GABAergic and glutamatergic identities of developing midbrain Pitx2 neurons

Abstract

Pitx2, a paired-like homeodomain transcription factor, is expressed in post-mitotic neurons within highly restricted domains of the embryonic mouse brain. Previous reports identified critical roles for PITX2 in histogenesis of the hypothalamus and midbrain, but the cellular identities of PITX2-positive neurons in these regions were not fully explored. This study characterizes Pitx2 expression with respect to midbrain transcription factor and neurotransmitter phenotypes in mid-to-late mouse gestation. In the dorsal midbrain, we identified Pitx2-positive neurons in the stratum griseum intermedium (SGI) as GABAergic and observed a requirement for PITX2 in GABAergic differentiation. We also identified two Pitx2-positive neuronal populations in the ventral midbrain, the red nucleus, and a ventromedial population, both of which contain glutamatergic precursors. Our data suggest that PITX2 is present in regionally restricted subpopulations of midbrain neurons and may have unique functions that promote GABAergic and glutamatergic differentiation.

Figure 1

PITX2 identifies GABAergic interneurons in an intermediate layer of the dorsal midbrain. (A) Cartoon showing sagittal view of an embryonic mouse brain with midbrain highlighted in pink and a dotted line indicating the location of coronal sections shown in C–T. (B) Cartoon of a coronal midbrain section highlighting the superior colliculus (SC), aqueduct (AQ), alar-basal boundary, and the ventricular (VZ), intermediate (IZ), and mantle zones (MZ). (C–H) E14.5 and P8 midbrains processed for immunofluorescence for PITX2 (red) and GABA (green). Boxes in E and H are enlarged in E′ and H′. At E14.5, PITX2-positive and GABA-positive cells are located in the intermediate and mantle regions of the superior colliculus, where most PITX2-positive cells are also GABA-positive (E′). White arrow in E′ indicates co-localization of PITX2 and GABA. Open arrow indicates a GABA-positive, PITX2-negative neuron. (F–H′) At P8, PITX2-positive cells occupy an intermediate GABAergic layer of cells where GABA-positive cytoplasm surrounds PITX2-positive nuclei. (I–N′) E14.5 and P8 midbrains processed for immunofluorescence for LHX1/5 and GABA. Boxes in K and N are enlarged in K′ and N′. At E14.5, LHX1/5-positive cells are distributed throughout the superior colliculus, whereas GABA-positive cells reside in the intermediate and mantle zones. At P8, some LHX1/5-positive cells are GABA-positive (white arrow in K′). The open arrow in K′ indicates a GABA-positive, LHX1/5-negative neuron. (O–Q′) At P8, PITX2-positive neurons are localized superficial to the LHX1/5-positive population. (R–T) Adjacent sections from P8 brains processed for acetylcholinesterase (AChE) staining and Pitx2 in situ hybridization shows Pitx2 mRNA and AChE strongly expressed in an intermediate superior colliculus layer, the stratum griseum intermedium (SGI). Dotted lines indicate the outline of the AChE-positive layer. Scale bar in C is 100 μm and applies to panels C–E and I–K. Scale bar in F is 100 μm and applies to panels F–H and L–Q. Scale bar in R is 250 μm and applies to panels R–T. Figures in L–N′ were imaged using confocal microscopy. Abbreviations: mantle zone (MZ); intermediate zone (IZ); stratum griseum superficiale (SGS); stratum opticum (SO); stratum griseum intermedium (SGI); stratum album intermedium (SAI).

Figure 2

Collicular glutamatergic neurons are BRN3A-positive and PITX2-negative. Coronal sections of E14.5 and P8 midbrains were processed for immunofluorescence for PITX2 and VGLUT2 (A–F). Boxes in C and F are enlarged in C′ and F′. Dotted lines indicate the midbrain pial surface. (A–C′) At E14.5, VGLUT2 is absent from the dorsal midbrain. (D–F′) At P8, PITX2-positive cells are located in the SGI and VGLUT2-positive neurons occupy the intermediate and deep layers of the superior colliculus; however, VGLUT2-positive staining does not circumscribe the PITX2-positive nuclei. (G–L′) Coronal sections of E14.5 and P8 midbrains processed for immunofluorescence for BRN3A and VGLUT2. Boxes in I and L are enlarged I′ and L′. At E14.5, BRN3A-positive cells are located in the deep and intermediate superior colliculus, and VGLUT2 is absent. (J–L′) At P8, VGLUT2 circumscribes collicular BRN3A-positive nuclei. (M–R) Coronal sections from E16.5 (M–O) and E18.5 (P–R) embryos labeled with antibodies against PITX2 and BRN3A show that PITX2 is present in an intermediate layer positioned between two BRN3A-positive layers. (P–R) At E18.5, PITX2 and BRN3A continue to be localized in separate tectal layers and the PITX2-positive layer appears more compact. Dotted lines indicate the outline of the PITX2-positive collicular layer. Scale bar in A is 100 μm and applies to panels A–C and G–1. Scale bar in D is 100 μm and applies to panels D–F and J–L. Scale bar in M is 100 μm and applies to panels M–R. Panels D–F′ and J–L′ were imaged using confocal microscopy. Abbreviations: mantle zone (MZ); intermediate zone (IZ); stratum griseum intermedium (SGI).

Figure 3

PITX2-positive cells represent a unique population of GABAergic dorsal midbrain precursors. Coronal sections of E12.5 and E14.5 midbrains were processed for double immunofluorescence with antibodies against markers of neuronal precursors. At E12.5 and E14.5, PITX2-positive cells (red) reside in the mantle layer of the superior colliculus. (A–B) GATA2-positive cells are located intermedially at E12.5 and extend throughout the superior colliculus at E14.5, but do not overlap with PITX2-positive cells. (C–D) LHX1/5 is located deep to PITX2 at E12.5 and by E14.5 LHX1/5-positive cells are found throughout the superior colliculus. At both timepoints, only a few neurons show co-localization of both markers (inserts in C′, D′). White arrows indicate cells with co-localization. (E–F) BRN3A and PITX2 are present in distinct cells at E12.5 and E14.5. (G–H) PAX3-positive cells are found throughout dorsal ventricular and intermediate zones and are PITX2-negative at E12.5. (H) At E14.5, PAX3-positive cells are PITX2-negative and ventricularly restricted. (I–J) PAX7-positive cells are broadly distributed throughout the superior colliculus and expanded superficially at E14.5 but do not co-localize with PITX2. Dotted lines indicate the pial surface. Scale bars in A and B are 100 μm and apply to panels A, C, E, G, I and B, D, F, H, J, respectively. Abbreviations: mantle zone (MZ); intermediate zone (IZ); ventricular zone (VZ).

Figure 4

PITX2 is required for GABAergic differentiation. Coronal sections of E14.5 Pitx2^Cre/+;NL and Pitx2^Cre/−;NL midbrains were processed for double immunofluorescence with antibodies against βGAL and GABA (A, B). Boxes in A and B are enlarged in A′ and B′. (A, A′) PITX2-lineage cells in the Pitx2^Cre/+;NL embryo are located near the pial surface in a strongly GABAergic layer and are GABA-positive. (B, B′) PITX2-lineage cells in the Pitx2^Cre/−;NL embryo are medially mislocalized and are GABA-negative. (C–L) Pitx2^Cre/+;NL and Pitx2^Cre/−;NL coronal E14.5 midbrain sections were processed for immunofluorescence with antibodies against PAX3, PAX7, GATA2, LHX1/5, or BRN3A. (C–D) PAX3 is restricted to the ventricular zone in both Pitx2^Cre/+;NL and Pitx2^Cre/−;NL embryos. (E–F) PAX7-positive cells are distributed throughout the colliculus of both Pitx2^Cre/+;NL and Pitx2^Cre/−;NL midbrains. (G–H) GATA2 is restricted to deep collicular cells and loss of PITX2 does not affect GATA2 patterning. (I–L) In both Pitx2^Cre/+;NL and Pitx2^Cre/−;NL midbrains, LHX1/5 and BRN3A-positive cells are spread throughout the superior colliculus. Panels A–L were imaged using confocal microscopy. Scale bars in A and C are 100 μm and apply to A–B and C–L, respectively.

Figure 5

PITX2 identifies restricted populations of ventromedial midbrain precursors. Immunofluorescence of E12.5 midbrain coronal sections processed with antibodies against PITX2 (red) and other ventral midbrain markers (green) and imaged with confocal microscopy. (A′) Cartoon showing coronal view of an embryonic mouse midbrain identifying two ventral PITX2-positive populations. Boxes indicate the location of the ventromedial (VM) and red nucleus PITX2-positive populations magnified in panels A–L and M–U, respectively. (A–C) FOXA2 marks precursors in the m6 domain and is co-localized with PITX2 in deep m6. (D–I) Most ventromedial PITX2-positive cells are positive for LHX1/5 and BRN3A, which mark glutamatergic precursors in the m6 domain. (J–L) NKX6.2-positive cells in deep m6 are also PITX2-positive. White arrows indicate transcription factor co-localization with PITX2, whereas open arrows indicate PITX2-positive, FOXA2, LHX1/5, or BRN3A-negative cells. Panels M–U focus on PITX2-positive cells in the red nucleus. (M–R) PITX2-positive cells in the red nucleus are FOXA2 and LHX1/5-positive. (S–U) Most but not all BRN3A-positive red nucleus cells are PITX2-positive. Scale bar in A applies to panels A–U and is 25 μm.

Figure 6

Ventral midbrain domains are delineated by transcription factor patterning. E12.5 coronal sections were processed for immunofluorescence with antibodies against PITX2 (red) and other ventral midbrain markers (green) and imaged with confocal microscopy. (A) NKX6.1 is restricted to m6 progenitors and a PITX2-negative region between deep m6 and the PITX2-positive red nucleus. (B) Ventromedial PITX2-positive cells in m6 reside near GATA2-positive cells in the m5 domain. (C) NKX2.2-positive cells occupy m4 and m5, but not the m6 domain. (D) ISL1-positive oculomotor neurons (OMN) are located superficial to the deep PITX2 population in m6. Scale bar in A is 50 μm and applies to panels A–D. Abbreviations: ventromedial population (VM), red nucleus (RN), and oculomotor nucleus (OMN).

Figure 7

Early ventral midbrain patterning is PITX2-independent. E12.5 coronal sections were processed for immunofluorescence with antibodies against BRN3A (green) and FOXA2, LHX1/5, NKX6.1, ISL1, or NKX6.2 (red) and imaged with confocal microscopy. (A–B) FOXA2-positive cells in m6 are localized in the ventricular zone and the BRN3A-positive red nucleus in both Pitx2^Cre/+ and Pitx2^Cre/− midbrains. (C–D) LHX1/5-positive cells are distributed from deep m6 to the red nucleus, where all LHX1/5-positive cells are also BRN3A-positive. LHX1/5 patterning appears unchanged in Pitx2^Cre/− midbrains. (E–F) Cells in the ventricular zone are weakly NKX6.1-positive and a second, more lateral population is strongly NKX6.1-positive. Neither NKX6.1-positive population displays co-localization with BRN3A and both are unchanged in the Pitx2^Cre/− midbrain. (G–H) In both Pitx2^Cre/+ and Pitx2^Cre/− tissues, ISL1 marks cells in the oculomotor nucleus which is surrounded by BRN3A-positive cells. (I–J′) NKX6.2-positive cells intermingle with BRN3A-positive cells and a few cells are also BRN3A-positive (white arrows) in both Pitx2^Cre/+ and Pitx2^Cre/− embryos. Scale bar in A is 100 μm and applies to panels A–J. Abbreviations: ventricular zone (VZ) ventromedial population (VM), and red nucleus (RN).

Figure 8

PITX2-lineage neurons are glutamatergic and sparse in the red nucleus. (A–F) Coronal sections of E14.5 midbrains processed for PITX2 and BRN3A (A–C) or GABA (D–F) immunofluorescence. Dotted areas demarcate cells in the PITX2-positive deep ventromedial population and the red nucleus. Boxes in C, F, I, and L are enlarged in C′, F′, I′, and L′. (A–C′) At E14.5, the red nucleus is composed of BRN3A-positive neurons, a few of which are PITX2-positive. White arrows indicate co-localization of BRN3A and PITX2, whereas open arrows indicate BRN3A-positive, PITX2-negative neurons. (D–F′) Ventral GABAergic neurons are generally restricted to the m5 domain, and are PITX2-negative. (G–L′) E18.5 Pitx2^Cre/+;NL coronal sections were processed for immunofluorescence for β-galactosidase (βGAL) and VGLUT2 or BRN3A and visualized with confocal microscopy. (G–I′) At E18.5, PITX2-lineage neurons are present in the red nucleus and are VGLUT2-positive. (J–L′) E18.5 red nucleus PITX2-lineage neurons are also BRN3A-positive. White arrows indicate co-localization of βGAL and BRN3A, whereas the open arrow indicates BRN3A-positive, βGAL-negative neurons. Dotted areas delimit the boundary of the red nucleus. Scale bars in A and G are 100 μm and 50 μm and apply to panels A–F and G–L, respectively. Abbreviations: ventromedial population (VM) and red nucleus (RN).

Figure 9

Pitx2 expression is transient in the ventral midbrain. (A–L) Adjacent coronal sections of Pitx2^Cre/+;NL midbrains processed for βGAL histochemistry (A, B, E, F, I, J), Pitx2 mRNA (C, G, K) or PITX2 immunofluorescence (D, H, L). The dotted line demarcates the red nucleus. (A) At E14.5, Pitx2-lineage neurons are located in the superior colliculus (SC) and red nucleus (RN). (B) High magnification of panel A shows only a few Pitx2-lineage cells in the red nucleus, despite high red nucleus Pitx2 mRNA (C). (D) Immunofluorescence indicates only a few PITX2-positive red nucleus cells. (E, F, I, J) At E16.5–E18.5, β-galactosidase activity shows Pitx2-lineage neurons in the superior colliculus and red nucleus. (G, H, K, L) The E16.5–E18.5 red nucleus continues to express Pitx2 mRNA, although PITX2 protein is absent. (M) Coronal section of an E14.5 Pitx2^Cre/+;ZsGrn midbrain showing ZsGrn fluorescence in the superior colliculus. Few cells are fluorescent in the ventral midbrain as seen in a high magnification image of the red nucleus (N). (O) A neighboring section to M processed for Pitx2 in situ hybridization shows Pitx2 mRNA in the superior colliculus, ventromedial population, and red nucleus. (P) Higher magnification of the red nucleus in O. Dotted areas denote the ventromedial population and red nucleus. Scale bar in A is 125 μm and applies to panels A, E, and I. Scale bar in B is 32μm and applies to panels B–D, F–H, and J–L. Scale bars in M and N are 100μm and 50 μm and apply to panels M, O and N, P, respectively.

Figure 10

Summary of Pitx2 expression in the developing dorsal and ventral midbrain. Schematic is based on previously published models (Nakatani et al., 2007; Kala et al., 2009), wherein early developmental transcription factors were mapped by domain. (A, C) Cartoons of typical E12.5 coronal sections showing PITX2-positive cells mapped onto the domain-delineated midbrain. PITX2-positive cells (red) are abundant in m1-m4 domains in the superior colliculus (SC) and in the m6 domain (C) containing a ventromedial (VM) population and the red nucleus (RN). PITX2-positive cells are sparse in m5 and m7. (B, D) Solid bars show areas with no overlap in marker expression with PITX2; hatched bars show areas of co-localization with PITX2. In m1–m4, PITX2-positive neurons express some markers of GABAergic precursors and neurons (LHX1/5 and GABA, respectively). In m6, PITX2 co-localizes with glutamatergic markers (D). PITX2 is co-localized with the m6 precursor markers FOXA2, LHX1/5, BRN3A, and NKX6.2 in the ventromedial population. PITX2 neurons in the early red nucleus are positive for FOXA2, LHX1/5, and BRN3A and red nucleus PITX2-lineage neurons are glutamatergic. Abbreviations: dopaminergic neurons (DA); glutamatergic neurons (GLUT); GABAergic neurons (GABA); ventricular zone (VZ); ventromedial population (VM); oculomotor nucleus (OMN); red nucleus (RN); mantle layer (ML).