Expression of ventral telencephalon transcription factors ASCL1 and DLX2 in the early fetal human cerebral cortex

Abstract

In rodent ventral telencephalon, diffusible morphogens induce expression of the proneural transcription factor ASCL1, which in turn induces expression of the transcription factor DLX2 that controls differentiation of cortical interneuron precursors and their tangential migration to the cerebral cortex. RNAseq analysis of human fetal samples of dorsal telencephalon revealed consistently high cortical expression of ASCL1 and increasing expression of DLX2 between 7.5 and 17 post‐conceptional weeks (PCW). We explored whether cortical expression of these genes represented a population of intracortically derived interneuron precursors. Immunohistochemistry revealed an ASCL1+/DLX2+ population of progenitor cells in the human ganglionic eminences between 6.5 and 12 PCW, but in the cortex there also existed a population of ASCL1+/DLX2– progenitors in the subventricular zone (SVZ) that largely co‐expressed cortical markers PAX6 or TBR2, although a few ASCL1+/PAX6– progenitors were observed in the ventricular zone (VZ) and ASCL1+ cells expressing the interneuron marker GAD67 were present in the SVZ. Although rare in the VZ, DLX2+ cells progressively increased in number between 8 and 12 PCW across the cortical wall and the majority co‐expressed LHX6 and originated either in the MGE, migrating to the lateral cortex, or from the septum, populating the medial wall. A minority co‐expressed COUP‐TFII, which identifies cells from the caudal ganglionic eminence (CGE). By 19 PCW, a significant increase in expression of DLX2 and ASCL1 was observed in the cortical VZ with a small proportion expressing both proteins. The DLX2+ cells did not co‐express a cell division marker, so were not progenitors. The majority of DLX2+ cells throughout the cortical plate expressed COUP‐TFII rather than LHX6+. As the VZ declined as a proliferative zone it appeared to be re‐defined as a migration pathway for COUP‐TFII+/DLX2+ interneurons from CGE to cortex. Therefore, in developing human cortex, ASCL1 expression predominantly marks a population of intermediate progenitors giving rise to glutamatergic neurons. DLX2 expression predominantly defines post‐mitotic interneuron precursors.

Multiple Origins of Secretagogin Expressing Cortical GABAergic Neuron Precursors in the Early Human Fetal Telencephalon

“…SCGN expression was also evaluated in the cortical wall of the human fetal brain around the mid-gestational period. A robust increase in the number of SCGN+ cells was observed in the cortical VZ at 19 PCW ( Figures 3A,B ) as has previously been described for DLX2+ and CalR+ neurons at this developmental stage (Alzu’bi and Clowry, 2019 ). SCGN+ cells in the SVZ mainly showed a migratory morphology that suggested they were most likely following a radial path from the VZ towards the CP; whereas a stream of cells at the interface between the IZ and SVZ were mainly horizontally oriented perhaps representing tangentially migrating cells ( Figures 3B–D ).…”

“…At 19 PCW several SCGN+ cells co-expressed ANXA5 ( Figure 5E ), with also a proportion of SCGN+ cells expressing MMP2 on their extracellular surface ( Figure 5J ) however these two types of double-labeled cells were only observed in the VZ/SVZ which becomes a major tangential migratory route for GABAergic neuron precursors (post-mitotic, migratory) at this stage of development (Alzu’bi and Clowry, 2019 ) suggesting that SCGN could also have a role in GABAergic neuron migration in the human cortex. However, it is worth mentioning that neither the expression of ANXA5 or MMP2 was specific to SCGN+ cells but that they were also expressed by other cell types including dorsal progenitor cells and post-mitotic neurons of the CP which indicates the widespread role of MMP2 activity, possibly in neural migration in the developing human cortex, but not limited to SCGN+ cells.…”

“…These results suggest that the origin of SCGN expressing GABAergic neurons in the fetal human neocortex is not restricted to the CGE/LGE; the subpallial septum and POA also contribute to a proportion of these populations. We have recently discovered that the septum is a major contributor of GABAergic neuron precursors to the human cortex via medial migration routes not observed in rodent models (Alzu’bi et al, 2017 ; Alzu’bi and Clowry, 2019 ). The subpallial septum can be divided into MGE-like and LGE-like domains, based on gene expression patterns (Alzu’bi et al, 2017 ; Clowry et al, 2018 ) and as would be predicted, SCGN+ interneuron precursors arose from the more dorsal LGE-like domain.…”

“…Previous studies have provided evidence that, at this stage of development, there is an increased incidence of neurogenesis of GABAergic neurons, particularly those expressing calretinin, within the cerebral cortex itself (Letinic et al, 2002 ; Petanjek et al, 2009 ; Jakovcevski et al, 2011 ; Hladnik et al, 2014 ) therefore it is possible that some of these SCGN+ cells could be proliferative, or derived from proliferating cells in the dorsal telencephalon. Even though there is still appreciable Ki67 expression in the VZ at mid-gestation (Petanjek and Kostović, 2012 ; Alzu’bi and Clowry, 2019 ) we would conclude that the proportion of Ki67+ cells that are GABAergic neuron progenitors must be quite small as at 19PCW nearly all SCGN+ cells express DLX2 (a marker for GABAergic neuron progenitors and neuroblasts, see Figure 3 ) but nearly all DLX2+ cells are KI67− (i.e., non-proliferative, Alzu’bi and Clowry, 2019 ).…”

New insights into the development of the human cerebral cortex

“…DLX2+, OLIG2+ and GAD67+ cells from the septum can enter the dorsal and ventral medial walls of the anterior cortical regions, some of which retain the potential to divide (Alzu'bi et al. ; Alzu'bi & Clowry, ).…”