they displayed coordinated movements of the limbs and trunk without apparent neurological abnormalities. During the second week, the homozygous mutant mice displayed a gradual growth reduction, and the first deaths of mice were observed. Three to 4 weeks after birth, the body weight of those homozygous mutants that survived was approximately one-third that of wild-type littermate controls (Fig. 3D,F) because they did not nurse properly. Deaths continued to be observed during this week and no homozygous mutant mouse survived beyond 5 weeks (Fig. 3E). An initial examination at around P12 of the behaviour of Mgn^tZ/tZ mice revealed the first signs of neurological impairment. Homozygous mutant mice retracted their fore/hindlimbs and digits when suspended by their tail - in contrast to wild-type mice, which extend them (Fig. 3A-C). Fore/-hindlimb clenching was consistently observed in all homozygous Mgn^tZ/tZ knockout mice, but not in the wild-type or heterozygous Mgn^+/tZ mice. Occasional convulsions in mice over 14 days of age preceded by a wild running phase, forelimb tonic-clonic spasms with hyperextended hindlimbs episodes, were observed in the homozygous mutant mice prior death, resembling a seizure-like phenotype. No spontaneous convulsions were observed in Mgn^+/tZ mice.

Mgn is essential for the development of GABAergic neurons of the SC
To ascertain the cause of death of Mgn^tZ/tZ mice, mutants and controls were prepared for morphological examination. The analysis of both embryonic and postnatal brain revealed no gross morphological alterations. Nissl and Luxol Fast Blue stains showed similar staining patterns in wild-type and homozygous mutant mice (data not shown). Given the expression of Mgn in the VZ/SVZ of the embryonic CNS and its striking spatiotemporal correlation with Gad65 and Gad67 markers (Fig. 2), we next addressed whether the expression of Mgn in the SVZ underlying GABAergic neurons has a role in the neuronal specification of this neurotransmitter population.

During postnatal stages, we found a complete loss of Gad65 and Gad67 expression in the SC of the mutant Mgn^tZ/tZ mice, indicating a loss of GABAergic neurons (Fig. 4A,B; see Fig. S1B,E in the supplementary material). In the inferior colliculus (IC), a partial but not a complete absence of Gad65 and Gad67 expression was observed (Fig. 4C,D; data not shown). This lack of GABAergic neurons takes places in the most rostral and most caudal part of the IC (see Fig. S1B,E in the supplementary material), where Mgn is normally expressed (Guimera et al., 2006). Strikingly, in the ventral part of the postnatal mesencephalon and in other brain areas outside the colliculi, no obvious changes of the GABAergic neurons could be detected as determined by the presence of Gad65 and Gad67 expression (see Fig. S1A-F in the supplementary material; data not shown). The cytoarchitecture of the SC appeared to be normal, as evidenced by immunohistochemical staining against the three Ca²⁺-binding proteins (parvalbumin, calretinin and calbindin), which still revealed the typical layered and patched structure of the SC (Fig. 4E-H; data not shown). Mgn^+/tZ animals were undistinguishable from wild type, indicating that one wild-type allele of Mgn is sufficient to promote Gad65 and Gad67 expression and a GABAergic phenotype.

These findings prompted us to address whether the induction of GABAergic neurons was affected. Analysis during early mouse embryonic development of Mgn^tZ/tZ mutant brains (Fig. 5A-L) and later embryonic stages (E18.5; Fig. 5M-O) also showed a complete lack of Gad65 and Gad67 expression in the SC when compared with wild-type or heterozygous brains. By contrast, Gad65 and Gad67-expressing cells are less numerous in the ventral aspect of the midbrain at E10.5-E12.5. Therefore, Mgn is the key regulator necessary for the proper specification of GABAergic neurons in the dorsal midbrain, as Gad65 and Gad67 are never expressed in the SC at any stage during development or postnatal brains.

View larger version (38K): [in this window] [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).

Furthermore, no changes in the density of differentiated neurons within the mantle zone of the developing SC of mutant animals could be detected using the marker NeuN, which is a specific marker for post-mitotic neurons in vertebrates (Fig. 6C,F). We could also detect no changes in the expression of Ebf2 (a general marker for neuronal maturation). In addition, the expression of Pitx2 [the expression of which essentially overlaps with that of Gad65 and Gad67 in the developing basal mesencephalon and SC (Katarova et al., 2000)], of Ca²⁺-binding proteins (which mark subpopulations of GAD expressing cells in differentiated GABAergic neurons of the SC) and of GABA_A transporter 1 (Gat1) was not altered (Fig. 7; see Fig. S1G-I in the supplementary material). These results indicate that the Mgn-expressing cells derived from the ventricular zone of the dorsal midbrain, which in the wild-type situation give rise to GABAergic neurons, are still present in the mutant but no longer express Gad65 and Gad67, and therefore are not capable of producing GABA. This observation is supported by the fact that the Gad65/Gad67 double-knockout mice completely lack GABA (Ji et al., 1999).

View larger version (88K): [in this window] [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.

Non-GABAergic systems are not disturbed in Mgn^tZ/tZ mice
As the lack of Mgn could have an effect in other neural lineages, we next investigated other neural subpopulations that, during development, arise close to the Mgn expression domain. To analyse an effect in the dopaminergic system, we studied the expression of Th, Dat, Pitx3 and Nurr1 (Nr4a2 - Mouse Genome Informatics) in embryos and postnatal tissue. The results revealed that all these four dopaminergic markers have no apparent change in expression in the Mgn^tZ/tZ mutants (see Fig. S3E-J in the supplementary material; data not shown). Other non-GABAergic cell types, such as glutamatergic or cholinergic neurons, did not appear to be disturbed in the homozygous mutant mice, as determined by expression of the markers vesicular glutamatergic transporter 1/2 (Vglut1/2) and choline acetyltransferase transporter (Chat1), respectively (see Fig. S1J-L,M-O in the supplementary material; data not shown). Furthermore, we examined the oligodendrocytes, as it is known that the oligodendrocyte lineage is dependent on Nkx2.2, the expression of which overlaps with Mgn expression in the midbrain as well as in the ZLI (Guimera et al., 2006). We found that the oligodendrocyte system is not altered in the homozygous mutant brains at P15 using Plp1, Olig1 and Olig2 as markers (see Fig. S3K-M in the supplementary material; data not shown).

In addition, we did not observe any changes in the morphology of other midbrain regions, such as the red nucleus (marked by Brn3a) and the nuclei occulomotorius (marked by Isl1) (Fig. 7M,N; see Fig. S3A-D in the supplementary material). Altogether, we conclude that Mgn does not have any general patterning capacities in the midbrain; rather its function seems to be crucial for the acquisition of the neuronal GABAergic neurotransmitter subtype.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We have identified and characterized a bHLH factor referred to as megane (Mgn) related to the Drosophila h/E(spl) proteins (Guimera et al., 2006). In this study, we describe the function of Mgn in vivo using a knockout approach in mouse embryonic stem cells (ES). Homozygous Mgn^tZ/tZ mutants are postnatal lethal between the second and fifth week of age. Histological analysis of the brain did not reveal any alteration. However, using GABAergic neuron specific markers, we could show that Mgn plays a key role controlling the acquisition of GABAergic neuronal identity in the SC.

Mgn protein and GABAergic neurogenesis
Expression of Mgn mRNA shows a specific and dynamic expression pattern in the embryonic CNS in a region-specific manner. In the midbrain, Mgn is expressed at a high level in the ventricular zone, next to the lumen, where neuroepithelial germinal cells proliferate to generate neural precursors, but it decreases rapidly as neural differentiation proceeds. These observations suggest Mgn might control early steps in the specification and/or differentiation of particular neural lineages in the CNS, rather than their maturation or functional maintenance. The developmentally controlled specific expression pattern of Mgn prompted us to investigate its correlation with GABA neurotransmitter markers and its function in cell differentiation. As determined by comparative expression analysis of in situ hybridization experiments on consecutive sections, Mgn mRNA expression was detected in a spatiotemporal correlation with Gad65- and Gad67-expressing cells. Gad65 and Gad67 expression appears when differentiating cells have migrated from the ventricular to the mantle layer. Mgn expression ceases once Gad65 and Gad67 expression becomes prominent in the mantle layer. Thus, Mgn and Gad expression are adjacent and not co-expressed. Expression of Mgn may demarcate neurogenic regions that give rise to GABAergic neurons. In postnatal brain, after neuronal differentiation has been completed, Mgn expression is strongly reduced in the CNS, and only maintained in scattered cells surrounding the lateral ventricles and olfactory ventricles.

View larger version (101K): [in this window] [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 this window] [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 this window] [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).

The presence of: (1) early neurogenesis markers (Mash1, Ngn1/2/3, Hes5 and Hey2); (2) neuronal markers (Ebf2 and NeuN); (3) GABAergic markers (Pitx2 and Gat1); and (4) lacZ-positive cells in the in the SC of homozygous mutant brains compared with their control littermates, as well as apoptosis and proliferation assays, indicates that Mgn-lacZ expressing cells are present in the Mgn^tZ/tZ mutants and do not undergo apoptosis, despite the fact that they do not express Gad65 and Gad67. The partial loss of dorsal midbrain GABAergic identity in the Mgn^tZ/tZ mice occurs from the onset of neuronal differentiation and is not due to alterations in cell death or cell proliferation during development. These results demonstrate that fully neural GABAergic identity specification failed in the SC of the Mgn^tZ/tZ mutants. Presumptive GABAergic neurons lack their fully GABA identity, without the gain of an alternative neurotransmitter phenotype during embryonic and postnatal stages, retaining some GABAergic markers such as Gat1 and Pitx2. Despite the hypothetical fact that a few postsynaptic membranes (p.e. astrocytes and glutamatergic neurons) may express Gat1 in the midbrain is still not reported, the observation that Gat1 expression is not reduced in the dorsal midbrain of the mutant mice suggests that the presumptive GABAergic neurons (the vast majority of Gat1-expressing cells in the wild-type midbrain - if not all) still express Gat1 in the mutant. This suggests that different pathways are involved in specific aspects of GABAergic fate and Mgn impairment is not sufficient to produce a neurotransmitter identity switch.

Interestingly, Mgn and Gad mRNAs are not co-expressed, suggesting that Mgn does not regulate the expression of Gad genes directly. Alternatively, it is likely that Mgn induces expression of unknown transcription factors in the ventricular zone that might, in turn, regulate, among others, the expression of Gad65 and Gad67 in the mantle zone (Fig. 8).

View larger version (32K): [in this window] [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.

The most likely explanation for this difference is that other transcription factors in the ventral midbrain, e.g. MashI (which is not misregulated in the Mgn^tZ/tZ mice; data not shown), are sufficient to compensate for the loss of Mgn in the ventral, but not in the dorsal, midbrain. Alternatively, Mgn and Mash1 proteins may collaborate with other transcription factors that modify their activity in controlling distinct genetic pathways for dorsoventral midbrain GABAergic neuron development. These observations support the hypothesis that the neuronal differentiation activity of the bHLH factors (e.g. Mash1, Ptf1a and Mgn) is strongly dependent on the regional and cellular context, and proneural information is combined with positional information to regulate different downstream genes to control the specification and differentiation of GABAergic neurons in distinct brain regions (Brunet and Ghysen, 1999; Bertrand et al., 2002; Miyoshi et al., 2004; Hoshino et al., 2005).

Mgn and mammalian neurogenesis
Mammalian neurogenesis depends essentially upon the balance between repressors of neurogenesis [represented mainly by the h/E(spl) factors] and activators of neurogenesis (represented mainly by the proneural genes). The level of expression of any of these two types of bHLH factors over the other one will determine whether the cells stay in an undifferentiated state or proceed with their cell-fate program (reviewed by Ross et al., 2003).

Despite the large number of transcription factors identified in the last years, vertebrate h/E(spl)-related genes with a specific role in the acquisition of neuronal identity have been shadowed by the role of the proneural genes. A major conclusion from our results is that h/E(spl) mammalian members are not only involved in controlling the proper number of precursor cells, but also in the acquisition of neuronal identity. Vertebrate h/E(spl)-related genes can no longer be regarded solely as a factors that confer generic neurogenic properties, but are also key components for the subtype-neuronal identity in the mammalian CNS.

The general function of h/E(spl) genes has been conserved from Drosophila to vertebrates. However, the unique expression pattern and the function of Mgn in vivo shows a previously unrecognized role for h/E(spl)-related genes in midbrain GABAergic cell differentiation. We failed to find any orthologue of Mgn in silico or by screening Drosophila cDNA libraries (Guimera et al., 2006). The molecular mechanisms in mammalian neurogenesis are largely controlled by genes related to those involved in Drosophila neurogenesis. However, neuronal development in mammals is still poorly understood compared with that of Drosophila, because, among other reasons, of the appearance of novel genes during vertebrate evolution that permit the addition of new functions during brain development. Therefore, bHLH mammalian factors with neuronal identity specification/differentiation activity without homologous counterparts in Drosophila should provide novel clues for understanding the genetic pathways of vertebrate neuronal development.

Supplementary material
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/19/3847/DC1

	ACKNOWLEDGMENTS

 
We thank L. Bally-Cuif, N. Prakash, J. Graw and A. Wizenmann for critical reading of the manuscript; S. Hoelter and D. Echevarria for helpful comments and suggestions during the course of this research; J. Favor for editing; T. Faus-Keßler for statistical analysis; and J. Rubenstein, A. Simeone and A. Wanaka for the Islet1, Brn3a and Chat1 plasmids. We also thank S. Promoli, S. Bourier and J. Bergold for excellent technical assistance. J. Guimera acknowledges support from an EU Marie-Curie fellowship. This work has been funded by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN), Förderkennzeichen 01GS0476 (W.W.), the BMBF Förderkennzeichen 01GN0512 (W.W.) and by European Union (Eumorphia) grants to W.W. The authors are responsible for the contents of this publication and declare that they have no competing financial interest.

	REFERENCES

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

Ajiro, K., Yoda, K., Utsumi, K. and Nishikawa, Y. (1996). Alteration of cell cycle-dependent histone phosphorylations by okadaic acid. Induction of mitosis-specific H3 phosphorylation and chromatin condensation in mammalian interphase cells. J. Biol. Chem. 31,13197 -13201.

Anderson, S. A., Eisenstat, D. D., Shi, L. and Rubenstein, J. L. (1997). Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes. Science 17,474 -476.

Asada, H., Kawamura, Y., Maruyama, K., Kume, H., Ding, R., Ji, F. Y., Kanbara, N., Kuzume, H., Sanbo, M., Yagi, T. et al. (1996). Mice lacking the 65 kDa isoform of glutamic acid decarboxylase (GAD65) maintain normal levels of GAD67 and GABA in their brains but are susceptible to seizures. Biochem. Biophys. Res. Commun. 24,891 -895.

Asada, H., Kawamura, Y., Maruyama, K., Kume, H., Ding, R. G., Kanbara, N., Kuzume, H., Sanbo, M., Yagi, T. and Obata, K. (1997). Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc. Natl. Acad. Sci. USA 10,6496 -6499.

Bertrand, N., Castro, D. S. and Guillemot, F. (2002). Proneural genes and the specification of neural cell types. Nat. Rev. Neurosci. 3, 517-530.[CrossRef][Medline]

Brunet, J. F. and Ghysen, A. (1999). Deconstructing cell determination: proneural genes and neuronal identity. BioEssays 21,313 -318.[CrossRef][Medline]

Condie, B. G., Bain, G., Gottlieb, D. I. and Capecchi, M. R. (1997). Cleft palate in mice with a targeted mutation in the gamma-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67. Proc. Natl. Acad. Sci. USA 14,11451 -11455.

Corbin, J. G., Nery, S. and Fishell, G. (2001). Telencephalic cells take a tangent: non-radial migration in the mammalian forebrain. Nat. Neurosci. 4,1177 -1182.

Dagerlind, A., Friberg, K., Bean, A. J. and Hokfelt, T. (1992). Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry 98,39 -49.[CrossRef][Medline]

Davis, R. L. and Turner, D. L. (2001). Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20,8342 -8357.[CrossRef][Medline]

Erlander, M. G. and Tobin, A. J. (1991). The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem. Res. 16,215 -226.[CrossRef][Medline]

Erlander, M. G., Tillakaratne, N. J., Feldblum, S., Patel, N. and Tobin, A. J. (1991). Two genes encode distinct glutamate decarboxylases. Neuron 7, 91-100.[CrossRef][Medline]

Fisher, A. and Caudy, M. (1998). The function of hairy-related bHLH repressor proteins in cell fate decisions. BioEssays 20,298 -306.[CrossRef][Medline]

Gossler, A. and Zachgo, J. (1993). Gene and enhancer trap screens in ES cell chimaeras. In Gene Targeting: A Practical Approach (ed. A. Joyner), pp.181 -227. Oxford: Oxford University Press.

Guillemot, F. (1999). Vertebrate bHLH genes and the determination of neuronal fates. Exp. Cell Res. 15,357 -364.

Guimera, J., Vogt Weisenhorn, D., Echevarria, D., Martinez, S. and Wurst, W. (2006). Molecular characterization, structure and developmental expression of Megane bHLH factor. Gene 377,65 -76.[CrossRef][Medline]

Heitzler, P. and Simpson, P. (1991). The choice of cell fate in the epidermis of Drosophila. Cell 22,1083 -1092.

Homanics, G. E., DeLorey, T. M., Firestone, L. L., Quinlan, J. J., Handforth, A., Harrison, N. L., Krasowski, M. D., Rick, C. E., Korpi, E. R. and Makela, R. (1997). Mice devoid of gamma-aminobutyrate type A receptor beta3 subunit have epilepsy, cleft palate, and hypersensitive behavior. Proc. Natl. Acad. Sci. USA 15,4143 -4148.

Hoshino, M., Nakamura, S., Mori, K., Kawauchi, T., Terao, M., Nishimura, Y., Fukuda, A., Fuse, T., Matsuo, N. and Sone, M. (2005). Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum. Neuron 21,201 -213.

Jan, Y. N. and Jan, L. Y. (1994). Genetic control of cell fate specification in Drosophila peripheral nervous system. Annu. Rev. Genet. 28,373 -393.[CrossRef][Medline]

Ji, F., Kanbara, N. and Obata, K. (1999). GABA and histogenesis in fetal and neonatal mouse brain lacking both the isoforms of glutamic acid decarboxylase. Neurosci. Res. 33,187 -194.[CrossRef][Medline]

Kageyama, R. and Nakanishi, S. (1997). Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system. Curr. Opin. Genet. Dev. 7, 659-665.[CrossRef][Medline]

Katarova, Z., Sekerkova, G., Prodan, S., Mugnaini, E. and Szabo. (2000). Domain-restricted expression of two glutamic acid decarboxylase genes in midgestation mouse embryos. J. Comp. Neurol. 4,607 -627.

Krajewska, M., Wang, H. G., Krajewski, S., Zapata, J. M., Shabaik, A., Gascoyne, R. and Reed, J. C. (1997). Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease. Cancer Res. 15,1605 -1613.

Lee, J. E. (1997). Basic helix-loop-helix genes in neural development. Curr. Opin. Neurobiol. 7, 13-20.[CrossRef][Medline]

Mahadevan, L. C., Willis, A. C. and Barratt, M. J. (1991). Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors. Cell 31,775 -783.[CrossRef]

Miyoshi, G., Bessho, Y., Yamada, S. and Kageyama, R. (2004). Identification of a novel basic helix-loop-helix gene, Heslike, and its role in GABAergic neurogenesis. J. Neurosci. 7,3672 -3682.

Nakada, Y., Hunsaker, T. L., Henke, R. M. and Johnson, J. E. (2004). Distinct domains within Mash1 and Math1 are required for function in neuronal differentiation versus neuronal cell-type specification. Development 131,1319 -1330.[Abstract/Free Full Text]

Nakatani, T., Mizuhara, E., Minaki, Y., Sakamoto, Y. and Ono, Y. (2004). Helt, a novel basic-helix-loop-helix transcriptional repressor expressed in the developing central nervous system. J. Biol. Chem. 16,16356 -16367.

Parnavelas, J. G. (2000). The origin and migration of cortical neurones: new vistas. Trends Neurosci. 23,126 -131.[CrossRef][Medline]

Parras, C. M., Schuurmans, C., Scardigli, R., Kim, J., Anderson, D. J. and Guillemot, F. (2002). Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity. Genes Dev. 1,324 -338.

Pinheiro, J. C. and Bates, D. M. (2000). Mixed-Effects Models in S and S-Plus. New York: Springer-Verlag.

Ross, S. E., Greenberg, M. E. and Stiles, C. D. (2003). Basic helix-loop-helix factors in cortical development. Neuron 3,13 -25.

Schwenk, F., Baron, U. and Rajewsky, K. (1995). A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 25,5080 -5081.

Sporle, R. and Schughart, K. (1998). Paradox segmentation along inter- and intrasomitic borderlines is followed by dysmorphology of the axial skeleton in the open brain (opb) mouse mutant. Dev. Genet. 22,359 -373.[CrossRef][Medline]

Tamamaki, N., Fujimori, K. E. and Takauji, R. (1997). Origin and route of tangentially migrating neurons in the developing neocortical intermediate zone. J. Neurosci. 17,8313 -8323.[Abstract/Free Full Text]

Theiler, K. (1989). The House Mouse: Atlas of Mouse Development. New York: Springer-Verlag.

Tsunekawa, N., Yanagawa, Y. and Obata, K. (2005). Development of GABAergic neurons from the ventricular zone in the superior colliculus of the mouse. Neurosci. Res. 51,243 -251.[CrossRef][Medline]

Verney, C., Zecevic, N. and Puelles, L. (2001). Structure of longitudinal brain zones that provide the origin for the substantia nigra and ventral tegmental area in human embryos, as revealed by cytoarchitecture and tyrosine hydroxylase, calretinin, calbindin, and GABA immunoreactions. J. Comp. Neurol. 1, 22-44.

Related articles in Development:

Megane: a basic requirement for GABAergic identity

Development 2006 133: e1905. [Full Text]  

This article has been cited by other articles:

				K. Hori, J. Cholewa-Waclaw, Y. Nakada, S. M. Glasgow, T. Masui, R. M. Henke, H. Wildner, B. Martarelli, T. M. Beres, J. A. Epstein, et al. A nonclassical bHLH Rbpj transcription factor complex is required for specification of GABAergic neurons independent of Notch signaling Genes & Dev., January 15, 2008; 22(2): 166 - 178. [Abstract] [Full Text] [PDF]

				T. Nakatani, Y. Minaki, M. Kumai, and Y. Ono Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon Development, August 1, 2007; 134(15): 2783 - 2793. [Abstract] [Full Text] [PDF]

This Article Summary Figures Only Full Text (PDF) Supplementary Material Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar