Introduction
The Substantia nigra (SN) is a complex nucleus located not only in the mesencephalic basomedial territory (Puelles
2007; Moreno-Bravo et al.
2012; Puelles et al.
2012) but it is also extended along the pretectum, thalamus and prethalamus (diencephalic prosomeres). It is divided into a pial superficial part, SN pars reticulata (SNR), constituted by GABAergic neurons (GABAn) and a more internal SN pars compacta (SNC), primarily containing dopaminergic neurons (Hanaway et al.
1970). There is, nonetheless, some intermixing of dopamine neurons within the SNR (González-Hernández and Rodríguez
2000). The dopaminergic neurons are a deeply studied population due to their implication in several motor syndromes such as Parkinson’s disease; however, the molecular diversity and regulation of GABAn development are only beginning to be understood. These GABAn control several aspects of behavior, play important roles in psychiatric diseases, susceptibility to drugs of abuse and are also important targets for several medical treatments for these diseases (Jhou et al.
2009; Vargas-Perez et al.
2009; Cohen et al.
2012).
The SNR and the internal segment of the globus pallidus provide the major output projections of the basal ganglia system where the final stage of information processing takes place. These cell groups are mainly composed of GABAn; they integrate inputs from all other components of the basal ganglia system (striatum, globus pallidus, subthalamic nucleus) and elaborate the message sent by this system to extrinsic structures (Rinvik et al.
1976). For this purpose, SNR neurons project to the superior colliculus, reticular formation and thalamus, mainly to the ventral lateral and ventral anterior region. SNR GABAn also issue local axon collaterals that carry out an important role of inhibition within the SNC itself. Therefore, the SNR constitutes one of the main output pathways of the basal ganglia system regulating mainly voluntary movements (Beckstead et al.
1979).
The mechanisms of GABAergic development in the midbrain have been, surprisingly, neglected until recently. On the one hand, Nakatani et al. (
2007) studied the spatial patterning relevant to the GABAergic neurogenesis. Seven distinct progenitor domains were identified along the midbrain neuroepithelium dorsoventral axis (m1–m7; Nakatani et al.
2007; renamed in Puelles et al.
2012). GABAn are originated from the domains m3 to m5 (corresponding to alar ventro-lateral, basal lateral and basal intermediate domains; Puelles et al.
2012), and later in development also from m1 and m2 (corresponding to alar dorsal and alar lateral domains).
On the other hand, Achim et al. (
2012) analyzed molecular regulation of ventral tegmental area (VTA) and SNR GABAn differentiation. They demonstrated that GABAn of these regions, mainly the caudal portion, were originated in rhombomere 1 (r1) and occupied their final destination by tangential migration. Nevertheless, the origin of the main rostral mes-diencephalic SNR GABAn population was not described.
Our previous data pointed out that
Nkx6.2 transcription factor plays an important role in the determination and differentiation of the mesencephalon and diencephalon ventral neuronal populations. We found a
Nkx6.2 dynamic expression pattern in the developing mes-diencephalic basal plate, with an early alar positive ventricular domain. However, later in development, only the pre-Edinger-Westphal remains
Nkx6.2 positive (preEW; described previously as Interstitial mesencephalic nucleus by Moreno-Bravo et al.
2010). In other regions of the brain,
Nkx6.2 positive ventricular territories give rise to a massive amount of derivatives which switch off its expression as they differentiate and migrate tangentially (Fogarty et al.
2007). This study prompted us to analyze the fate of the mesencephalic
Nkx6.2 derivatives. We found out that they contribute to several basal populations, being the SNR among them. With the aim to verify their mesencephalic origin, we selected
Six3, a positive marker of SNR (Conte et al.
2005). This transcription factor belongs to the sine oculis family (Oliver et al.
1995) and it already has been involved in GABAn development (Virolainen et al.
2012). It has a complex expression pattern restricted to the fore- and midbrain (Conte et al.
2005). Summarizing, our working hypothesis postulates a complex multiple origin of the SNR neurons. We demonstrate, using the transcription factors
Nkx6.2 and
Six3, the mesencephalic neuronal contribution to the SNR. The GABAn generated in the
Nkx6.2 positive ventricular domain populated the SNR in a rostrocaudal gradient.
Discussion
The SN GABAn origin is still not completely unveiled despite the studies developed in the last years. Here, we planned to find out the origin of the rostral SNR GABAn. We demonstrated that the rostral SNR is colonized by alar mesencephalic Nkx6.2 derivatives. These tangentially migrated neurons populate mainly the rostral diencephalic part of the SNR, but are also present in the caudal mesencephalic part.
We hypothesized that the rostral SNR had an alar mesencephalic origin. There were several preliminary data in the literature that supported our hypothesis. Fate map analysis of the mesencephalic basal plate
Shh positive derivatives demonstrated that the SNR is derived from a
Shh negative territory (Joksimovic et al.
2009; Achim et al.
2012). Therefore, the generation of the SNR GABAn in the
Shh negative r1 basal plate or in the mesencephalic alar plate appeared as plausible hypothesis. It has been also proven that the SNR GABAn are partially derived from r1 (Achim et al.
2012). These authors do not exclude a mesencephalic or diencephalic origin of the rostral SNR as the rhombomeric originated GABAn described concentrate in the caudal SNR (Achim et al.
2012).
Our previous studies of
Nkx6.2 expression pattern showed that this transcription factor has a ventricular positive domain adjacent to the alar–basal boundary. This domain gives rise to the preEW, a neuronal population that maintains
Nkx6.2 expression and migrates tangentially into the basal plate.
Nkx6.2 derived neurons usually display tangential migration events. The analysis of the cortical GABA interneurons origin demonstrated that the
Nkx6.2 positive ventricular domain in the medial ganglionic eminence gives rise to a huge amount of GABAn that switch off the expression of the gene as they differentiate and migrate into the cortex (Fogarty et al.
2007). This result supported our hypothesis that the
Nkx6.2 positive ventricular domain could contribute neurons to different mesencephalic populations, by tangential migration, as proposed by Verney et al.
2001.
The use of a transgenic mouse line (
Nkx6.2
tmcre/+;
tdTomato
flox/+;
Gad67
gfp+/−) allowed us to label all the derivatives generated from
Nkx6.2 positive neuroblasts (RFP+) and also to distinguish the GABAn (GFP+) among them. This analysis demonstrated the colonization of basal neuronal structures by these derivatives. The SNR was among these neuronal populations. We found RFP+ neurons distributed in a rostrocaudal gradient along the SNR. This distribution was opposed and complementary to the r1 derived GABAn described by Achim et al. (
2012).
The distribution of these two subpopulations is translated in neuronal morphological differences, the rostrolateral SNR is populated by fusiform GABAn with major cellular diameter and the caudomedial SNR by elongated GABAn with minor diameter (González-Hernández and Rodríguez
2000). These differences have been also illustrated by SNR projections labeling. Both territories project to the same thalamic areas but the rostral SNR also projects to the centrolateral and thalamic reticular nucleus (Gulcebi et al.
2012).
Due to the proximity of the territories involved (r1, isthmus and midbrain), we confirmed the mesencephalic origin of the RFP+; GFP+ neurons using
Six3 as specific mesencephalic marker. It reported its expression in the SNR (Conte et al.
2005) and it is never expressed along development in the hindbrain (Oliver et al.
1995). Therefore, taking into account of all these data, we can postulate that the SNR GABAn are originated, at least, from two different sources, r1 and midbrain. The GABAn originated in these two territories are distributed in two opposite rostrocaudal gradients and certainly present neuronal morphological, projections and functional differences.
The molecular regulation of the GABAn differentiation associated with the populations described is distinct from the rest of mesencephalic GABAn (Lahti et al.
2013). In the
Gata2
cko
mutant, all the midbrain GABAn populations were transformed to a glutamatergic phenotype, except for the SNR and mRf (Lahti et al.
2013). This information together with our data allowed us to postulate that
Nkx6.2 and
Six3 must participate sequentially in the genetic cascade responsible of rostral SNR and mRf neuronal differentiation program.
It has been described that the GABAn development in the different regions of the central nervous system is regulated by diverse genetic mechanisms. Transcription factors, such as
Ascl1,
Helt or
Gata2, have been shown to be selectively required for the development of midbrain GABAn. However, GABAn associated with the dopaminergic nuclei in the VTA and SN do not require any of them (Peltopuro et al.
2010). Indeed, as they develop independently of the known transcriptional regulators, the VTA and SNR GABAn appear molecularly distinct (Guimera et al.
2006; Kala et al.
2009) and therefore likely to have a different origin.
Strikingly, in the
Gata2
cko
mutant, all the midbrain GABAn subpopulations were transformed to a glutamatergic phenotype, except for the GABAn associated with the DA neurons in the VTA and SNR, indicating that the remaining mesencephalic GABAn could be born in a region of the midbrain that does not require
Gata2 (Kala et al.
2009). During postmitotic differentiation,
Gata2 controls the expression of downstream GABAn-specific genes and transcription factors (Virolainen et al.
2012), but in
Gata2
cko
embryo the expression of
Six3 is altered but does not disappear (Peltopuro et al.
2010).
In the last years,
Tal2 has been identified as a firm candidate to control the differentiation of the SNR GABAn. Together with
Gata2, it is expressed in all GABAergic precursors in the area spanning from zona limitans to the midbrain–hindbrain boundary (Achim and Salminen
2014). This coexpression does not imply a direct interaction since
Tal2 expression does not require
Gata2 function (Virolainen et al.
2012). The analysis of the
Tal2 lack of function corroborated its role in SNR GABAn differentiation. In the
Tal2 mutant, the
Six3 expression is completely lost and
Gad1 expression, and therefore GABAn differentiation, is absent specifically in the BL domain of the midbrain (location of the
Nkx6.2+ ventricular domain; Achim et al.
2013). As expected, the generation of the SNR is strongly affected.
All this data support the hypothesis that Tal2 regulates the differentiation of the SNR GABAn. This regulation takes place in the BL mesencephalic domain where Nkx6.2 is expressed in the ventricular neuroblasts and Six3 is expressed in the early-differentiated neurons in the mantle layer. These early GABAn migrate tangentially until their final destination in the SNR.
Another important conclusion to highlight from our data is that we have identified an alar ventricular domain in the mesencephalon able to give rise to different neuronal types. Early in development, it produces glutamatergic neurons that tangentially colonize the preEW nucleus. Later, the Nkx6.2 positive neuroblasts switch and generate GABAn that tangentially and radially populate the SNR and mRf, respectively.
Finally, the midbrain dopaminergic neurons (SNC and VTA) and their development have been under intensive research due to their relation to Parkinson’s disease. However, importance of the VTA- and SN-associated GABAn for the activity of dopaminergic pathways and behavioral control has become increasingly evident (Vargas-Perez et al.
2009). In fact, GABAn in the ventral mesodiencephalic region are highly important for the function of dopaminergic pathways that regulate multiple aspects of behavior and movement control. These complex morphological and functional structures display intricate developmental processes with multiple origins and migratory routes. Consequently, all our results contribute to implement our knowledge of how these important GABAergic populations are generated.
Material and methods
Mouse strains
The mouse lines used and their genotyping have been described previously:
Nkx6.2 cre ER
T2
(Feil et al.
1997; Sousa et al.
2009),
GAD67-
GFP (Tamamaki et al.
2003),
R26R-
CAG-
tdTomato, obtained from Jackson Laboratories (strain 007905). A loxP-flanked STOP cassette prevents transcription of the downstream RFP variant (tdTomato) in the TdTomato reporter mice.
Nkx6.2
cre/+; tdTomato
flox/+; are generated by crossing homozygous mouse males (Nkx6.2cre/cre) with homozygous reporter females (tdtomato
flox/flox
). The triple mutant embryos, Nkx6.2
cre/+; tdTomato
flox/+; Gad67
gfp/+ were generated by crossing homozygous mouse males (Nkx6.2
cre/cre
) with double heterozygous females (tdTomato
flox/+; Gad67
gfp/
+
). For tamoxifen induction, we administer 4 mg of tamoxifen (Sigma, T-5648) (20 mg/ml dissolved in corn oil, Sigma C-8267) per 30 g of pregnant mouse with a gavage needle.
For staging, the day of vaginal plug was counted as embryonic day 0.5 (E0.5). For immunochemistry and in situ hybridization, embryos were fixed in 4 % paraformaldehyde in PBS overnight and completely dehydrated for storage at −20 °C. Samples were paraffin embedded and sectioned at 7 µm or agarose embedded (1 %) and sectioned at 150 µm.
All mouse experiments were performed according to protocols approved by the Universidad Miguel Hernandez OEP committee.
Immunohistochemistry and in situ hybridization
IHC was performed as described (Moreno-Bravo et al.
2014). The following antibodies were used: Rabbit α-RFP IgG (MBL Cat. No. PM005; 1:100), Mouse α-GAD67 IgG (Millipore Cat. No. MAB5406; 1:300), Rabbit α-TH IgG (Institute Jacques Boy Cat. No. 268020234; 1:1,000), Rabbit α-PAX2 IgG (Zymed 71-6000; 1:5), Sheep α-BrdU IgG (Abcam ab1893; 1:150), Guinea pig a α-SIX3·IgG (Rockland 200-201-A26; 1:200.)
In situ hybridization analyses on paraffin sections were performed as previously described (Moreno-Bravo et al.
2014) using digoxigenin-labeled RNA probes. Mouse cDNA probes used for in situ hybridization analysis were
Six3 (P. Gruss)
, Gad67 (W. Wurst)
, Nkx6.2 and
Otx2 (A. Simeone).
Birth dating by BrdU labeling
For detection of the peak of neurogenic proliferation, BrdU was administered intraperitoneally to the pregnant females (3 mg/100 g body weight) every 2 h, for a period of 10 h (five injections in total) starting at desired stages.
Time lapse
For the time-lapse experiments, the embryos were extracted and dissected in cold PBS. Samples were embedded in low melting point agarose (4 %) and sectioned at 250 µm. The sections were collected using Krebs 1X medium (Krebs, glucose, NaHCO3, Hepes 1 M 1 %, penicillin/streptomycin 1 %, Gentamicina 0.2 %) at 4 °C. The selected slice was placed in a polycarbonate membrane (MilliCell PICMORG50) with neurobasal medium and incubated during the experiment (37 °C, 5 % CO2).
For confocal imaging, a Leica SPE-II DM5550 laser scanning confocal microscope was used. A TCS-SP2-AOBS laser scanning spectral inverted confocal microscope (fitted with temperature and CO2 control; Leica Microsystems) was used for live imaging of brain slice culture. Images were collected every 20 min during 16 h. All the focal planes were merged to visualize the maximum projection. Videos were processed with Imaris and ImageJ software.
Microscopy and quantification
IHC and ISH staining on paraffin and vibratome sections were visualized under fluorescence automated DM6000B microscope and MZ16FA Fluorescence Stereomicroscope (for wide-field microscopy), running Leica Application Suite (LAS) AF6000 Software (version 2.0.2), equipped with a DFC350-FX (monochrome) or DC500 (color) digital cameras. Images were processed and assembled with Adobe Photoshop software.
For quantification, cells were counted only from the rostrocaudal SNR domain. A fix area (275 µm × 687.5 µm) in this region was used to count GABA and Nkx6.2 positive neurons and then compare rostrocaudal SNR axis. A standard Student’s t test was used for comparing the mean values of the data sets.