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First published online 15 December 2008
doi: 10.1242/dev.029900

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Gata2 is a tissue-specific post-mitotic selector gene for midbrain GABAergic neurons

¹ Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
² Division of Biochemistry, Faculty of Veterinary Medicine, University of Helsinki, 00014 Helsinki, Finland.
³ Department of Developmental Biology and Department of Physiology, University of Tartu, 50090 Tartu, Estonia.
⁴ Helmholtz Zentrum München, Institute of Developmental Genetics, D-85764 Neuherberg, Germany.

View larger version (110K): [in this window] [in a new window]   Fig. 1. Gata2 and Gata3 expression in embryonic mouse midbrain compared with expression of neural subtype markers. (A-R) In situ hybridization (ISH) with Gata2, Gata3, Gad1, Pou4f1, Isl1 and Lmx1b probes on coronal sections of wild-type embryos. The embryonic stages are indicated at the top of each column. Black arrows point to the negative areas within the expression domains of Gata2, Gata3 and Gad1. (S-X) Co-immunostaining for Gata2 (red) and Lhx1, Nkx6-1, Nkx2-2, Pax6 or Olig2 (green). The boxed area in S indicates the region enlarged in T-X. White arrows indicate examples of Gata2 and Nkx6-1 (U), and Gata2 and Nkx2-2 (V), coexpressing cells. Scale bars: 100 µm.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Expression of Gata2 during GABAergic differentiation. (A-F'') Relationship between Gata2, Helt and Ascl1 expression in GABAergic progenitors analyzed by immunohistochemical co-staining. The boxed area in A-C is enlarged in D-H''. Nuclei at the apical (Ap) side of the ventricular zone abundantly coexpress Helt (D') and Ascl1 (D''). Nuclei near the basal (Ba) side of the ventricular zone coexpress Ascl1 (F') and Gata2 (F''). Nuclei coexpressing Helt (E') and Gata2 (E'') are detected mostly in the medial ventricular zone. (G-H'') Analysis of post-mitotic differentiation of the Gata2-positive cells. Both HuC/D-positive (arrows) and -negative (arrowheads) cells expressing Gata2 can be detected (G). Ventricular zone cells expressing Gata2 do not incorporate BrdU during a 1.5-hour labeling pulse (H). Scale bars: 100 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Gata2 and GABAergic marker gene expression in Helt-null and Ascl1-null mouse embryos. (A-F) ISH analysis of Gata2, Gad1 and Ascl1 expression on coronal sections of E11.5 WT and HeltKO midbrains. Arrows indicate the ventral population of GABAergic neurons that still develop in the Helt-null mutants. (G-J) Immunohistochemistry (IHC) of Gata2 and Helt expression in E11.5 wild-type (WT) and Ascl1-null embryos. (K,L) Sox2 and HuC/D co-IHC demonstrates delayed neurogenesis in the Ascl1-null embryo, especially in the m3 GABAergic domain. The borders of the m3 region were deduced from Nkx2-2 IHC signal on adjacent sections. Scale bar: 100 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 4. Fate transformation of midbrain GABAergic neuron precursors in Gata2cko mouse embryos. (A-P) ISH analysis of Gad1, Slc17a6, Gata3, Pou4f1, Helt, Nkx2-2, Ascl1 and Ngn2 expression on coronal sections of E11.5 wild-type (WT) and Gata2cko midbrains. (A'-D') Higher magnification views from the GABAergic neurogenesis domain. Black arrows point to the small group of Slc17a6-expressing glutamatergic neurons in m4 (C,C') surrounded by Gad1-expressing GABAergic neurons (A,A') in WT embryos. (Q-X) IHC analysis of transcription factors Nkx6-1, Lhx1 and Pax6 in the m3-m5 domains of E11.5 WT and Gata2cko midbrain. The borders of the m4 domain (S-X) were deduced from the borders of Nkx2-2 ISH or IHC signal on adjacent sections. The ventral border of m5 was deduced from the ventral border of Helt expression. The border between m3 and m1-2 (W) was deduced from the ventral border of Pou4f1 IHC on an adjacent section. White arrows (W,X) point to the dorsal part of m4 (m4-D). Scale bars: 100 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Induction of GABAergic differentiation in chick embryonic midbrain by ectopic Gata2. (A-I) The expression patterns of Helt (ISH), cGad1 (ISH), cNgn2 (ISH), Gata2 (IHC), cGata3 (ISH), Lhx1 (IHC) and cSlc17a6 (ISH) in coronal sections of E3.5 (A-F) and E4 (G-I) embryonic chick midbrain (c, chicken). Arrows in C point to cNgn expression in the marginal zone of dorsal midbrain. Similar to in mouse midbrain, a gap was observed in ventrolateral cGad1 expression (arrowhead in H). Scattered cGad1 expression appears in the dorsal midbrain by E4 (arrows in H). (J-Q) Electroporation of Gata2-HA expression vector into chick dorsal midbrain. Expression of the electroporated Gata2 is detected by anti-HA antibody (J,O). Expression of cGad1 (M,P), cGata3 (L) and cNgn2 (N,Q) in the electroporated area was detected by ISH. Induction of Lhx1 was detected by IHC (K). Arrowheads (J-N) mark the electroporated area expressing ectopic Gata2. Red arrows (O-Q) point to the cells that express ectopic Gata2 (HA-positive). Scale bars: 100 µm.

View larger version (116K): [in this window] [in a new window]   Fig. 6. Development of r1 GABAergic and serotonergic neurons in Gata2cko mouse embryos. ISH with Gata3 (A,B) and Gad1 (E,F) probes on adjacent coronal sections of E11.5 wild-type (WT) and Gata2cko embryos. Midbrain (mb) and rhombomere 1 (r1) regions are indicated. The arrowhead points to Gata3 expression in the serotonergic neuron precursors (A). This Gata3 expression domain is lost in the Gata2cko mutants (arrowhead in B). Anti-serotonin (5-HT) IHC on coronal sections of E11.5 WT (I) and Gata2cko (J) embryos. Analysis of the serotonergic neuron markers Lmx1b (C,D, ISH), Fev (G,H, ISH) and 5-HT (K,L, IHC) on adjacent coronal sections of E13.5 WT and Gata2cko mutants. In I-L, higher magnification views from the r1 area are presented. Scale bars: 100 µm.

View larger version (144K): [in this window] [in a new window]   Fig. 7. Analysis of midbrain GABAergic neurons in Gata2cko mouse mutants at late embryonic stages. (A-H) IHC analysis of tyrosine hydroxylase (TH) and ISH with Gad1, Gata3 and Slc17a6 probes on adjacent coronal sections of E18.5 wild-type (WT) and Gata2cko midbrains. (I-P) TH, Gad1, Gata3 and Slc17a6 expression on adjacent coronal sections of E15.5 WT and Gata2cko midbrains. SN, substantia nigra; SNpr, substantia nigra pars reticulata; VTA, ventral tegmental area. The dashed line delineates the TH-positive SNpr-VTA area. Scale bars: 100 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 8. Model for patterning and neurotransmitter identity determination in the midbrain. (A) Patterns of gene expression in the ventricular zone proliferative progenitors and post-mitotic precursors in the midbrain of wild-type (WT) and Gata2cko mouse embryos. The m4 domain is divided into dorsal (D) and ventral (V) parts. (B) Model of the transcription factor functions regulating GABAergic neurogenesis. Cell cycle exit of GABAergic progenitors is primarily regulated by Ascl1, whereas the neurotransmitter identity is independently controlled by sequential functions of Helt and Gata2. In m5, Helt is not essential for Gata2 expression, which may be activated by additional mechanisms.

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