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First published online 4 July 2007
doi: 10.1242/dev.02870

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Helt determines GABAergic over glutamatergic neuronal fate by repressing Ngn genes in the developing mesencephalon

Tomoya Nakatani^*, Yasuko Minaki, Minoru Kumai and Yuichi Ono^*,

KAN Research Institute Inc., KobeMI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.

View larger version (107K): [in this window] [in a new window]   Fig. 1. Helt is selectively expressed in GABAergic progenitors in the developing mouse mesencephalon. (A) Comparison of the expression pattern of Helt with those of transmitter markers in the mesencephalon at E11.5 and E12.5. The lower panels show the dorsal mesencephalon region at E12.5. Helt is selectively expressed in GABAergic domains and excluded from glutamatergic domains. In the dorsal m1 domain, only glutamatergic neurons are generated and Helt is not expressed in the VZ at E11.5. At later stages, both GABAergic and glutamatergic neurons are generated, and Helt is expressed by subpopulations of the dorsal progenitors. (B) Domain structure of the developing mesencephalon. The expression patterns of transcription factors in the E11.5 mesencephalon are shown. Each domain is indicated as m1 to m7 (summarized in Fig. 2). Helt is expressed in the ventricular zone (VZ) of the m2 to m7 domains. Note that some Helt+ cells are observed in the ventral-most region of the m1 domain at E11.5. These Helt+ cells in the m1 domain are not obvious in embryos at a slightly earlier stage (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Gene expression map of the mouse anterior mesencephalon at E12.5. Summary of the gene expression pattern and transmitter phenotype of each domain (m1 to m7), obtained from the results shown in Figs 1, 5 and 7. In the m1 and m2 domains, both GABAergic and glutamatergic neurons emerge. The hatched lines indicate expression in subpopulations within each domain (i.e. expression of Helt and Ngns, Lim1 and Brn3a, and of Gad1 and Vglut2, is mutually exclusive). Note that the motoneurons of the oculomotor complex are not shown in this map as their generation is completed at an earlier stage and the motoneuron progenitor domain changes to the RN domain at E11.5 (data not shown). RP, roof plate; VZ, ventricular zone; ML, mantle layer; Glut, glutamatergic; DA, dopaminergic.

View larger version (89K): [in this window] [in a new window]   Fig. 3. Helt selects the GABAergic versus glutamatergic phenotype in the mesencephalon. (A) Gene targeting strategy. All exons encoding the Helt ORF were replaced with a GFP cDNA, and recombination was confirmed by Southern blotting (data not shown). The null phenotype in homozygous mutant mouse embryos was confirmed by complete loss of Helt expression (right-hand panels). (B) Helt is required for induction of GABAergic neurons and suppression of glutamatergic differentiation. In the Helt-/- mutant mesencephalon, Gad1+ neurons are only detected in the ventral-most m5 region of the presumptive GABAergic domains, whereas Vglut2+ neurons are generated in essentially all domains of the mesencephalon at E11.5. At E12.5, although Gad1+ neurons start to emerge in a broad area of the ventral GABAergic domains (m3 to m5), the frequency is still lower than that in wild-type control embryos and ectopic Vglut2+ neurogenesis continues. Dorsal Gad1+ neurons are completely absent in the mutants. (C) Helt suppresses glutamatergic neurogenesis and induces GABAergic neurons. In the transgenic embryos expressing Helt under the control of the nestin enhancer, the number of Vglut2+ cells is decreased and the number of Gad1+ cells is increased instead. Note that GABAergic neurons are efficiently generated and glutamatergic neurons are suppressed in the ML just outside of the VZ positive for exogenous Helt, suggesting a cell-autonomous effect of Helt.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Helt determines the transmitter phenotype without affecting neuronal subtype identity specification. (A) Progenitor domain formation is not affected by the Helt-null mutation. Note that Nkx2.2 expression in the VZ is not changed by the Helt mutation, although postmitotic expression of this factor in the m2 domain is lost in the mutants. By contrast, expression of Nkx2.2 in m4 neurons is maintained in the mutants. (B) Conversion of the transmitter phenotype in Nkx2.2+ m4 neurons by the Helt-null mutation. In wild-type mouse embryos, most Nkx2.2+ neurons in the anterior mesencephalon express Gad1 but not Vglut2. By contrast, in the Helt mutants, most Nkx2.2+ neurons express Vglut2, although some populations maintain Gad1 expression.

View larger version (118K): [in this window] [in a new window]   Fig. 5. Identification of postmitotic transcription factors selectively expressed in GABAergic and glutamatergic neurons in the mesencephalon. (A,B) Selective expression of Lim1 and Brn3a by GABAergic and glutamatergic neurons in the developing mouse mesencephalon. (A) Lim1 and Brn3a are mutually exclusively expressed, with the exception of the RN (m6) domain. (B) Brn3a is coincidently expressed by Vglut2+ neurons, although Vglut2 is also expressed by ventral midline dopaminergic neurons and not by Gad1+ neurons. By contrast, Lim1 is selectively expressed by Gad1+ neurons, with the exception of coexpression with Vglut2 and Brn3a in the RN (m6) domain.

View larger version (102K): [in this window] [in a new window]   Fig. 6. Helt induces Lim1+ neurons and suppresses Brn3a+ neurogenesis. (A) Effects of the Helt-null mutation on Lim1 and Brn3a expression. In the Helt-/- mutant at E11.5, Lim1 expression is lost in the m2 to m4 domains, whereas expression in the m5 and m6 domains is unaffected. At E13.5, Lim1 expression in the dorsal m1 domain is completely lost. By contrast, Brn3a is ectopically expressed in the m2 to m4 domains of the mutants at E11.5. In the m1 domain, virtually all the emerging neurons are positive for Brn3a at E13.5 and coexpression of GFP and Brn3a is observed. (B) Exogenous Helt suppresses Brn3a+ neurogenesis and induces Lim1+ neurons. In the area efficiently expressing exogenous Helt, many Lim1+ neurons are generated, but fewer Brn3a+ neurons emerge compared with wild-type control mouse embryos. Exogenous Helt-induced Lim1+ neurons express Gad65.

View larger version (80K): [in this window] [in a new window]   Fig. 7. Helt represses Ngn genes. (A) Expression patterns of proneural genes in the developing mouse mesencephalon. Expression of Ngns and Helt is mutually exclusive in all the domains. By contrast, Mash1 is expressed in essentially all domains and is coexpressed with Helt. (B) Ngn genes are selectively expressed in glutamatergic progenitors. Ngn1 is coincidently expressed in the VZ, where Brn3a+ neurons emerge. Ngn2 is selectively expressed by glutamatergic progenitors as well as dopaminergic progenitors. (C) Helt represses Ngn expression in the mesencephalon. In transgenic embryos expressing Helt under the control of the nestin enhancer, expression of Ngn1 and Ngn2 is repressed. By contrast, Mash1 expression is unaffected. (D) Ngns are ectopically expressed in presumptive GABAergic domains in the Helt-null mutant. At E11.5, Ngn1 and Ngn2 are ectopically expressed in the m2 to m4 domains in the Helt-/- mutant. At E12.5, the Ngn expression in the dorsal m1 domain is upregulated and coexpression of GFP and the Ngns is observed (inset).

View larger version (70K): [in this window] [in a new window]   Fig. 8. Ngn1 promotes glutamatergic differentiation. (A) Ngn1 induces ectopic Brn3a+ neurons in the ventral Helt+ domains. In transgenic mouse embryos expressing Ngn1 under the control of the nestin enhancer, Brn3a+ neurons are generated at E11.5 despite the fact that Helt expression is not repressed (arrowheads). Note that most of the exogenous Ngn1-expressing neurons in the ML express Brn3a. (B) Ngn1 determines glutamatergic fate in the m1 domain. Shown are the dorsal m1 domains of wild-type control embryos and transgenic embryos expressing Ngn1 and IRES-controlled GFP under the control of the nestin enhancer at E12.5. Neurons generated from transgene-expressing progenitors are marked by GFP expression. Virtually all the GFP+ neurons express Brn3a and do not express Lim1.

View larger version (38K): [in this window] [in a new window]   Fig. 9. Model for transmitter phenotype determination in the developing mesencephalon. (A) Summary of the phenotype of Helt-null mutant mouse embryos. (B) Model for transmitter phenotype determination by the Helt and Ngn genes. Helt determines the GABAergic phenotype by repressing the Ngn genes, which induce the glutamatergic pathway. Mash1 might also be involved in the GABAergic determination pathway (Miyoshi et al., 2004). GA, GABAergic; GL, glutamatergic; DA, dopaminergic; VZ, ventricular zone; ML, mantle layer.

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