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. 2014 Aug 13:8:82.
doi: 10.3389/fnana.2014.00082. eCollection 2014.

The complexity of the calretinin-expressing progenitors in the human cerebral cortex

Nevena V Radonjić  1 , Juan A Ortega  2 , Fani Memi  2 , Krista Dionne  2 , Igor Jakovcevski  3 , Nada Zecevic  2
Affiliations

The complexity of the calretinin-expressing progenitors in the human cerebral cortex

Nevena V Radonjić et al. Front Neuroanat. .

Abstract

The complex structure and function of the cerebral cortex critically depend on the balance of excitation and inhibition provided by the pyramidal projection neurons and GABAergic interneurons, respectively. The calretinin-expressing (CalR(+)) cell is a subtype of GABAergic cortical interneurons that is more prevalent in humans than in rodents. In rodents, CalR(+) interneurons originate in the caudal ganglionic eminence (CGE) from Gsx2(+) progenitors, but in humans it has been suggested that a subpopulation of CalR(+) cells can also be generated in the cortical ventricular/subventricular zone (VZ/SVZ). The progenitors for cortically generated CalR(+) subpopulation in primates are not yet characterized. Hence, the aim of this study was to identify patterns of expression of the transcription factors (TFs) that commit cortical stem cells to the CalR fate, with a focus on Gsx2. First, we studied the expression of Gsx2 and its downstream effectors, Ascl1 and Sp8 in the cortical regions of the fetal human forebrain at midgestation. Next, we established that a subpopulation of cells expressing these TFs are proliferating in the cortical SVZ, and can be co-labeled with CalR. The presence and proliferation of Gsx2(+) cells, not only in the ventral telencephalon (GE) as previously reported, but also in the cerebral cortex suggests cortical origin of a subpopulation of CalR(+) neurons in humans. In vitro treatment of human cortical progenitors with Sonic hedgehog (Shh), an important morphogen in the specification of interneurons, decreased levels of Ascl1 and Sp8 proteins, but did not affect Gsx2 levels. Taken together, our ex-vivo and in vitro results on human fetal brain suggest complex endogenous and exogenous regulation of TFs implied in the specification of different subtypes of CalR(+) cortical interneurons.

Keywords: GABA; Gsx2; cortical development; cortical interneurons; transcription factors.

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Figures

FIGURE 1
FIGURE 1
Gsx2 in the human and mouse developing brain at mid-gestation. Expression of Gsx2 mRNA (A) and protein (B) in the human fetal cortex. In situ hybridization in E12.5 mouse with Gsx2 anti-sense (C) and sense (D) probe. (E) ISH in human fetal cerebral cortex (18 gw); insets- high magnification of Gsx2+ cells; (F) Representative Gsx2+ cells in subplate (FISH) for quantification (23 gw); percentage of Gsx2+ cells from total cells is shown in the histogram in the different stages of development (18–24 gw). (G) In situ signal for Gsx2+ cells (red) co-labeled with anti-Ki67 antibody (green) in cortical VZ (optical sectioning); (H) CalR+ cells (red) in subplate colabeled with Gsx2 (green; 18 gw). VZ, ventricular zone; iSVZ, inner subventricular zone; oSVZ, outer subventricular zone; IZ, intermediate zone; SP, subplate; CP, cortical plate; Cx, cortex; LV, lateral ventricle; CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence. Scale bars 10 μm.
FIGURE 2
FIGURE 2
Ascl1 in the human and mouse developing brain at mid-gestation. Expression of Ascl1 mRNA (A) and protein (B) in the human fetal cortex. Immunohistochemistry in E12.5 mouse with anti-Ascl1 antibody (C). (D) Representative immunolabeled Ascl1+ in human fetal cerebral cortex (24 gw). (E) Percentage of Ascl1+ cells in all nuclei (labeled with bis-benzimide) in the human fetal neocortex at midgestation (21–24 gw). Data are presented as mean values + standard error of the mean (minimum three sections from two brains were studied). VZ/SVZ, ventricular/subventricular zone; IZ, intermediate zone; SP, subplate; CP, cortical plate; Cx, cortex; LV, lateral ventricle; CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence. Scale bar 50 μm.
FIGURE 3
FIGURE 3
Sp8 in the human and mouse developing brain at mid-gestation. Expression of Sp8 mRNA (A) and protein (B) in the human fetal cortex. Immunohistochemistry in E12.5 mouse with anti-Sp8 antibody (C). (D) Representative immunolabeled Sp8+ cells in the human fetal cerebral cortex (15–20 gw). Sp8+ cells (green) co-labeled with (E) CalR (red) and (F) Gsx2 (red) in cortical plate and subplate. VZ, ventricular zone; iSVZ, inner subventricular zone; oSVZ, outer subventricular zone; IZ, intermediate zone; SP, subplate; CP, cortical plate; Cx, cortex; LV, lateral ventricle; CGE, caudal ganglionic eminence; MGE, medial ganglionic eminence. Scale bar, 50 μm.
FIGURE 4
FIGURE 4
Differentiation of RGCs enriched from cortical or GE regions. (A) Markers of RGCs, projection neurons and interneurons expressed in cortical and GE enriched RGCs after 7 DIV. (B) Representative staining of CalR+ (red) and GABA+ (green) cells after 7 DIV in cortical and GE RGCs. (C) Histogram shows the percentage of CalR+ and GABA+ cells from the total cells differentiated after 7 DIV in cortical and GE RGC cultures. *p < 0.05, **p < 0.01; t-test. Scale bar, 20 μm.
FIGURE 5
FIGURE 5
Effect of Shh signaling on Gsx2, Ascl1, and Sp8 in cortical RGC cultures after 7 DIV. (A) Representative immunoblots and (B) quantification on the protein expression and (C) mRNA levels after treatment with PMM/Shh and cyclopamine. *p < 0.05, t-test.
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