Y2H screening and 2-protein interaction assays

The Y2H screen, yeast strains, vectors, growth media, and standard methods for manipulating yeast cells were performed according to the manufacturer’s recommendations (Matchmaker GAL4, Clontech) with the following steps: for large-scale screening, the Mgn coding sequence (GenBank accession no. DQ294234) was subcloned into a pGBKT7 plasmid (tryptophan, Trp⁺) to express to a transcription factor-binding domain (BD) of the GAL4-DNA vector (bait placed at the C-terminus). A ds-cDNA library was directionally subcloned into the C-terminus of the pGADT7 activator domain (AD) vector (leucine, Leu⁺) using mRNA from E9–E10.5 mouse embryos (Matchmaker library construction kit, Clontech). The 2 Saccharomyces cerevisiae yeast strains used in the screen, AH109 and Y187, were auxotrophic for several amino acids (Trp; Leu; histidine, His; and adenosine, Ade^-). The AH109 strain transformed with BD-MGN fusion protein did not grow on SD/His3⁻ plates and was negative for β-gal activity, excluding the possibility of host autoactivation.

BD-MGN and all bait plasmids used for the Y2H and 2-protein interaction screen appeared not to be toxic to the yeast cells when plated on SD/Trp⁻ plates. Therefore, BD-MGN plasmids were used to screen our primary cDNA library (not amplified). Basal His3 gene expression was inhibited with 3-amino-triazole (3-AT) in a pilot transformation before performing the large-scale screening experiment. Competent AH109 cells (yeast transformation kit, YEASTMAKER #K1606-1) were co-transformed with bait and prey vectors using the lithium acetate-mediated method. After electroporation, only transformants where protein-protein interaction between the bait and prey took place, activation of His and Ade promoters occurred and were able to grown on synthetic dropout medium (SD) lacking tryptophan (Trp⁻), leucine (Leu⁻), histidine (His⁻) and adenosine (Ade⁻) (SD/Trp⁻Leu⁻His⁻Ade⁻). Approximately 2.8 × 10⁶ co-transformants were screened and subsequently selected for growth on SD/Ade⁻His⁻Leu⁻Trp⁻ medium containing 3 mM of 3-AT. Co-electroporation of the bait and prey vectors resulted in the isolation of 160 putative clones.

In addition to a nutritional selection of the His3 gene, we performed color-based assay to detect the transcription LacZ reporter, because β-galactosidase activity allows a more stringent assay than the His3 gene in the GAL4 system (less likely to give false positives). After 7–15 days of growth at 30°C on SD/Ade⁻His⁻Leu⁻Trp⁻ plates with 3 mM 3-AT (high stringency selection), colonies were replicated onto plates containing X-gal substrate by the colony-lift filter assay. Positive interactors that showed β-galactosidase activity when co-expressed with the bait, but not when co-expressed with the bait-plasmid control, were considered bait-dependent transformants. Co-transformants that survived the HIS3⁻ growth selection and blue-colored colonies were selected.

The identity of the interactors was determined by sequencing. To test paired genes in the 2-protein interaction assay in yeast, the pGBKT7-BD expressing Mgn–Mash1 or Ngn2 as the bait and the pGADT7 vector harboring the coding sequence of Mash1, Ngn1, Ngn2, Ngn3, and Mgn as preys were used to transform haploid yeast AH109 (Mat-a) and Y187 (Mat-α) strains, respectively. Yeast strains were mated and diploid cells were plated on SD/Ade⁻His⁻Leu⁻Trp⁻ media. To quantify the relative strength of the protein–protein interaction in the 2-protein interaction assay, quantitative β-galactosidase assays were performed on liquid cultures using X-Gal (Sigma #B4252500MG). After cell lysis, a pellet was resuspended in water and mixed with PBS (pH 7.4) containing 500 μg/mL of X-gal, 0.5% agarose, and 0.05% β-mercaptoethanol, followed by incubation at room temperature. The p53/SV40 were diluted 1/5 to adjust the signal to the linear range due to their strong β-galactosidase activity.

Five independent transformants were assayed per interaction pair. The results were normalized against cell density and dilution. In parallel experiments, measurement of lacZ activity by OD was performed using ONPG as a substrate. The reaction was stopped with chilled 1 M Na₂CO₃ and absorbance was determined at 420 nm according to the manufacturer’s recommendations (Yeast Protocols Handbook, PT3024-1, Clontech).

Mouse lines and breeding

Mgn and Mash1 mouse strains and their genotyping have been described previously ([9,13], respectively). The strains were maintained at the Helmholtz Zentrum München. Mgn/Mash1 compound mutants were obtained from intercrosses of Mgn^−/+ and Mash1^−/+ inbreed mice that had been consecutively backcrossed for eight generations to C57BL/6. Upon crossing double-heterozygous mice (Mgn^−/+/Mash1^−/+ x Mgn^−/+/Mash1^−/+), a myriad of compound mutants could be found. The term “single mutant” is reserved for Mgn^−/−/ Mash1^+/+or Mgn^+/+/Mash1^−/− genotypes. The term “double mutant” is reserved for Mgn^−/−/Mash1^−/− genotype. The term “compound mutant” refers to any other genotype in general, otherwise specified. The compound mouse Mgn^−/+/Mash1^−/+ deserves, in this article, a specific mention, given its particular phenotypic traits that differentiate them from the rest of compound mice. Therefore, it is referred as “double heterozygous mice. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the European Union and of the Federal Republic of Germany (TierSchG). The protocol was approved by the Helmholtz Zentrum München Institutional Animal Care and Use Committee (ATV). Surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Histological analysis

Noon on the day when a vaginal plug was detected was considered E0.5. Embryos were staged precisely by counting somites. Embryos and dissected brains from different stages (E9.5–E18.5) were fixed in 4% paraformaldehyde overnight at 4°C, paraffin embedded, cut on a microtome at 4–6 μm, and hybridized with radioactive ((α-³⁵S)-UTP) in situ probes (see also oligonucleotides and ISH probes in S1 Material and Methods). Following in situ hybridization (ISH), paraffin sections were counterstained with cresyl violet. All analyses were confirmed using 3 embryos from different litters.

Immunohistochemistry and colocalization

A monoclonal mouse anti-MGN antibody (clone JG35) was raised against the MGN protein (GenBank accession no. ABB96784.1) and purified by protein A/G affinity chromatography. The primary antibodies were as follows: rabbit anti-β-galactosidase (AbD Serotec #AHP1292; 1:100); mouse anti-BRN3a (Santa Cruz #sc-8429; 1:200); mouse anti-calbindin (Swant #300; 1:500); rabbit anti-calretinin (Swant #7699/4; 1:2000); rabbit anti-cleaved caspase-3 (cCASP3; Cell Signalling #9661; 1:1200); rabbit anti-GABA (Sigma #A2052; 1:300); mouse anti-NeuN (Chemicon #MAB377; 1:1000); mouse anti-NKX2-2 (DSHB #74.5A5-c; 1:250); rabbit anti-phospho-histone H3 (pHH3; Upstate #06–570; 1:500); and rabbit anti-tyrosine hydroxylase (TH; Merck Millipore #AB152; 1:150). For colocalization studies in the MB, mouse anti-MASH1 isotype IgG1 (BD Pharmingen #556604; 1:300) and mouse anti-MGN (# JG35; 1:5000) were used. Secondary antibodies were goat anti-mouse IgG-1 Alexa Fluor 594 (Invitrogen #A21125; 1:500) and goat anti-mouse IgG-2b Alexa Fluor 488 (Invitrogen #A21141; 1:500), counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or coupled with horseradish peroxidase (HRP) and detected using the Vectastain ABC Elite Kit (Vector Laboratories/ USA). IHC and ISH paraffin sections were visualized with a Zeiss Axiovert-200M microscope, Stemi SV6 stereomicroscope, and Olympus IX81-Fluoview FV100 confocal laser-scanning microscope.

Mammalian cell culture and co-immunoprecipitation (Co-IP)

For the 2-protein Co-IP validation assays in mammalian cell culture, HEK293 cells were transiently co-transfected (FuGENE-6 transfection kit; ROCHE #1815091) with the coding region of Mgn or Mash1 (N-terminal FLAG-tagged) and Mash1 or Mgn (N-terminal myc-tagged) subcloned into pcDNA3.1 vectors. The transfected cells were incubated at 37°C for 24–48 h. The pull-down assay was performed using the Protein G immunoprecipitation kit (Sigma #IP50). Total protein concentration was measured with the Pierce BCA protein assay kit (Thermo #23227) and the products were analyzed on 10% NuPAGE (Novex #NP0301). The blots were blocked with 4% skimmed milk in TBST, followed by immunoblot analysis with anti-c-myc antibody (Dianova #9E10.3; 1:8000), anti-FLAG antibody (Sigma #F1804; 1:50000), and HRP-conjugated goat anti-mouse IgG (H+L) (Jackson #115-035-003; 1:1000) secondary antibody. For the MGN/MASH1 heterodimer and homodimer Co-IP assays, dMB, vMB, and the rest of the body from E12.5 wild-type (WT) embryos as well MB tissue from E12.5 Mgn^−/− and Mash1^−/− mutant embryos were dissected and collected. Brain tissues homogenized in RIPA buffer were lysed in the presence of protease inhibitor cocktail (Complete, Roche #04693124001) with DNases and RNases and then incubated with MASH1 or MGN antibodies covalently coupled and cross-linked with G-beads (Pierce Crosslink Magnetic IP/CO-IP Kit, Thermo scientific #88805). The protein lysate was incubated over night with cross-linked magnetic beads washed with low ionic strength buffer (120 mM NaCl) and eluted with mildly acidic buffer (100 mM glycine-HCL (pH 2.5) for 10 min). The products were verified by classical Western blot analysis using anti-MGN (Abcam; #ab101842; 1:300, anti-MASH1 (BD Pharmingen #556604; 1:300), and peroxidase-conjugated antibodies. Detection was performed using an ECL substrate (Amersham #RPN2232) and exposure to Hyperfilm ECL (GE Healthcare). Mouse embryonic stem (ES) cells were electroporated with a pCDNA3-1 vector harboring the cDNA-coding region of Mgn and/or Mash1 genes. The protein lysate was extracted 24–48 h later for Co-IP assays. Mouse anti-β-actin (Abcam #ab6276; 1:5000) was used as a quality control prior to Co-IP.

Real-time quantitative PCR (qPCR) and protein quantification

RNA was extracted from 3 E9.5–E10 mouse embryos for each genotype (RNeasy Kit, Qiagen #74104). Residual DNA was further removed from all extracts by DNase digestion and ds-cDNA was synthetized with d(T)_12–18 oligos (SuperScript, Invitrogen #11904–018). Amplification and real-time measurement was performed on a LightCycler real-time PCR system (Roche) using 10 μL of 2× Fast SYBR Green Real-Time PCR Master Mix (Life Technologies #4385610). The relative expression ratio of Mgn and Mash1 genes among genotypes was computed and analyzed according to the crossing point and threshold values with kinetic PCR efficiency correction method [14]. The ß-actin gene was probed to be unaffected among genotypes by comparing the values with other housekeeping controls (Gapdh, β-tubuline, and Pgk1) and therefore used as a loading control for normalization. The PCR length was 70–100 bp according to the Bustin’s guidelines [15] (MIQE compliant).

Proliferation and cell death studies

Pregnant females carrying E9.5–E13.5 embryos were intraperitoneally injected with a single BrdU injection (50 μg/g body weight) at 15 min and at 1 h before they were sacrificed. Incorporation and detection of BrdU in cellular DNA were performed with the BrdU labeling and detection kit II (Roche, #1299964). For the terminal deoxynucleotidyl nick end labelling (TUNEL) assay, apoptosis was detected using the In Situ Cell Death Detection kit-Fluorescein (Roche, #1684795). Apoptosis in the mouse embryo limbs served as the positive control for the cCASP3 and TUNEL assays. The cells were counterstained with DAPI and analyzed under a fluorescence microscope (Zeiss Axiovert-200M).

Organotypic explant culture and micro-electroporation

Organotypic WT and Mash1^−/− mouse anterior neural tube explants (ONTCs) were collected and opened along the dorsal midline prior to electroporation, as described previously [16]. Electroporation assays were performed using a square pulse generator device. Plasmid (Mgn-IRES-EGFP) (1 μg/μl in Fast Green, Sigma) was injected into the neuroepithelium using a pressure injector and constantly visualized by fast green. Electroporation was performed by applying 5 pulses of 10 ms at a current of 350 μA with intervals of 50 ms. After electroporation, each explant was cultured ex vivo at 37°C up to 24 h and then fixed in 4% paraformaldehyde before further processing for ISH in whole-mount embryos (WISH).

Cell counting and statistical analysis

For the calculation of NeuN and Calbindin positive cells, we sampled three 0.02 mm² bins in the dorsal, mediolateral, and ventrolateral regions of the MB. A total of three cases were counted in sections for each region. For the calculation of proliferative cells at E10.5 and E12.5, all PHH3+ cells were counted from six slices taken from rostral to caudal MB/group (n = 3). In all cases, the experimenter was blind during sampling, image analysis, data collection and statistical analysis. Statistical analysis was performed with the GraphPad Prism v6.04 software by pair-wise comparisons using Student’s t-test and the significance level was taken as p < 0.05. Data are represented as mean ± SEM, unless otherwise specified.

Identification of MASH1 as the binding partner of MGN protein by the Y2H screen

To find novel factors involved in the MB-GABAergic pathway, we used MGN as a bait in a Y2H assay to screen a cDNA library of E10.5 mouse embryos to identify protein–protein interactions at the time when MB-GABAergic neurogenesis begins. Expression of the fusion protein in yeast was confirmed (S1 Fig) and the absence of self-activation was verified. Each interaction was further assessed by positive β-galactosidase activity. Protein–protein interactions were confirmed individually with a second Y2H assay by α/a mating between MGN-bait and each segregated positive clone. The 160 clones were sequenced and represented 25 unique proteins (S1 Table). Among the protein interactors obtained in the Y2H assay, MGN and MASH1 factors were identified. As further indication of the validity of the assay, we confirmed binding specificity between MGN and MASH1 proteins using a mammalian cell line. Co-IP assays using MGN and MASH1 epitope-tagged fusion proteins confirmed the data from our yeast screening results (Fig 1A). MGN can form both homodimers (MGN/MGN), as previously reported by in vitro experiments in 293E cells [7], and heterodimers with MASH1 (MGN/MASH1). Based on the protein–protein interaction between MGN and the proneural MASH1, we tested if MGN interacted with other bHLH members of the proneural family, such as the neurogenin proteins. The results from the 2-protein assay in yeast and from further tests by Co-IP in mammalian cell cultures revealed that MGN was unlikely to form dimers with the neurogenin proteins NGN1 (Neurog1_MGI), NGN2 (Neurog2_MGI), and NGN3 (Neurog3_MGI) (Fig 1B and 1C), as suggested by our Y2H screen data.

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Fig 1. MGN formed homodimers and heterodimers with MASH1 in yeast.

(A) HEK293 cells were co-transfected, immunoprecipitated (IP), and analyzed by western blotting (WB) with plasmids and antibodies indicated in the IP and WB lines, respectively. The amounts of MGN, MASH1, and NGN2 were verified in total cell lysates (—-) as a control test. Anti-HA antibody (α-HA) was used as a negative control. (B) Y2H analysis of the interactions between MGN and different proneural proteins. AH109 yeasts were co-transformed with a bait and prey plasmid (bait/prey) before being plated on synthetic dropout medium (SD/Leu⁻Trp⁻). (C) The same yeast colonies were plated in the same order on SD/Ade⁻His⁻Leu⁻Trp⁻ (to select positive protein–protein interactions) and used for the ß-galactosidase activity assay (D). Binding domain (BD) alone and activator domain (AD) alone plasmids; BD–human Lamin C provided a control for the AD–prey interaction. P53/SV40 interaction was used as positive control (P53/SV40 was diluted to 1 in 5 in D).

https://doi.org/10.1371/journal.pone.0127681.g001

MGN and MASH1 form heterodimers in vivo

Mash1 was expressed in a manner similar to Mgn (i.e., at high levels in the vlMB area at E10.5 when compared with the low levels in the dMB at E12.5). Furthermore, both Mash1 and Mgn have been proved to function as key factors for the specification of dMB-GABAn by loss-of-function experiments in mice [9,10,11]. Therefore, we focused on the role of these two factors and the mechanisms underlying their role in MB-GABAergic neurogenesis.

Colocalization studies between MGN and MASH1 were mandatory because of the so-called time/space constraints, according to which two proteins may never be in close proximity to each other within the cell even though they are otherwise able to interact. Given that MGN and MASH1 colocalize in most vlMB cells [8] and that the GABAergic phenotype of single knockout mice is circumscribed in the dMB, we studied colocalization in that area. Although the MASH1 domain was broader (also found in postmitotic neuronal progenitors) than the MGN domain (restricted exclusively to mitotic neuronal progenitors of the VZ), all MGN⁺ cells were positive for MASH1 in VZ of the dMB at E12.5 (the onset of dGABAn) (Fig 2A). Co-IP analysis of endogenous protein extracts derived from WT using specific antibodies recognizing each protein indicated that MGN and MASH1 form heterodimers in the dorsal and ventral aspect of the MB at the onset of GABAergic neurogenesis in vivo (Fig 2B and 2C). This interaction does not take place in single mutants. Embryonic stem (ES) cells were used as control to validate the protein-protein interaction observed in the immunoprecipitation analysis from mouse brain tissue. Heterodimer formation was visualized only in lysed ES cells expressing MGN and MASH1 proteins. Contrary, MGN/MASH1 heterodimerization does not take place in wild-type cells or ES cells expressing either Mgn or Mash1, indicating a specific interaction.

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Fig 2. Colocalization and co-immunoprecipitation of MGN and MASH1 in mice.

Immunohistochemical staining of coronal sections from E12.5 wild-type mouse embryos. (A) Representative confocal photomicrographs of immunohistochemical staining with anti-MASH1 (A2) and anti-MGN (A3) from the dMB (squared area). Nuclei stained with DAPI are shown in blue (A1). A merged picture of A2 and A3 indicating the colocalization of MASH1 and MGN is shown in A4. The MGN⁺ subpopulation represented approximately 50% of the total MASH1⁺ cells of the VZ at this stage. (B-C) MASH1 is an interaction partner of MGN in physiological conditions. Endogenous MGN was IP from the dMB and vMB of E12.5 wild-type mouse embryos with anti-MASH1 antibody and detected by western blotting with anti-MGN antibody (B) and vice versa (C). Lysates from 129SV embryonic stem cells (ES) electroporated with the indicated plasmids, the embryo body (where Mgn is not expressed) and the MB from Mgn^−/− and Mash1^−/− mutants were used as controls. Equal input and quality of the protein lysate prior to immunoprecipitation was shown by immunostaining with anti-ß-ACTIN antibody. Scale bars: 100 μm.

https://doi.org/10.1371/journal.pone.0127681.g002

MGN and MASH1 heterodimers are needed for proper dMB-GABAn specification in vivo

The analysis of Mgn single knockout mice (Mgn^−/−) revealed a GABAergic phenotype very similar to that of the Mash1 single knockout mice (Mash1^−/−). Both single mutants have complete depletion of GABAn in the SC and induction of dMB-GABAn does not take place at any time during development [9,10,11]. However, induction of vlGABAn does occur and, although a slight delay in neurogenesis is observed between E10.5 and E14.5, vlMB-GABAergic induction is clearly observed. A puzzling phenotypic feature of Mgn and Mash1 single mutants is that the GABAergic phenotype is circumscribed to the domain where Mgn and Mash1 are expressed at low levels compared with their ventrolateral expression. Therefore, a key developmental question arose: is there a third redundant factor specifying vlMB-GABAn at E10.5 or does MB-GABAergic neurogenesis rely on different mechanisms? Concomitant loss of function of Mgn and Mash1 (Mgn^−/−Mash1^−/−) revealed that in contrast to Mgn and Mash1 single mutants, GABAergic fate is severely compromised in Mgn^−/−Mash^−/− embryos and most GABAn of the MB are absent (Fig 3A). Time-course analysis showed that the induction of GABAn does not take place at any time during MB development, as determined by the absence of the GABAergic markers Gad65 (Gad2_MGI), Gad67 (Gad1_MGI), and GABA in Mgn^−/−Mash1^−/− embryos. Therefore, complete ablation of GABAn was observed in both the dMB and vlMB, and only few GABAn were detected in the vlMB m5 domain of double mutant mice (Fig 3B4 and 3C4). This phenotype occurred with 100% penetrance and persisted to death, indicating that there is no other code involved in their specification except for a small number of cells located within the m5 domain (intermediate basal plate). However, double heterozygotes (Mgn^+/−Mash1^+/−) showed normal GABAergic induction (dorsally and ventrally) compared with WT animals (Fig 4B, 4D2 and 4E) and it is maintained until postnatal stage 0 (P0) (Fig 4F and 4G). We therefore tested whether or not a compensatory mechanism existed between the levels of mRNA expression of Mgn and Mash1 in double heterozygous mice. Reverse transcription-qPCR analysis revealed that Mgn mRNA expression was not upregulated in Mash1^−/− mice and vice versa (Fig 5A). This indicated that the expressions of Mgn and Mash1 were independent and occurred without compensatory mechanisms in single or compound mutants. Therefore, MGN/MASH1 heterodimers appear necessary for dMB-GABAn induction. This novel finding prompted us to hypothesize that heterodimer formation may have a higher efficiency than homodimer formation in dMB-GABAergic specification. One appealing feature of the Y2H system is that the strength of interaction predicted by the 2-hybrid approach essentially correlates with that determined in vivo [17]. We quantified the difference in relative strength between heterodimers and homodimers in a double manner. On one hand, we increased the stringency of the growing conditions by adding 3-AT to the media, an inhibitor of HIS3 synthesis, to discriminate different degrees of affinity interaction. On the other hand, we measured β-galactosidase activity in liquid cultures because of its higher sensibility and reproducibility. The 3-AT analysis showed that only yeasts forming MGN/MASH1 heterodimers were able to grow in higher levels of 3-AT, whereas yeasts forming MGN/MGN or MASH1/MASH1 homodimers were not (Figures A, B and C in S2 Fig). Consistent with these results, the β-galactosidase assays showed that the strength of the MGN/MASH1 protein interaction was 4.4 and 4.0 times higher than that for the MGN/MGN and MASH1/MASH1 homodimers, respectively (Figures D and E in S2 Fig, and Fig 1D). Thus, MGN/MASH1 heterodimers were more efficient in activating HIS3 and LacZ gene transcription than their homodimers.

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Fig 3. Mgn and Mash1 were necessary and sufficient to induce GABAn in the MB.

Phenotypic analysis of Mgn^−/−Mash1^−/− (−−/−−) mice by ISH with the Gad67 riboprobe on sagittal (A) and coronal (B-C) sections, showing the mesencephalon at E12.5 and E14.5 from both wild-types (A1–A4; B1 and B2) and double mutants (with B5–B7 being representative slides from anterior to posterior). The arrows in A14, B4, and C4 indicate the presence of few Gad67 cells in the m5 domain (m5) at E12.5 and E14.5. Scale bars: 500 μm. Abbreviations: HB, hindbrain; Is; isthmus; SC, superior colliculus; SNpr, substantia nigra pars reticulata; vMB, ventral midbrain; VTA, ventral tegmental area.

https://doi.org/10.1371/journal.pone.0127681.g003

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Fig 4. MB GABAergic pathway was severely compromised in the double and compound mutants.

Expression of GABAergic-specific genes on the coronal sections of E12.5 in wild-type and single knockout embryos (A), as well as in compound and double knockout mutants (B). MB GABAergic induction occurred in double heterozygous mice based on the presence of GABAergic markers (Gad67 and Tal1). (A-E) The GABA phenotype observed at E12.5 in the vlMB of Mgn^−/−Mash1^−/− (−−/−−) mice (C2 and D3) was recovered in Mgn^+/−Mash1^+/− mice (D2 and E). The intermediate zone of m3 domain is magnified in E. GABAn of double heterozygous embryos were maintained at E18.5 (F) and until postnatal death P0 (G). DAPI, NKX2-2, TH, and β-Gal staining were used as morphological markers). Arrows indicate the loss of postmitotic NKX2-2 neuronal subpopulation in the m2 domain, but not in the m4 domain (C). Scale bars: 500 μm in A and G, and 50 μm in E.

https://doi.org/10.1371/journal.pone.0127681.g004

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Fig 5. Endogenous and ectopic expression of Mgn.

(A) Relative expression levels of Mgn and Mash1 in mutant to wild-type mice. The bar height and error bars show the mean and standard deviation, respectively. (B) Mgn mRNA was expressed at high levels in the vlMB of E12.5 mouse embryos compared with the low level of expression in the dMB determined by ISH, LacZ staining of Mgn^tLacZ mice, and IHC of wild-type embryos with the anti-MGN antibody on coronal sections. The lines indicated the borders of m3-m5 domains. MASH1 protein is also expressed at higher levels in the vlMB compared with its dorsal expression, as determined by IHC with anti-MASH1 antibody. (C) WISH was performed with Gad67 probe in ONTC of wild-type E11.5 embryos with Mgn cDNA electroporated in one of the sides (right side), whereas the other side served as a control (left side). The neural tube is opened along the ventral anterior-posterior axis. Lightning bolt icons point to the ectopic expression of Gad67 after Mgn electroporation. Subdomains are shown according to the prosomeric model [21]. (D) WISH was performed with a Gad67 probe in the ONTCs of E11.5 wild-type mouse embryos after 10 h of incubation. The red dashed line delineates the alar/basal (a/b) boundary. The black dashed lines delineate the transversal neural tube domain boundaries. (E and F) E11.5 Mash1^−/− ONTCs 24 h after micro-electroporation in the mesencephalic alar plate with the Mgn-IRES-EGFP plasmid. (E) A fluorescence signal showing the electroporated location. (F) Induction of Gad67 expression after electroporation of Mgn cDNA in Mash1^−/− background embryos. (F1) A merged profile of the fluorescence signal and Gad67 induction. Scale bars: 500 μm. Abbreviations: a/b, alar/basal plate boundary; d/m, diencephalic–mesencephalic boundary; Hy, hypothalamus; Is, isthmus; p1–p3, prosomeres 1 to 3 in diencephalon; PcP, precommissural domain in the pretectum (p1); rp, roof plate; Zli, zona limitans intrathalamica.

https://doi.org/10.1371/journal.pone.0127681.g005

We next investigated why MGN/MASH1 heterodimers, but not their homodimers, are sufficient for GABAergic induction. Phenotypic analyses of single mutants (where heterodimer formation cannot take place) and double heterozygous mice revealed that the presence of a single WT allele from each locus was sufficient to promote dMB-GABAergic induction, whereas two Mgn or two Mash1 alleles alone were not. These observations suggested that heterodimer formation is essential for proper d-GABAergic neurogenesis but not for vl-GABAn, where only a single allele from the Mgn or Mash1 loci was sufficient to trigger GABAergic induction. Therefore, MGN and MASH1 cooperate synergistically for dMB-GABAergic neurogenesis.

A dose-dependent mechanism for vl-GABAn

The absence of GABAn in the double knockout mice suggested that dorsal and vlMB-GABAergic induction were controlled by the same codes, raising questions about the intrinsic cell mechanisms that govern the differential development of GABAn in the dMB and vlMB. Dorsally, analysis of compound mice showed that both genes do not have a predominant role over the other and that both alleles contribute equally to the MB-GABAergic phenotype. One allele per locus was required for the formation of MGN/MASH1 heterodimers and for the specification of d-GABAn. Ventrolaterally, the presence of two alleles (regardless of whether they come from the Mgn or Mash1 locus) was sufficient to determine the GABAergic fate; that is, heterodimers were not required because GABAergic induction took place in Mgn^+/+Mash1^−/− or Mgn^−/−Mash1^+/+ mice. Furthermore, the presence of a single allele (Mgn^+/−Mash1^−/− or Mgn^−/−Mash1^+/− mice) was sufficient to trigger GABAergic induction in the vlMB, although a strong reduction in GABAn was observed (Fig 4B). Thus, the severity of the vlMB-GABAergic phenotype increases in a dose-dependent manner with the number of mutant alleles from the Mgn and Mash1 loci. The phenotypic severity culminates in the absence of GABAn in the double knockout mice (Fig 4C2 and 4D3), except for a small number of residual GABA⁺ cells in the ventrolateral m5 domain. These data support a model that requires both MGN/MASH1 heterodimers and dose dependency as mechanisms operating at the dMB and vlMB, respectively. Consistent with this finding, the high level of Mgn expression observed in the vlMB (Fig 5B) may compensate for the lower efficiency of homodimers compared with that of heterodimers. MAHS1 protein is also not evenly distributed throughout the MB but highly expressed in the vlMB domain (Fig 5B). This result is in good agreement with published data from Partanen’s group [11] where Mash1 mRNA expression is markedly higher in the vlMB compared with its dorsal expression. This hypothesis was later tested using gain-of-function experiments. Ectopically expressed Mgn cDNA into organotypic cultures of WT mouse embryos at E10.5 induced Gad65/67 expression (two specific and independent enzymes for GABA synthesis) throughout the nervous system (Fig 5C; additional data not shown). The ectopic GAD65/67 expression observed in mouse brain regions upon Mgn electroporation suggests that Mgn, when expressed at high levels, does not need to cooperate with additional cell type-specific transcription factors to execute its function in a cell-autonomous manner. Moreover, we investigated if high expression of Mgn cDNA electroporated into the dMB of Mash1^−/− embryos can initiate GABAergic cell fate in the absence of Mash1 (and therefore in the absence of MNG/MASH1 heterodimers). In the Mash1^−/− E12.5 embryos electroporated with Mgn cDNA, the GABAergic fate was induced as determined by the in novo expression of Gad65/67 (Fig 5F).

Impaired neurotransmitter specification in the d/vlMB of Mgn^−/−Mash1^−/− mice

The depletion of GABAn engendered the need for cell proliferation, programmed cell death, and cytoarchitectural studies to investigate the cellular basis of the lack of d/vl-GABAn in double knockout mice. No reduction in cell proliferation was identified in the d/vlMB of double mutants at E10.5–E14.5 as revealed by immunohistochemical staining of mitotic cells surrounding the lumen of the neural tube with pHH3 (Figure A in S3 Fig). Cell death was analyzed by IHC to detect cleaved caspase 3 (cCASP3). There was no evidence of increased apoptosis in the MB of Mgn^−/−Mash^−/− embryos when compared with the littermate controls at the time of GABAergic neurogenesis (Figure B in S3 Fig). We next confirmed the results of proliferation and apoptosis using BrdU incorporation and TUNEL assays, respectively. New cells from VZ, time-traced with BrdU, showed no proliferation deficits in the MB of double mutants, and the BrdU-labeled progenitors migrated radially to the mantle zone, as in the littermate controls (Fig 6A). TUNEL assays revealed no apoptotic cells among genotypes on serial samples of coronal sections at E10.5–E13.5 (Figure C in S3 Fig). Furthermore, in the developing MB of double mutants, cytoarchitecture studies revealed no significant difference in the cell density of differentiated neurons within the intermediate and mantle zones (compared with WT mice. This was determined by immunocytochemistry for Neuronal Nuclei (NeuN), a specific marker of postmitotic neurons in vertebrates (Fig 6B). Calcium-binding protein (calretinin and calbindin) marking of subpopulations of GAD⁺ cells in differentiated GABAn indicated that the MB-GABAergic cytoarchitecture of double mutants is not affected at the gross morphological level (Fig 6B). Moreover, we traced lacZ⁺ cells outside the VZ neuroepithelium, expressed under the endogenous Mgn regulatory elements in mice with Tau-lacZ knocked in the Mgn locus (Mgn^TlacZ/TlacZMash1^−/− mice). Consistent with previous studies in single knockout mice [9], the same pattern of lacZ staining was observed in compound and double mutants, showing that the Mgn^−/−Mash^−/− lacZ-stained cells were positioned in stream-like routes and initiated their radial migration toward the marginal zone through the intermediate zone (Fig 6C and 6D) as neurogenesis ensued [18]. MGN and MASH1 pathway control GABAergic neurotransmitter identity without regulating neurogenesis.

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Fig 6. Depletion of dMB and vlMB GABAn in the Mgn^−/−Mash^−/− mice occurred through GABAergic identity failure.

(A) Immunohistochemical staining of BrdU on coronal sections showing the distribution of BrdU⁺ cells in the developing dMB and vMB of wild-type (WT) and double mutant mice (−−/−−). (B) Neuronal cell density and cytoarchitectural analysis on the coronal sections of E12.5 embryos using NeuN and the Ca²⁺-binding proteins Calretinin and Calbindin, respectively. There were no significant difference between mean WT and double mutant (NeuN: dorsal WT = 64.22±2.08; dorsal −−/−− = 61.33±2.69; n = 18; p = 0.40. Medial WT = 52.28±3.60; medial −−/−− = 56.72±1.97; n = 18; p = 0.29. Ventrolateral WT = 46.44±2.36; ventrolateral −−/−− = 43.67±2.52; n = 18; p = 0.43); (Calbindin: dorsal WT = 43.28±6.20; dorsal −−/−− = 37.50±5.54; n = 18; p = 0.49. Medial WT = 52.22±5.32; medial −−/−− = 46.56±2.69; n = 18; p = 0.35. Ventrolateral WT = 47.39±3.19; ventrolateral −−/−− = 46.17±3.92; n = 18; p = 0.81). (C) LacZ staining of coronal sections from double mutants and controls at E12.5; C2 and C4 show prolonged staining times. Owing to the long half-life of lacZ protein, signal can also be seen in the intermediate and mantle zones. (D) Immunohistochemical staining with anti-LacZ (in green) and anti-BRN3a (Pou4f1_MGI) (in red, as a morphological marker) antibodies that shows radial migration of GABA-defective neurons through the intermediate zone (IZ) toward the mantle zone (MZ) in double mutants. Scale bars: 100 μm in A, and 200 μm in B-C.

https://doi.org/10.1371/journal.pone.0127681.g006

MGN and/or MASH1 dimers essentially activate the same MB-GABAergic neurogenesis pathway

We looked for downstream GABAergic markers to explore if MGN or MASH1 homodimers in the corresponding single knockout conditions were able to activate equivalent genetic programs compared with the MGN/MASH1 heterodimers present in WT animals. Genetic analyses of specific markers of the GABAergic pathway suggested that the MGN/MASH1 heterodimer and homodimers essentially activate the same pathway. Expression of postmitotic GABAergic terminal selector genes were largely downregulated. Thus, the zinc-finger Gata2, Gata3, and the bHLH Tal1 transcription factors, which are essentially co-expressed in the MB GABAn at E10.5-E12.5, follow the dorsal/ventrolateral Gad65 and Gad67 phenotypes of single, double, and compound knockout mutants. In particular, the dorsal expression of Gata2, Gata3, and Tal1 is donwregulated in single, double, and compound mice, except for the double knockout heterozygotes, where MGN/MASH1 heterodimers are present (Figures A and B in S4 Fig, Fig 4A and 4B, respectively). Ventrally, their downregulation follows the dose-dependent mechanism according to the Mgn and Mash1 genotype and their expression is also absent in the double knockout conditions, showing a total dependence on Mgn and Mash1 genes (Figures A6 and B9 in S4 Fig). Another observation from of our studies is that Gata2, Gata3, and Tal1 expression occurred only in few cells in the m5 domain of double knockout mutants in the same subdomain as Gad65/67 expression. The GABAergic related LIM1 (Lhx1_MGI) transcription factor is also misregulated in the vlMB. Lim1 expression depended on the MGN/MASH1 heterodimers in the m3 and m4 domains only, because LIM⁺ cells were largely absent in these subdomains of all compound mutant mice where heterodimer formation was not possible, and was independent of Mgn and Mash1 expression in the m5 and m6 domains, because normal Lim1 expression occurred in the double mutants (Figure A in S5 Fig). Unlike Tal1 expression, which was absent in the double mutants, Tal2 expression was totally dependent on the MGN/MASH1 heterodimers in the m5 domain, yet independent of Mgn and Mash1 expression in the m1–m4 domains (Figure A in S6 Fig).

Because TAL factors, particularly TAL2, appear to operate as postmitotic selectors between GABA and glutamatergic neurons [19], we investigated transpecification of defective GABAn. Consistent with the GABAergic to glutamatergic neurotransmitter switch observed in single knockouts [10,11], upregulation of Vglut2 (Slc17a6_MGI)-expressing cells can also be observed in the dMB of Mgn/Mash1 double mutants, concomitantly with a delay in neurogenesis at E12.5. Ventrolaterally, upregulation of Vglut2 expression depended on Mgn; however, it was not dependent on either Mash1 expression or in the formation of MGN/MASH1 heterodimers, because Mash1 single mutants and +−/−− compound mice showed no upregulation of Vglut2, respectively (Figure B in S6 Fig).

The observation that Mgn and Mash1 both control equivalent programs of MB-GABAergic neurogenesis during this stage of development should not be taken to imply that Mgn and Mash1 have the same function. The ectopic expression of Vglut2 depending on Mgn but not on Mash1 suggest that these factors have also unique targets. On the other hand, beyond the GABAergic pathway, these particular bHLH codes have distinct roles during development.

Different dorsal/ventrolateral mechanisms for GABAergic neurogenesis operating at MB

Two genes, Mgn and Mash1, function as selectors of GABAergic identity in the vlMB during the first wave of GABAergic neurogenesis at E10.5. This occurs through a mechanism different from that operating in the dMB during the second wave of GABAergic neurogenesis at E12.5. From the comparative phenotypic analysis of Mgn and Mash1 in single, double, and compound mutant mice, we make the following conclusions. First, neither MGN nor MASH1 homodimers can compensate for the loss of MGN/MASH1 heterodimer function in the dMB, possibly because of the low expression levels of Mgn and Mash1 mRNAs in this domain. Second, MGN/MASH1 heterodimers have an instructive role in specifying the identity of dMB-GABAn. It is possible that the higher efficiency of MGN/MASH1 heterodimers over homodimers compensates for the low levels of Mgn and Mash1 expression in the dMB compared with the vlMB. Third, either MGN or MASH1 homodimers are sufficient to trigger vlMB-GABAn at physiological protein levels. Finally, although a gradual phenotype appears in a dose-dependent manner in compound mice, MGN/MASH1 heterodimers are not needed for the induction of vl-GABAn (heterodimer formation is prevented in single knockout mice). In the absence of MGN, MASH1 can take over the function of determining GABAergic cell fates, but only in the vlMB, and vice versa. This is because at these developmental stages (E9.5–10–5), the high level of expression probably circumvents the lack of MNG/MASH1 heterodimer formation in single mutants.

An example that best supports our hypothesis can be seen in the phenotypic analysis of double heterozygous and compound mice, as well as the gain-of-function experiments. In these experiments, Mgn was overexpressed in the dMB in the absence of Mash1 (Mash1^−/− mice) at levels comparable to those of endogenous Mgn expression in the vlMB. Higher levels of MGN homodimers under these conditions were able to trigger the expression of GABAergic markers (Gad65/67) in the dMB despite the absence of MGN/MASH1 heterodimers. Thus, high levels of MGN protein in the dorsal aspect overcome the lower efficiency of homodimers and mimic the in vivo vlMB conditions of Mash1^−/− mutants where MGN homodimers are sufficient to induce GABAergic fate (neither MGN/MASH1 heterodimers nor MASH1 homodimers can be formed in Mash1^−/− mice). Together, these data indicate a novel mechanistic action in the dorsal/ventrolateral areas governing the induction of MB GABAn based on the differential expression of Mgn and Mash1 at this stage (Fig 7A). Later, at E14.5–E15.5, a conspicuous subpopulation of GABAn was detected in the vmMB in the Mgn^−/−Mash1^−/− double mutants (domains m6–m7). An explanation for this is that this subpopulation of GABAn associated with dopaminergic nuclei (substantia nigra pars reticulata (SNpr) and ventral tegmental area (VTA)) has its developmental origin outside the MB. These neurons migrate out of the ventral rhombomere 1 neuroepithelium in the HB [20], where the GABAergic fate is independent of Mgn (Mgn is not expressed in the HB during development; Fig 5B).

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Fig 7. Summary model of the dorsal/ventral mechanisms underlying MB GABAergic neurogenesis.

(A) Two genes, Mgn and Mash1, functioned as selectors of GABAergic identity in the MB through different dorsal/ventral mechanistic actions at different times. The MGN/MASH1 heterodimers (green shape coupled with yellow) appear essential for the induction of dorsal GABAn (m1 and m2 domains) that generate GABAn in the colliculi, laterodorsal periaqueductal gray area (ldPAG) and the ventrolateral periaqueductal gray area (vPAG). MGN or MASH1 homodimers (shapes coupled with the same color) are not sufficient to trigger GABAergic identity, and synergistic cooperation between MGN and MASH1 factors to form heterodimers is essential for the acquisition of dorsal GABAergic identity. The mechanism operating in the vlMB area (m3, m4, and m5 domains), which gives rise to the ventral PGA and reticular formation nuclei (MBRf) GABAn, appeared to be different from that in the dorsal area. In this model, the MGN/MASH1 heterodimer is not a prerequisite for the induction of ventrolateral GABAn, with either homodimer being sufficient alone and following a dose-dependent mechanism. The GABAergic phenotype observed in mutant mice is depicted with a green tick or red cross to denote the presence or absence of GABAn, respectively. (B) MB GABAn arise during three waves of neurogenesis, at both specific developmental times and specific locations. The earliest wave of GABAergic neurogenesis (1^st) starts at E10.5 in the presence of Mgn and/or Mash1, without preference. The second phase (2^nd) takes place two days later at E12.5 in the dMB. The third population (3^rd), corresponding to the GABAn of the SNpc and VTA, appears at E14.5. These different mechanisms define a new model for dorsal and ventrolateral GABAergic neurogenesis. Red lines delimitate the embryonic MB into seven subdomains (m1-m7) along the dorsal/ventral axis, characterized by the expression of specific transcription factors [10] and their corresponding MB plates, established by [22]. (−−/++; ++/−−) stand for Mgn and Mash1 single knock-out embryos, respectively. (−+/−+; −−/−+; −+/−−) stand for compound embryos; (−−/−−) stands for double knock-out embryos and ++++/−− stands for overexpression of Mgn cDNA in Mash1^−/− genetic background embryos. Abbreviations: f, floor plate; r, roof plate; Dap, dorsal alar plate; Lap, lateral alar plate; VLap, ventrolateral alar plate; Lbp, lateral basal plate; Ibp, intermediate basal plate; Mbp, medial basal plate.”

https://doi.org/10.1371/journal.pone.0127681.g007

Taken together, the present data provide compelling evidence that the MB has adopted two crucial factors (MGN and MASH1) and three mechanisms for the acquisition of MB-GABAn during the three distinct phases of development, which are separated in time and place as follows. The earliest wave of GABAergic neurogenesis corresponds to the highest expression level of Mgn and Mash1 mRNA in the vlGABAn at E10.5 and occurs indistinctly in the presence of Mgn and/or Mash1 in a dose-dependent manner. At this stage, MGN or MASH1 homodimers are sufficient to induce GABAn, and heterodimers are not required. The second phase occurs 2 days later (E12.5) in the dMB, where the levels of Mgn and Mash1 expression are lower than in the ventrolateral, which makes the formation of MGN/MASH1 heterodimers a prerequisite for induction, with neither homodimer being sufficient alone. The third population corresponds to GABAn of the SN and VTA and appears at E14.5 in the medial basal/floor plate (domains m6–m7, the vmMB). The fact that none of the Mgn/Mash1 single, double, or compound mutant embryos show a GABAergic phenotype in the SN/VTA indicates that neither MGN/MASH1 heterodimers nor homodimers are necessary for their specification or migration. This explains why Mgn, a specific marker for MB-GABAergic neurogenesis, is not expressed in the medial basal/floor plate of the MB at any developmental stage [6]. These different mechanisms define a new model for dorsal/ventrolateral GABAergic neurogenesis (Fig 7B).

No proliferative function is attributed to Mgn or Mash1 in the vlMB

A role of Mgn and Mash1 in generating the proper number of GABAergic precursor cells in the vlMB cannot be excluded. Given that Mgn and Mash1 can take over each other’s function in d/vMB-GABAergic specification, redundancy may also mask a proliferative function. Extensive analyses performed at the time of GABAergic neurogenesis and later in development indicate that the MB neuronal cytoarchitecture of double mutant mice appears normal despite GABAn constituting a major neuronal population in this region. Several lines of research indicate that Mgn^−/− Mash^−/− neurons are generated at proper numbers in VZ of d/vlMB during the first two waves of GABAergic neurogenesis and that the progenitors that should become GABAn do not enter into mutant-specific apoptotic programs but migrate toward the mantle zone. The neuronal citoarchitecture and the molecular analysis of double mutants show that defective neurons are not arrested into a progenitor-like state, since neuronal density of differentiated neurons and expression markers for mature neurons appear to be normal among genotypes. Although some defective cells express glutamatergic markers at the time of GABAergic neurogenesis (Figure B in S2 Fig), it is not clear whether this cells acquire a full glutamatergic identity. Electrophysiological studies are needed to determine whether there is a complete switch of neurotransmitter identity or just a transient acquisition of some glutamatergic markers like Vglut2, due to misregulation of GABAergic neurogenesis. Defective GABAn persist during development in double mutant mice and continue to be maintained without expressing GABA until birth. Therefore, massive depletion of d/vlMB-GABAn occurs in Mgn^−/−Mash^−/− mutants at the onset of neurogenesis through failure to achieve GABAergic identity rather than deficits in the number of precursor cells or migration cues.

Downstream GABAergic markers follow the dorsal/ventrolateral context mechanisms

Because all MGN⁺ cells co-express MASH1 in VZ, thereby sharing the pool of presumptive GABAergic precursor cells, it is considered that the high levels of Mgn and Mash1 expression in the ventrolateral domain most likely reflects the limited efficiency of their homodimers to trigger GABAergic neurogenesis rather than an inherent functional difference between heterodimers and homodimers. Consistent with this, the genetic network behind MGN/MASH1 heterodimers and homodimers is essentially maintained along the d/vlMB axis, because both appear to activate the same downstream genetic cascade for neuronal differentiation. Upon ablation of Mgn and Mash1 expression, defective postmitotic GABAn downregulate the expression of transcription factors that are predictive of their GABAergic commitment (Gad65, Gad67, Gata2, Gata3, and Tal1) and appear to follow the GABA phenotype and the dorsal/ventrolateral context mechanisms proposed above. The Tal2 gene, expressed in MB-VZ and involved in the GABA pathway, as shown by the lack of GABAn in the Tal2^−/− mutant [19], appears to function regardless of Mgn and Mash1 genes in the m1-m4 domains. This is particularly evident in the MB of Mgn^−/−Mash1^−/− mice, where Tal2 is expressed without any detectable downregulation, suggesting an independent pathway. Conversely, Tal2 expression depends on MGN/MASH1 heterodimer in the m5 domain, but not on their homodimers. The m5-GAD⁺ phenotype observed in the Mgn^−/−Mash1^−/− mice resembles that of Tal2^−/− mutants, where few cells in the m5 domain express GAD markers [19].

In summary, we have provided molecular, genetic and phenotypic evidence about two cooperating bHLH factors in the MB for the acquisition of GABAergic identity. We showed distinct mechanisms by which MGN and MASH1 trigger the GABAergic cell fate, which suggest a model for d/vlMB-GABAergic neurogenesis during mouse development. Transcription factors belonging to the hairy/E(spl) and proneural families have long been believed to counterpart each other’s function. This work uncovers a synergistic cooperation between these two families, outlines the underlying different dorsal/ventrolateral mechanisms, and provides a novel paradigm for how they cooperate for the acquisition of MB GABAergic neuronal identity. Finally, an important consideration in MB GABAergic neurogenesis is that these results can help our understanding of the molecular mechanisms underlying neurogenesis in other brain areas and should allow us to understand the functional diversity of these neurons more clearly. Thus, they may facilitate the development of novel insights into the molecular etiologies underlying neurological and psychiatric disorders in which the GABAergic system is disturbed.