Distinct cortical and sub-cortical neurogenic domains for GABAergic interneuron precursor transcription factors NKX2.1, OLIG2 and COUP-TFII in early fetal human telencephalon
The extent of similarities and differences between cortical GABAergic interneuron generation in rodent and primate telencephalon remains contentious. We examined expression of three interneuron precursor transcription factors, alongside other markers, using immunohistochemistry on 8–12 post-conceptional weeks (PCW) human telencephalon sections. NKX2.1, OLIG2, and COUP-TFII expression occupied distinct (although overlapping) neurogenic domains which extended into the cortex and revealed three CGE compartments: lateral, medial, and ventral. NKX2.1 expression was very largely confined to the MGE, medial CGE, and ventral septum confirming that, at this developmental stage, interneuron generation from NKX2.1+ precursors closely resembles the process observed in rodents. OLIG2 immunoreactivity was observed in GABAergic cells of the proliferative zones of the MGE and septum, but not necessarily co-expressed with NKX2.1, and OLIG2 expression was also extensively seen in the LGE, CGE, and cortex. At 8 PCW, OLIG2+ cells were only present in the medial and anterior cortical wall suggesting a migratory pathway for interneuron precursors via the septum into the medial cortex. By 12 PCW, OLIG2+ cells were present throughout the cortex and many were actively dividing but without co-expressing cortical progenitor markers. Dividing COUP-TFII+ progenitor cells were localized to ventral CGE as previously described but were also numerous in adjacent ventral cortex; in both the cases, COUP-TFII was co-expressed with PAX6 in proliferative zones and TBR1 or calretinin in post-mitotic cortical neurons. Thus COUP-TFII+ progenitors gave rise to pyramidal cells, but also interneurons which not only migrated posteriorly into the cortex from ventral CGE but also anteriorly via the LGE.
KeywordsGanglionic eminences Inhibitory interneurons Neurodevelopment Neuronal fate specification Pallium Subpallium
Chicken ovalbumin upstream promotor-transcription factor 2
Distal-less homeobox 2
NK2 homeobox 1
Oligodendrocyte lineage transcription factor 2
Paired box 6
- TBR1 and TBR2
T-box brain 1 and 2
Caudal ganglionic eminence
Lateral ganglionic eminence
Medial ganglionic eminence
Humans have considerably expanded cognitive abilities compared to all other species which may be dependent on the evolution of a greater interconnectedness of a larger number of functional modules (DeFelipe 2011; Buckner and Krienen 2013). This not only depends on the physical presence of neurons, axon pathways, and synapses, but also on synchronicity of neural activity between cortical areas binding together outputs of all neurons within a spatially distributed functional network (Singer and Gray 1995; Fries 2009). The synchronicity essential to higher order processing is dependent on the activity of gamma-aminobutyric acidergic (GABAergic) interneurons (Whittington et al. 2011; Buzsaki and Wang 2012), and we might predict a more sophisticated functional repertoire for interneurons in higher species (Ballesteros-Yáñez et al. 2005; Molnár et al. 2008; DeFelipe 2011; Povysheva et al. 2013; Clowry 2015).
Is this expanded repertoire of functional types matched by an evolution of their developmental origins? It is well established in rodents that GABAergic interneurons are born almost entirely outside the neocortex in the ganglionic eminences and associated structures (such as the preoptic area) from which they migrate tangentially into the cortex (De Carlos et al. 1996; Parnavelas 2000; Marín and Rubenstein 2001; Welagen and Anderson 2011). The ganglionic eminences are divided into three main neurogenic domains: the lateral, medial, and caudal ganglionic eminences (LGE, MGE, and CGE, respectively). The LGE is the origin of the striatal projection neurons and a small population of olfactory bulb interneurons (Waclaw et al. 2009). The MGE and the CGE are the major sites of cortical interneurogenesis (Xu et al. 2004; Butt et al. 2005).
Recent studies support the idea that generation of interneurons in the ventral telencephalon may be more complicated in primates, which have evolved a large and complex outer subventricular zone in the ganglionic eminences (Hansen et al. 2013). In addition, proportionally, more interneurons appear to be produced in the CGE, the majority of which populate the superficial layers of the cortex (Hansen et al. 2013; Ma et al. 2013). Whether or not the cortical proliferative zones are a source of interneurogenesis, to what extent and significance, is a contentious issue (Molnár and Butt 2013; Clowry 2015). Some researchers have proposed that primates generate interneurons in the proliferative zones of the cortex (Letinic et al. 2002; Petanjek et al. 2009; Zecevic et al. 2011; Radonjic et al. 2014a; Al-Jaberi et al. 2015) as well as in the ganglionic eminences. Other groups have convincingly argued that interneuronogenesis is essentially the same in primates as in rodent models (Hansen et al. 2013; Ma et al. 2013; Arshad et al. 2016). As there is growing evidence that conditions, such as autism, schizophrenia, and congenital epilepsy, may have developmental origins in the failure of interneuron production and migration (De Felipe 1999; Lewis et al. 2005; Uhlhaas and Singer 2010; Marín 2012), it is important that we understand fully the similarities and differences between human development and that in our animal models.
Therefore, we have carried out a detailed study of expression of three transcription factors expressed by interneuron progenitors, NKX2.1, OLIG2, and COUP-TFII. We looked between the ages of 8–12 post-conceptional weeks (PCW) which have been a relatively neglected period of development in the previous studies of interneurogenesis. NKX2.1 is considered the key regulator of MGE-derived GABAergic interneuron specification (Sussel et al. 1999; Xu et al. 2004; Butt et al. 2008; Du et al. 2008) and the only transcription factor that distinguishes the MGE from other subcortical domains, including in the human embryo at 7 PCW (Pauly et al. 2014). In mice, following the early loss of Nkx2.1 function, the MGE acquires an LGE-like molecular specification (Sussel et al. 1999), whereas the late conditional loss of function switches the MGE to CGE in character (Butt et al. 2008). In rodents, the MGE is the major source of cortical GABAergic interneurons and Nkx2.1 expression in this region is required for the specification of somatostatin and parvalbumin expressing interneurons from MGE progenitors (Xu et al. 2004; Butt et al. 2008; Du et al. 2008).
In the rodent, Olig2 expressing progenitors in the MGE give rise to GABAergic interneurons at an earlier stage of development and oligodendrocytes at later stages (Miyoshi et al. 2007). In the human, OLIG2 has been detected principally in the proliferative zones of the ganglionic eminences between 5–15 PCW, prior to significant expression of markers for oligodendrocyte precursors, with a spread into the cortex by 20 gestational weeks, along with co-expression of OLIG2 with markers for immature neurons, neurogenic radial glia, and intermediate progenitor cells (Jakovceski and Zecevic 2005; Jackocevski et al. 2009).
COUP-TFII, in rodents, is preferentially expressed in the CGE as well as in interneurons migrating into the cortex (Kanatani et al. 2008; Lodato et al. 2011). CGE-derived interneurons migrate caudally to the most posterior part of the telencephalon (Yozu et al. 2005; Faux et al. 2012), and COUP-TFII is essential to establish this caudal migratory stream (Kanatani et al. 2008). Reinchisi et al. (2012) have provided some evidence that COUP-TFII may play a similar role in human forebrain development. However, the precise roles of COUP-TFs in specifying the CGE-derived interneurons are still unclear.
Expression of the transcription factor PAX6 was also investigated alongside the three GABAergic markers. PAX6 is considered a marker for dorsal telencephalic radial glia giving rise to glutamatergic neurons in rodents (Hevner et al. 2006), and in human, PAX6 expression delineates the cortical ventricular and subventricular proliferative zones (Bayatti et al. 2008a). However, in humans, it is also known to be expressed in a gradient across the proliferative layers of the LGE revealing its boundary with the MGE (Pauly et al. 2014; Harkin et al. 2016).
The present study aimed to map and quantify the expression of the three interneuron progenitor markers in the human telencephalon at an important stage of development prior to the arrival of thalamic innervation to delineate the extent of cortical interneurogenesis. This aim was achieved, demonstrating distinct neurogenic domains for all three including expression in the developing cortex. In addition, careful observation of these expression patterns revealed a complex organization for the CGE and provided evidence for anterior and medial migration pathways for interneuron precursors into the cortex from the ganglionic eminences in addition to the more generally recognised lateral and posterior pathways.
Methods and materials
Human fetal tissue from terminated pregnancies was obtained from the joint MRC/Wellcome Trust-funded Human Developmental Biology Resource (HDBR, http://www.hdbr.org; Gerrelli et al. 2015). All tissues were collected with appropriate maternal consent and approval from the Newcastle and North Tyneside NHS Health Authority Joint Ethics Committee. Fetal samples ranging in age from 8 to 12 PCW were used. Ages were estimated from the measurements of foot and heel to knee length compared with the fetal staging chart as described by Hern (1984). Brains were isolated and fixed for at least 24 h at 4 °C in 4% paraformaldehyde dissolved in 0.1 M phosphate-buffered saline (PBS) (PFA; Sigma Aldrich). Once fixed, whole or half brains (divided sagittally) were dehydrated in a series of graded ethanols before embedding in paraffin. Eight brain samples were cut at 8 μm section thickness in three different planes; horizontally, sagittally, and coronally, mounted on slides and used for haematoxylin and eosin staining (H&E) and immunostaining.
Primary antibodies used in this study
Dako, Ely, UK
Abcam, Cambridge, UK
Cambridge Bioscience, Cambridge, UK
R&D Systems, Abingdon, UK
Merck Millipore, Watford, UK
Swant, Marly, Switzerland
Immunofluorescence (double and triple labelling)
We used a novel immunofluorescent staining method, Tyramide Signal Amplification (TSA), that permits sequential double and triple staining using antibodies from the same species without cross-reactions (Goto et al. 2015; Harkin et al. 2016). Sections were treated as described above until the secondary antibody stage, then they were incubated with HRP-conjugated secondary antibody for 30 min [ImmPRESS™ HRP IgG (Peroxidase) Polymer Detection Kit, Vector Labs], washed twice for 5 min in TBS, and incubated in dark for 10 min with fluorescein tyramide diluted at 1/500 in 1× Amplification buffer (Tyramide Signal Amplification (TSA™) fluorescein plus system reagent, Perkin Elmer, Buckingham, UK). Tyramide reacts with HRP to leave fluorescent tags covalently bound to the section.
Prior to starting the second round of staining, sections were first washed in TBS and boiled in 10 mM citrate buffer to remove all antibodies and unbound fluorescein from the first round. Sections were then incubated in 10% normal serum before incubating with the second primary antibody for 2 h at room temperature. Following washing, sections were again incubated with ready to use HRP-conjugated secondary antibody and then incubated with CY3 tyramide for 10 min [Tyramide Signal Amplification (TSA™) CY3 plus system reagent, Perkin Elmer]. The same steps were repeated for the third round of staining (if triple labelling was needed) using CY5 Tyramide (Tyramide Signal Amplification (TSA™) CY5 plus system reagent, Perkin Elmer). Sections were washed before applying 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher Scientific, Cramlington, UK) and mounted using Vectashield Hardset Mounting Medium (Vector Labs).
All immunoperoxidase staining figures presented in this study were captured using a Leica slide scanner and Zeiss Axioplan 2 microscope. The double immunofluorescent figures were obtained with a Zeiss Axioimager Z2 apotome and Triple Immunofluorescent images were obtained with a Nikon A1R confocal microscope. Processing of images, which included only adjustment of brightness and sharpness, was achieved using the Adobe Photoshop CS6 software.
Cell counts in proliferative zones of 12 PCW fetus
Density (cells/mm3 × 103)
Density (cells/mm3 × 103)
Density (cells/mm3 × 103)
Examination of our immunoperoxidase labelled sections for various markers at low magnification revealed details of the characteristics of the CGE and septum in human not fully reported on before in detail. Therefore, we have begun the results section by describing these regions, before moving on to describe the level of expression of each GABAergic interneuron precursor transcription factor in different parts of the telencephalon, aided by a more detailed knowledge of CGE and septal sub-compartments.
The position and subdivisions of the caudal ganglionic eminence (CGE)
The position of the CGE can be determined with respect to other subcortical landmarks in H&E stained sections (Suppl. Figure 1). For example, in the horizontal plane, at the level of the internal capsule, the MGE and LGE appeared as prominent bulges into the lateral ventricles, and in a rostral position relative to the internal capsule. The CGE can be seen as the part of the GE positioned caudally to the internal capsule immediately adjacent to the ventral/temporal cortex (Suppl. Figure 1a). In sagittal sections, the most dorsal part of the CGE appeared as well-defined protrusion into the lateral ventricle, close to the hippocampus (Suppl. Figure 1b). The central part lies next to the narrow ventral extension of the lateral ventricles (Suppl. Figure 1c). In a coronal plane at the level of the rostral half of thalamus, parts of MGE and LGE can be seen dorsal to the internal capsule (Suppl. Figure 1d and e) and only the most ventral part of the CGE was observed ventral to the internal capsule and close to the ventral/temporal cortex (Suppl. Figure 1e). At the level of the caudal half of thalamus and caudal to the internal capsule, the CGE was present but not the MGE and LGE (Suppl. Figure 1f).
Interestingly, these two domains could not be distinguished in the most ventral part of CGE, which was located immediately adjacent to the ventral/temporal cortex (Figs. 1e, f, 2a″, b″). Thus, we propose that there is a third compartment to the CGE in human, “ventral CGE” (vCGE), which was characterized by strong immunoreactivity for PAX6, but not for NKX2.1. Despite the high PAX6 expression in the vCGE, there was still a distinct boundary between it and the adjacent cortex, characterized by a thicker cortical VZ compared with more condensed PAX6+ cells in the SVZ of the vCGE (Fig. 2a″, b″). As the CGE is recognised as the birth place of calretinin (CalR) expressing interneurons in rodents (Nery et al. 2002; Butt et al. 2005), we studied the expression of CalR in the three defined compartments of the CGE. CalR was preferentially expressed in cells of the VZ and SVZ of the lCGE and vCGE. Only scattered cells were observed in mCGE. CalR immunostaining also revealed a distinct boundary between the vCGE and the ventral cortex. In vCGE, CalR was expressed in the VZ and SVZ, but in the ventral cortex, expression of CalR was mainly confined to the SVZ with only scattered CalR+ cells in the VZ (Fig. 2c, c′, c″).
Subdivisions of the septum
Similar to the ganglionic eminences, the septum is a subcortical structure that exhibited complementary expression of PAX6 and NKX2.1. The most ventral part of septum could be defined as MGE-like septum characterized by strong immunoreactivity for NKX2.1 but not PAX6 expression. More dorsally, we found LGE-like septum, which was characterized by moderate expression of PAX6 but not NKX2.1. The most dorsal part of septum had a cortical rather than sub-cortical identity, manifested by higher PAX6 expression in the VZ and SVZ and expression of TBR1 by post-mitotic cells in the SVZ, IZ, and cortical plate (Fig. 1e–h; Suppl. Figure 2).
Expression of NKX2.1 in human fetal telencephalon 8–12 PCW
However, at 12 PCW, we found scattered NKX2.1+ cells in the cortical VZ and SVZ sometimes far removed from the ganglionic eminences and septum (Fig. 3b), but no NKX2.1 positive (NKX2.1+) cells were observed to co-express KI67 (not shown) a marker for active cell division (Scholzen and Gerdes 2000). We next quantified the average density of NKX2.1+ in the proliferative zones of the MGE, septum, LGE, vCGE, and the cortex of a 12PCW brain cut in the coronal plane (Fig. 3c; Table 2), and estimated that 93% of NKX2.1 cells in proliferative layers were found in the MGE and ventral septum, 4.6% in the LGE and vCGE, and only 2.4% in the cortex.
Expression of OLIG2 in human fetal ventral telencephalon 8–12 PCW
Since OLIG2 was highly expressed in the NKX2.1-expressing neurogenic domain (MGE and ventral septum), we examined the cellular co-localization of these two markers in the MGE at 8 and 12 PCW. Interestingly, we found three population of cells, NKX2.1-/OLIG2+ , NKX2.1+/OLIG2−, and NKX2.1+/OLIG2+ (Fig. 5a). To confirm that OLIG2+ cells at this stage of human forebrain development (8–12 PCW) were GABAergic interneuron precursors, we performed OLIG2 and GAD65/67 double labelling, and found most of OLIG2+ cells expressed GAD65/67 (Fig. 5b, c). Furthermore, a proportion of cells in the MGE were triple labelled with OLIG2, NK2.1, and GAD65/67 (Fig. 5d–g). However, although both OLIG2 and CalR were expressed in lCGE and mCGE, no double labelling for these two markers was detected (Suppl. Figure 3S).
Expression of OLIG2 in human fetal dorsal telencephalon 8–12 PCW
At 12 PCW, a proportion of OLIG2+ cells in the cortex were also found to co-express KI67 (Fig. 6b). However, OLIG2+ cells were not double-labelled with either PAX6 or TBR2 (Fig. 6c, d), showing that OLIG2 is not expressed by typical cortical radial glial progenitors or intermediate progenitors (Bayatti et al. 2008a; Lui et al. 2011). OLIG2+ cells populated the whole cortex and were mainly seen in the SVZ and IZ; nevertheless, scattered positive cells were sometimes observed in the VZ and the cortical plate (CP; Fig. 6e–i). We quantified the average density of OLIG2+ cells in the proliferative zones of the four different cortical regions. A higher density was found in the medial cortex with a decreasing gradient to the latero-ventral regions (Fig. 6k). Overall, OLIG2 expression was far less confined to the MGE than NKX2.1, with approximately 38% of OLIG2+ cells in proliferative layers found in the MGE and ventral septum, 50% in the LGE and vCGE, and 12% in the cortex (Fig. 6j; Table 2).
Expression of COUP-TFII in the ventral telencephalon 8–12 PCW
Expression of COUP-TFII in the dorsal telencephalon 8–12 PCW
We found a distinct distribution of COUP-TFII+ cells between the anterior and posterior cortex at 8 PCW. In the anterior cortex, COUPT-FII protein was localized to all layers. Although most of COUP-TFII+ cells were located in the SVZ and IZ, a considerable number of cells were also observed in the VZ and CP (Fig. 7c, e). A different distribution of COUP-TFII+ cells was observed in the posterior cortex, where cells were restricted to what appeared to be two migratory streams; a major one in the SVZ, and a less defined one in the nascent pre-subplate at the border between the IZ and the CP (Fig. 7c, d). No COUP-TFII positive cells were found in the VZ (Suppl. Figure 5d).
All these findings suggest that the neurogenic domain for COUPT-FII precursors in the vCGE extends into the cortical wall of the ventral cortex, but not dorsal cortex, and includes radial glial progenitor cells that generate glutamatergic neurons. We quantified the average density of COUP-TFII+ cells in the proliferative zones across the cortex (Fig. 9a–f) and found a decreasing gradient of density from higher gradient in the ventral cortex to a lower gradient more dorsally. In a recent study, Reinchisi et al. (2012) reported that COUP-TFII+ cells are more abundant in the temporal/caudal cortex of human fetal brain, which was attributed to a caudal migratory stream from the CGE. However, our results suggest, in humans, the presence of an additional anterior migratory stream, and provide evidence that the neurogenic domain of COUP-TFII expressing progenitor cells is not confined to the CGE, but extends to the ventral cortex, including the frontal lobe (Fig. 9g). In addition, we found most COUP-TFII+ cells in the ventral cortex to be double-labelled with CalR. However, a proportion of COUP-TFII+ cells in all other regions of the cortex were also shown to be a subpopulation of CalR expressing cells (Suppl. Figure 5c–e).
We have described the expression patterns of three transcription factors important to the generation of cortical interneurons in the early fetal human telencephalon and demonstrated that they occupy distinct (although overlapping) neurogenic domains which can extend into the cortex. NKX2.1 was very largely confined to the MGE, mCGE, and ventral septum, and at this stage of development, these observations support the previous studies suggesting that interneuron generation from NKX2.1 positive cells may be identical in nature with the process occurring in rodents (Hansen et al. 2013; Ma et al. 2013; Arshad et al. 2016). OLIG2 was expressed by cells in the proliferative zones of the MGE, mCGE, and septum, co-expressed with GAD65/67, but not necessarily co-expressed with NKX2.1, and was also extensively expressed in the LGE, lCGE, and in dividing cells in the cortex; observations previously unreported at this key stage of development. Within the ganglionic eminences, dividing COUP-TFII precursors were localized to the vCGE as previously described (Hansen et al. 2013) but were also numerous in adjacent regions of ventral cortex. From careful examination of multiple expression patterns, we have been able to more accurately compartmentalise the CGE than has previously been attempted, and describe additional ventral to dorsal migratory streams for interneuron precursors not previously reported in rodents, as will be discussed in more detail below. Further evidence for interneuron generation in the human dorsal telencephalon has been presented.
Anatomical and molecular subdivisions of the CGE
In rodents, the CGE has been identified as a source of specific GABAergic interneuronal subtypes different from those generated from the MGE (Miyoshi et al. 2010; Rudy et al. 2011). Some researchers have concluded that the CGE comprises caudal extensions of LGE and MGE, respectively, by virtue of its gene expression patterns (Corbin et al. 2003; Flames et al. 2007). However, as additional transcription factors, such as COUP-TFI and COUP-TFII, are enriched in CGE, it is possible that the CGE has evolved as a distinct neurogenic domain separate from the MGE and LGE (Kanatani et al. 2008). This study has revealed that in the developing human brain, the lateral and medial portions of the CGE share the expression patterns of PAX6, OLIG2, and NKX2.1 of the LGE and MGE, respectively. However, in addition to these lateral and medial portions of the CGE, the extension of the CGE along the lateral ventricle into the greatly enlarged temporal lobe has produced a third compartment distinguishable by its characteristic co-localisation of intense COUP-TFII and PAX6 expression in the proliferative layers. Dividing COUP-TFII+ cells were confirmed as being confined to this ventral region of the CGE (Hansen et al., 2013). In addition, unlike the dorsally located lateral and medial portions, almost no NKX2.1+ cells were found in the vCGE. These findings suggest that the anatomical and molecular boundaries of the CGE should be defined carefully and separately, with the dorsal region formed from caudal extensions of the LGE and MGE, albeit with a higher density of post-mitotic COUP-TFII and CalR positive cells, and the ventral region having its own molecular signature including COUP-TFII positive progenitor cells.
Are anterior and medial migratory streams prominent in the human telencephalon?
Quantitative PCR, microarray, in situ hybridisation, and immunohistochemical studies between 8–12 PCW have previously identified an anterior-to-posterior gradient of expression of multiple genes identified with GABAergic interneurons and GABAergic neurotransmission, including transcription factors characteristic of interneuron precursors, isoforms of GAD, GABA receptor sub-units, and calcium-binding proteins (Bayatti et al. 2008a; Ip et al. 2010; Al-Jaberi et al. 2015) seemingly at odds with the accepted lateral (MGE derived) and posterior (CGE derived) pathways of migration for interneuron precursors from ventral to dorsal telencephalon (Wonders and Anderson 2006). This led to speculation that the anterior cortex in particular may be a novel site for generation of interneurons in the primate telencephalon, perhaps, to populate the enlarged prefrontal lobes of the primate brain (Al-Jaberi et al. 2015; Clowry 2015). This study offers up the alternative explanation that migrating interneurons may more rapidly invade the anterior than the posterior cortex, even from apparently caudal structures, such as the vCGE. We saw evidence of a rostral migratory stream of COUP-TFII and CalR expressing cells from the vCGE, where COUPTFII expressing progenitors exclusively underwent division, to the anterior cortex via the lCGE, LGE, and ventral pallium (Fig. 9). Such cells were more numerous in the anterior than posterior cortex, as previously described for CalR+ neurons (Bayatti et al. 2008a). Examination of our 3D reconstructions of the 12 PCW fetal brain confirmed that this path length is similar or even shorter than that from the vCGE to the dorso-posterior cortex via the temporal lobe (Fig. 9g). Although rostral migratory streams from the LGE to olfactory bulbs are well described in mammals (Corbin et al. 2001; Waclaw et al. 2009), a rostral migratory stream from the GE to the rostral pallium has only been reported in shark (Quintana-Urzainqui et al. 2015) and so may be overlooked or missing in rodent models.
In addition, we have observed increased expression of OLIG2 in the anterior compared to posterior cortex in agreement with the previous studies (Ip et al. 2010; Al-Jaberi et al. 2015) particularly at 8 PCW where there was also a distinct medial to lateral gradient of OLIG2 expression. In this case, the migratory stream appeared to derive from progenitor cells in the MGE and sub-cortical septum, and enter the cortex via the medial wall. This is in direct contradiction to what has been reported in rodents where interneurons populating medial wall-derived structures, such as the hippocampus, are described as deriving from the MGE and CGE via lateral migration (Pleasure et al. 2000; Wonders and Anderson 2006; Morozov et al. 2009; Faux et al. 2012). In our preparations, we found evidence that OLIG2+ and NKX2.1+ progenitors reside in the septum and OLIG2+ cells, at least, migrate medially to the cortex. Again, this is in disagreement with findings in rodents, where septum derived cells were reported not to enter the cortex at all (Rubin et al. 2010). Thus, we propose that the human or primate brain possesses an additional medial migratory pathway for GABAergic interneurons populating frontal and medial areas of the cerebral cortex. The much larger human cortex may require additional migratory pathways compared to smaller mammalian brains. However, it is worth noting that a medial migratory pathway for Nkx2.1 positive precursors from the MGE to the medial pallium has recently been reported in the shark (Quintana-Urzainqui et al. 2015); therefore, such a pathway cannot be proposed as evolutionarily novel to the human brain. Instead, we might speculate that this is missing or relatively small and overlooked in rodent compared to other vertebrate species.
Potential dorsal telencephalic origin of GABAergic interneurons
Based on studies carried out principally around mid-gestation, Radonjić et al. (2014a) proposed that three mechanisms exist for the production of cortical interneurons in primates: generation in the ventral telencephalon followed by migration to the cortex, precursors arriving in the cortex from the ventral telencephalon, and undergoing further division intra-cortically, and cortically derived progenitors giving rise to interneurons. The last two proposals are controversial, being firmly rejected by recent influential and persuasive studies (Hansen et al. 2013; Ma et al. 2013; Arshad et al. 2016). However, our present study found clear evidence for the second mechanism. OLIG2+ precursors appeared to follow migratory paths into the cortex; however, OLIG2+ cells were also shown to be undergoing proliferation and these OLIG2+ cells did not co-express any markers of cortically derived progenitors, such as PAX6 or TBR2 (although such double-labelling has been reported at later stages of human development; Jakovceski and Zecevic 2005). This firmly suggests that OLIG2 is not immediately downregulated in cells entering the cortex from subcortical structures, unlike NKX2.1, and that these cells may retain the ability to divide within the cortex, preferentially within anterior and medial locations, where the highest density of such cells was found. However, there also remains the possibility that OLIG2+/TBR2- intermediate progenitor cells are generated by cortical radial glial progenitor cells which go on to produce GABAergic interneurons.
It is also clear that in the more ventral areas of the anterior and temporal cortex, there is high expression of COUP-TFII expressing progenitor cells and post-mitotic neurons. These progenitor cells co-express either PAX6 or TBR2 and post-mitotic cells co-expressing TBR1 and COUPTFII were also observed, which demonstrates that in the cortex, dividing COUP-TFII+ progenitors give rise to glutamatergic neurons. Although there are also COUP-TFII+/CalR+ presumptive interneurons present, it is impossible to judge whether these have migrated in from the adjacent CGE, or been generated intra-cortically. However, a neuronal progenitor marker GSX2, expressed upstream of COUP-TFII, which localises to the LGE and CGE in rodent (Hsieh-Li et al. 1995; Wang et al. 2013), has been found to be expressed in cells undergoing division in the VZ/SVZ of the human fetal cortex (Radonjić et al. 2014b) making intra-cortical generation a possibility.
Whether or not proliferative NKX2.1+ progenitor cells are present in the cortex is contentious. Our observation at 12 PCW of NKX2.1+ cells throughout the latero-medial extent of the cortical wall, making up about 2.4% of all NKX2.1+ cells in the proliferative zones of the telencephalon at this time, is in conflict with Hansen et al. (2013) who reported nearly no NKX2.1+ cells in the cortex and only close to LGE/lateral cortex border. However, our findings are in partial agreement with Radonjić et al. (2014a) who found NKX2.1+ cells in the cortical wall of human and macaque monkey fetal forebrains (at later stages of development, 15–22 PCW for human) undergoing active division, as did Arshad et al. (2016) in human between 16–28 PCW although in very small numbers. As no NKX2.1+ cells were seen in the cortex at 8PCW in agreement with the previous studies (Hansen et al. 2013; Pauly et al. 2014), we propose that with age, the incidence of NKX2.1+ cells in the cortex gradually increases, along with the capacity to undergo proliferation. Whether these cells are generated in the cortex or have migrated there from the ventral telencephalon without downregulating NKX2.1 remains a question for further investigation.
OLIG2 and COUP-TFII as regulators of cortical arealisation
The division of the cerebral cortex into functional areas (the cortical map) differs little between individuals in any given species (Rakic et al. 2009). The previous work on rodent development has identified certain transcription factors (e.g., PAX6, SP8, EMX2, and COUP-TFI) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area specific thalamocortical afferent projections (López-Bendito and Molnár 2003; O’Leary et al. 2007; Rakic et al. 2009). There may be common mechanisms between species, as the developing human neocortex displays counter-gradients of PAX6 and EMX2 at the early stages of cortical development (Bayatti et al. 2008b). However, the human cerebral cortex is composed of different and more complex local area identities and so might be specified by a wider range of transcription factor gradients; for instance, an anterior-to-posterior gradient of CTIP2 expression has been observed in human early fetal cortex (Ip et al. 2011). In this study, we observed a prominent anterior-to-posterior gradient of OLIG2 expression, and a ventral-to-dorsal gradient of COUP-TFII expression. In both the cases, the transcription factors are also expressed at moderate levels in the cortical plate as well as the proliferative zones, suggesting that areal specification mechanisms in cells extend into the post-mitotic period. The extent to which these gradients interact with interneuron precursors is not known, but we might speculate that OLIG2 or COUP-TFII controls expression of cell adhesion molecules locally that attract migrating cells expressing the same transcription factors, setting up the migratory pathways into the cortex for interneurons arriving medially via the septum (OLIG2+) or laterally via ventral anterior or temporal cortex (COUP- TFII+).
Evidence continues to accumulate that cortical GABAergic interneuron production in primates differs in certain details from what has been learnt from our rodent models. A higher proportion of interneurons arise from the CGE in primates and we provide a description of the compartmentalisation of the CGE. This study presents further evidence that interneuron precursor cells may undergo division in the cortex, although it remains to be proven whether they are originally generated in the dorsal telencephalon. Finally, whereas in rodents, interneuron precursors are believed to enter the cortex from the ganglionic eminences exclusively via lateral and posterior routes, in human, we provide evidence of pathways via the anterior and medial cortex.
The human embryonic and fetal material was provided by the jointly funded Human Developmental Biology Resource (http://www.hdbr.org) MRC/Wellcome Trust grant# 099175/Z/12/Z). Ayman Alzu’bi is in receipt of a studentship from Yarmouk University, Jordan. We are grateful to Dr Peter Thelwall for capturing the MRI scans and Xin Xu for her help with slide scanning.
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