Extended Production of Cortical Interneurons into the Third Trimester of Human Gestation

Abstract

In humans, the developmental origins of interneurons in the third trimester of pregnancy and the timing of completion of interneuron neurogenesis have remained unknown. Here, we show that the total and cycling Nkx2.1(+)and Dlx2(+)interneuron progenitors as well as Sox2(+)precursor cells were higher in density in the medial ganglionic eminence (MGE) compared with the lateral ganglionic eminence and cortical ventricular/subventricular zone (VZ/SVZ) of 16-35 gw subjects. The proliferation of these progenitors reduced as a function of gestational age, almost terminating by 35 gw. Proliferating Dlx2(+)cells were higher in density in the caudal ganglionic eminence (CGE) compared with the MGE, and persisted beyond 35 gw. Consistent with these findings, Sox2, Nkx2.1, Dlx2, and Mash1 protein levels were higher in the ganglionic eminences relative to the cortical VZ/SVZ. The density of gamma-aminobutyric acid-positive (GABA(+)) interneurons was higher in the cortical VZ/SVZ relative to MGE, but Nkx2.1 or Dlx2-expressing GABA(+)cells were more dense in the MGE compared with the cortical VZ/SVZ. The data suggest that the MGE and CGE are the primary source of cortical interneurons. Moreover, their generation continues nearly to the end of pregnancy, which may predispose premature infants to neurobehavioral disorders.

Keywords: Dlx2; GABAergic; Nkx2.1; Sox2; ganglionic eminence; interneuron; subventricular zone.

Figure 1.

VZ and SVZ in the GE diminished in width as gestational age increased. (a) Representative immunofluorescence of coronal sections from the MGE of a 20-gw fetus labeled with antibodies specific to Sox2, Dlx2, and Ki67. Scale bar, 50 µm. Bar graph shows mean ± SEM (n = 4 brains each group). The VZ and SVZ thickness reduced as a function of gestational age. *P < 0.05 and **P < 0.01 for MGE versus cortical SVZ; ^#P < 0.05 for LGE versus cortical SVZ. (b) Cresyl violet staining of coronal section through the right cerebral hemisphere at the level of the head of caudate nucleus showing cortical SVZ, MGE, and LGE of 20 gw fetus (left). Scale bar, 0.25 cm. A typical triple immunolabeling of coronal brain section from 22 gw human fetus with Tbr2, Sox2, and Ki67 antibodies. Note the presence of Tbr2 immunoreactivity (arrows) in the cortical SVZ, but absence in the LGE. Scale bar, 50 µm. D, dorsal; V, ventral; M, medial; L, lateral. (c) Nkx2.1 immunolabeling of the MGE of 22 gw fetus showing Nkx2.1⁺ cells are densely packed in the MGE, but sparse in the LGE. Scale bar, 100 µm. (d) Representative immunofluorescence of cryosections from the CGE of a 19-gw fetus labeled with Coup-TFII- and Dlx2-specific antibodies. Coup-TFII⁺ cells and Dlx2⁺ cells are densely arranged in the CGE (both single- and double-labeled images are from the same field of view). Scale bar, 100 µm. Inset shows that coronal section of this 19 gw fetus stained with cresyl violet depicting the CGE. cSVZ, cortical SVZ.

Figure 2.

Nk2.1⁺ progenitors densely packed in the MGE of 16–28 gw fetuses and markedly reduced in 29–35 gw. (a) Representative immunofluorescence of coronal sections from a 20-gw fetus and a 25-gw premature infant labeled with Nkx2.1- and Ki67-specific antibodies. Inset shows a high power view of the boxed region in the image. Note abundance of both non-proliferating (arrows) and proliferating (arrowheads) Nkx2.1⁺ cells in the MGE compared with the LGE and cortical SVZ. Scale bar, 50 μm. Shown in the lower panel, above and to the right of the image, are orthogonal views in x–z and y–z planes of a composite z-stack of a series of confocal images taken 0.8 µm apart; the images depict Ki67-stained nucleus embedded within Nkx2.1⁺-stained cells (arrowhead) in a 25-gw infant. Scale bars, 20 µm. (b) A typical triple immunolabeling of coronal brain section from 20 and 25 gw subjects labeled with Nkx2.1- and Ki67-specific antibodies and DAPI. The non-proliferating (arrows) and proliferating (arrowheads) Nkx2.1⁺ cells are shown. Note the percentage of Nkx2.1⁺ cells (Nkx2.1⁺/DAPI⁺) was higher in the MGE relative to the LGE and cortical SVZ. Scale bar, 20 µm. (c) Bar diagram shows mean ± SEM (n = 5 each gestational group). Total Nkx2.1⁺ cells were higher in number in the MGE compared with the LGE and cortical SVZ for all ages, except for 29–25 gw. *P < 0.001 MGE versus LGE and ^#P < 0.001 MGE versus cortical SVZ within gestational group comparison. ^†P < 0.001, ^‡P < 0.001, and ^ψP < 0.001 16–22, 23–25, and 26–28 versus 29–35 gw in the MGE, respectively. (d) Bar chart shows mean ± SEM (n = 5 each group). Nkx2.1⁺Ki67⁺ cells were almost absent in the LGE and cortical SVZ and reduced with advancing gestation. *P < 0.001 MGE versus LGE and ^#P < 0.001 for MGE versus cortical SVZ for within gestational group comparison. ^†P < 0.001, ^‡P < 0.001 16–22 versus 26–28 and 29–35 gw in the MGE, respectively. ^ψP < 0.001 23–25 versus 29–35 gw in the MGE. cSVZ, cortical SVZ.

Figure 3.

Dlx2⁺ progenitors were higher in density in the MGE relative to the LGE and cortical SVZ, and disappeared by 35 gw. (a) Representative immunolabeling of coronal sections from a 20-gw fetus and a 25-gw preterm infant stained with Dlx2- and Ki67-specific antibodies. Dlx2⁺ progenitors were more abundant in the MGE compared with the LGE and cortical SVZ. Inset shows a high power view of the boxed region in the image. Note total (arrow) and cycling (arrowhead) Dlx2⁺ cells. Shown in the lower right panel, above and to the right of the image, are orthogonal views in x–z and y–z planes of a composite z-stack of a series of confocal images; the images depict Ki67-stained nucleus embedded within Dlx2-stained cells (arrowhead) in a 25-gw infant. Scale bar, 20 µm. (b) Representative immunofluorescence of coronal sections from 20 and 25 gw fetuses labeled with Dlx2- and Ki67-specific antibodies and DAPI. Note the percentage of Dlx2⁺ cells (Dlx2⁺/DAPI⁺) was higher in the MGE relative to the LGE and cortical SVZ. Proliferating (arrowhead) and non-proliferating (arrow) cells shown. Scale bar, 20 µm. (c) Bar chart shows mean ± SEM (n = 5 each group). The density of Dlx2+ cells was reduced in 29–35 gw relative to other groups. *P < 0.001 for MGE versus LGE and ^#P < 0.001 for MGE versus cortical SVZ within gestational group comparisons. ^†P < 0.001, ^‡P < 0.001 and ^ψP < 0.001 for 29–35 versus 16–22, 23–25, and 26–28 gw in the MGE, respectively. (d) Data are mean ± SEM (n = 5 each). Cycling Dlx2⁺ cells were more numerous in the MGE relative to the LGE and cortical SVZ. *P < 0.001 for MGE versus LGE and ^#P < 0.001 for MGE versus cortical SVZ within gestational group comparisons. ^†P < 0.001, ^‡P < 0.001, and ^ψP < 0.01 for 29–35 versus 16–22, 23–25, and 26–28 gw within the MGE, respectively. (e) Bar chart shows mean ± SEM (n = 5 each group). The percentage of Dlx2⁺ cells (Dlx2⁺/DAPI⁺ cells) was higher in the MGE compared with the LGE and cortical SVZ. *P < 0.001 for MGE versus LGE and ^#P < 0.001 for MGE versus cortical SVZ within gestational group comparisons. ^†P < 0.001, ^‡P < 0.001 and ^ψP < 0.01 for 29–35 versus 16–22, 23–25, and 26–28 gw in the MGE, respectively. ^φP < 0.01 for 23–25 versus 26–28 in the MGE.

Figure 4.

Sox2⁺ and Sox2⁺Nkx2.1⁺ cells were more abundant in the MGE relative to other germinal zones. (a) Representative triple labeling of coronal sections from the MGE of 20 and 25 gw subjects stained with Sox2-, Nkx2.1-, and Ki67-specific antibodies. Note abundance of Sox2⁺Ki67⁺ and Sox2⁺Nkx2.1⁺ cells in the VZ and SVZ of the MGE (lower magnification). Scale bar, 50 µm. (b) Typical triple immunolabeling of coronal sections from 20 and 25 gw subjects (higher magnification) using Sox2-, Nkx2.1-, and Ki67-specific antibodies. Note fewer Sox2⁺Nkx2.1⁺ (arrow) and Sox2⁺Ki67⁺ (arrowheads) cells in the LGE and cortical SVZ relative to the MGE. In 25 gw infants, clusters of non-proliferating Nkx2.1⁺Sox2⁻ cells (shown by an oval outline) were surrounded by both proliferating and non-proliferating Sox2⁺ cells. Scale bar, 20 µm. (c) Data are mean ± SEM (n = 5 each group). The total Sox2⁺ cells were reduced in 29–35 gw relative to other groups in all 3 regions. *P < 0.05 for 29–35 versus 16–22, 23–25, and 26–28 gw in the MGE; ^#P < 0.05 for 29–35 versus 16–22, 23–25, and 26–28 gw in the LGE; ^†P < 0.05 for 29–35 versus 16–22, 23–25, and 26–28 gw in the cortical SVZ. (d) Bar charts are mean ± SEM (n = 5 each group). ^†P < 0.001, ^‡P < 0.001 for 16–22 versus 26–28 and 29–35 gw, respectively, in the MGE; ^αP < 0.001, ^βP < 0.001 for 16–22 versus 26–28 and 29–35 gw, respectively, in the LGE; ^φP < 0.001, ^ψP < 0.001 for 16–22 versus 26–28 and 29–35 gw, respectively, in the cortical SVZ. *P < 0.05, ^#P < 0.01 for MGE versus cortical SVZ for 16–22 and 23–25 gw, respectively. (e) Bar charts are mean ± SEM (n = 3–5 brains each group). Sox2⁺Nkx2.1⁺ were abundant in the MGE and relatively few in the LGE and cortical SVZ. *P < 0.001 MGE versus LGE and ^#P < 0.001 for MGE versus cortical SVZ for within gestational group comparison. ^†P < 0.001 ^‡P < 0.001 for 16–22 gw versus 26–28 and 29–35 gw within the MGE; ^φP < 0.001, ^ψP < 0.001 for 29–35 gw versus 23–25 and 26–28 gw within the MGE.

Figure 5.

Sox2⁺Dlx2⁺ cells were more abundant in the MGE relative to other germinal zones, and Sox2 protein was elevated in the GE relative to the cortical SVZ. (a) Representative immunofluorescence of coronal sections from the MGE of a 20- and 25-gw subject labeled with antibodies to Sox2, Dlx2, and Ki67. Note abundance of Sox2⁺Ki67⁺ and Sox2⁺Dlx2⁺ cells and their distribution in the SVZ of the MGE (lower magnification). Scale bar, 50 µm. (b) Typical triple immunolabeling of coronal sections from the MGE, LGE, and cortical SVZ (higher magnification) for 20 and 25 gw subjects using Sox2-, Dlx2-, and Ki67-specific antibodies. Note fewer Sox2⁺Dlx2⁺ in the LGE and cortical SVZ relative to the MGE. Scale bar, 20 µm. (c) Bar charts are mean ± SEM (n = 5 each group). Sox2⁺ Dlx2⁺ cells were fewer in the LGE and cortical SVZ relative to the MGE. *P < 0.05 MGE versus cortical SVZ for 23–25 gw. ^†P < 0.01, ^‡ P < 0.01 for 16–22 and 23–25 gw versus 29–35 gw in the MGE. (d) Representative western blot analyses for Sox2 performed on homogenates of tissues from cortical SVZ and GE. Each lane represents one brain region from one brain. Bar charts are mean ± SEM (n = 7 brains each group). Data were normalized to β-actin. Sox2 protein levels were higher in the GE compared with the cortical SVZ. *P < 0.05 for cortical SVZ versus GE.

Figure 6.

CGE exhibited Coup-TFII and Dlx2 proliferation, which reduced with advancing gestational age. (a) Representative immunofluorescence of coronal sections from the CGE of 17 and 24 gw subjects labeled with antibodies to Coup-TFII and Ki67. Shown in the second and fourth image, above and to the right, are orthogonal views (as in Figs 2 and 3), depicting Ki67-stained nucleus embedded within the Coup -TFII⁺-stained cells (arrow) in 17 and 24 gw infants. Proliferating cells are shown by arrowheads. Scale bars, 20 µm. (b) Typical immunofluorescence of cryosections from the MGE of 17 and 24 gw subjects labeled with antibodies to Dlx2 and Coup-TFII. Shown in the second and fourth micrograph of the lower panel, above and to the right of the image, are orthogonal views depicting complete overlap of Dlx2 and Coup-TFII-stained cells (arrows) in the CGE of 17 and 24 gw subjects. Proliferating cells are shown by arrowheads. (c) Bar charts are mean ± SEM (n = 3–5 each group). Coup-TFII⁺ and Coup-TFII⁺Ki67⁺ cells reduced as a function of increasing gestational age. *P < 0.05, ^#P < 0.01 for 16–22 versus 29–35 and 35–40 gw, respectively. ^†P < 0.01, ^‡P < 0.01 for 23–25 versus 29–35 and 36–40 gw, respectively. ^ψP < 0.01, ^φP < 0.01 for 16–22 versus 29–35 and 36–40 gw, respectively. (d) Bar charts are mean ± SEM (n = 3–5 each group). *P < 0.05, ^#P < 0.01 for 16–22 versus 29–35 and 35–40 gw, respectively. ^‡P < 0.01 Dlx2⁺ cells for 23–25 versus 36–40 gw, respectively. ^ψ P < 0.01, ^φ P < 0.01 for 23–25 versus 29–35 and 36–40 gw, respectively. ^αP < 0.05 for 16–22 versus 36–40 gw. (e) Coup-TFII⁺Dlx2⁺ cells diminish with advance in gestational age. ^ψP < 0.01, ^φP < 0.05 for 16–22 versus 29–35 and 36–40 gw, respectively. ^α P < 0.05 for 23–25 versus 36–40 gw.

Figure 7.

Pan-Dlx, Nkx2.1, and Mash1 protein levels were higher in the GE compared with the cortex and white matter; these molecules were more abundant in the GE compared with the cortical SVZ. (a) Representative western blot analyses of Pan-Dlx, Nkx2.1, and Mash1 performed on homogenates form tissues taken from GE, cortex (cortical plate), and white matter (WM, intermediate zone). Rat brain (P5) was used as a positive control. Pan-Dlx, Nkx2.1, and Mash1 were predominantly expressed in the GE and weakly in the cortex and white matter. Each lane represents one brain region. Bar charts are mean ± SEM (n = 4 brains each gestational group). Data normalized to β-actin. *P < 0.001 **P < 0.01 for cortex versus GE within the gestational group; ^#P < 0.001 ^##P < 0.01 for WM versus GE within the gestational group. ^†P < 0.001, ^††P < 0.05 for 16–22 versus 29–35 gw in the GE; ^‡P < 0.001 for 23–25 versus 29–35 gw in the GE; ^ψP < 0.05 for 26–28 versus 29–35 gw in the GE. ^φP < 0.05 for 23–25 versus 26–28 gw in the GE; ^αP < 0.001 for 26–28 versus 23–25 gw in the GE; ^βP < 0.05 for 23–25 versus 29–35 gw in the GE. (b) Typical western blot analyses for Pan-Dlx, Nkx2.1, and Mash1 in the cortical SVZ and GE. Bar charts are mean ± SEM (n = 7 each group). Data normalized to β-actin.

Figure 8.

GABAergic cells were higher in number in the cortical SVZ compared with the MGE and LGE. (a) Representative immunostaining of coronal cyrosections from 17 and 24 gw subjects with GABA-specific antibody. Lower panel images are high magnification of the boxed area in the upper panel. Note more abundant GABA⁺ cells in the cortical SVZ (cSVZ) relative to the MGE. GABA⁺ cells were almost absent in the VZ of the MGE. In the cSVZ, GABA⁺ cells exhibited processes (arrowheads), and there were separated by intercellular spaces. Conversely, GABA⁺ cells were densely-packed, confluent, and lacking process in the MGE (arrows). Scale bar, 50 µm (upper panels), 20 µm (lower panels). (b) Bar charts are mean ± SEM (n = 5 each). The density of GABA⁺ cells was higher in the cSVZ compared with MGE and LGE for all ages, except for 19–35 gw. *P < 0.001, ^#P < 0.001 for MGE versus cSVZ and LGE versus cSVZ within the gestational group, respectively. ^†P < 0.05, ^‡P < 0.05 16–22 versus 29–35 within the cSVZ and LGE, respectively. (c) Bar charts are mean ± SEM (n = 5 each group). The percentages of GABAergic cells (density of GABA⁺/density of DAPI⁺) were also elevated in the cSVZ compared with the MGE and cSVZ. *P < 0.001, ^#P < 0.001 for MGE versus cSVZ and LGE versus cSVZ within the gestational group, respectively. ^†P < 0.05 16–22 versus 29–35.