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Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences

Nature Neuroscience volume 16, pages 1576–1587 (2013)Cite this article

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Abstract

GABAergic cortical interneurons underlie the complexity of neural circuits and are particularly numerous and diverse in humans. In rodents, cortical interneurons originate in the subpallial ganglionic eminences, but their developmental origins in humans are controversial. We characterized the developing human ganglionic eminences and found that the subventricular zone (SVZ) expanded massively during the early second trimester, becoming densely populated with neural stem cells and intermediate progenitor cells. In contrast with the cortex, most stem cells in the ganglionic eminence SVZ did not maintain radial fibers or orientation. The medial ganglionic eminence exhibited unique patterns of progenitor cell organization and clustering, and markers revealed that the caudal ganglionic eminence generated a greater proportion of cortical interneurons in humans than in rodents. On the basis of labeling of newborn neurons in slice culture and mapping of proliferating interneuron progenitors, we conclude that the vast majority of human cortical interneurons are produced in the ganglionic eminences, including an enormous contribution from non-epithelial SVZ stem cells.

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Figure 1: Developmental expansion of the OSVZ in the human ganglionic eminences.
Figure 2: Neural stem cells are abundant in the MGE OSVZ.
Figure 3: Most OSVZ stem cells in the ganglionic eminences lack M phase fibers and are randomly oriented.
Figure 4: Segregation of progenitor cells and neurons in the MGE OSVZ.
Figure 5: Destinations of human MGE-derived neurons.
Figure 6: Patterns of COUP-TFII expression in the CGE and LGE.
Figure 7: Production of cortical interneurons in the dorsal LGE.
Figure 8: Lack of DLX2+ cell production in cortical slice cultures.

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Acknowledgements

We thank the staff at San Francisco General Hospital Women's Options Center for their consideration in allowing access to donated human fetal tissue. We thank S. Davis and co-workers at the California National Primate Research Center for providing fixed brain tissue from fetal macaques. We also thank W. Walantus, K. Wang, S. Wang, Y. Wang and other University of California at San Francisco personnel for technical and administrative support. We are very grateful to J. Kohtz (Northwestern University) for sharing her final aliquot of pan-Dlx antibody. The NKX2-2 antibody developed by T. Jessell and S. Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa Department of Biology. This work was supported by grants from the National Institute of Neurological Disorders and Stroke and the National Institute of Mental Health of the US National Institutes of Health, California Institute for Regenerative Medicine, John G. Bowes Research Fund and from Bernard Osher.

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Author notes

  1. David V Hansen & Pierre Flandin

    Present address: Present addresses: Neuroscience, Genentech, South San Francisco, California, USA (D.V.H.), Quanticel Pharmaceuticals, San Francisco, California, USA (P.F.).,

Authors and Affiliations

  1. Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, USA

    David V Hansen, Jan H Lui, John L Rubenstein, Arturo Alvarez-Buylla & Arnold R Kriegstein

  2. Department of Neurology, University of California, San Francisco, San Francisco, California, USA

    David V Hansen, Jan H Lui & Arnold R Kriegstein

  3. Department of Psychiatry, University of California, San Francisco, San Francisco, California, USA

    Pierre Flandin & John L Rubenstein

  4. Laboratory of Regulation of Neuronal Development, Institute for Protein Research, Osaka University, Suita, Osaka, Japan

    Kazuaki Yoshikawa

  5. Department of Neurosurgery, University of California, San Francisco, San Francisco, California, USA

    Arturo Alvarez-Buylla

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Contributions

D.V.H. and J.H.L. acquired and processed tissue, cultured slices, and performed immunostains, confocal imaging and cell counts. P.F. performed LHX6 in situ hybridizations and imaging. K.Y. provided guinea pig antibody to Dlx2. D.V.H. wrote the manuscript. J.L.R., A.A.-B. and A.R.K. provided guidance and conceptual support and edited the manuscript.

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Correspondence to John L Rubenstein, Arturo Alvarez-Buylla or Arnold R Kriegstein.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Developmental timelines of human and macaque gestation.

Human and macaque tissues from postconception week (PCW)10 and gestational day (GD)65, respectively, are temporally and developmentally equivalent. Both tissues are in the tenth week post-conception and have ganglionic eminences of comparable dimensions. From this, we infer that the GD55 macaque tissue in main Fig. 1b is roughly equivalent to human PCW8 tissue. In previous manuscripts, we employed the clinical term 'gestational week' (GW), which is two weeks greater than postconception week since it is determined from the patient's last onset of menses. D, V, R, C: dorsal, ventral, rostral, caudal.

Supplementary Figure 2 Progenitor cell organization in the macaque MGE.

Cellular components and cytoarchitecture of macaque MGE at gestational day 65 (equivalent to human PCW10), sagittal views. Magnified regions 1 and 2 show the three MGE progenitor cell compartments. The ventricular zone (VZ) consists mainly of undifferentiated progenitor cells (radial glia) that express SOX2 and OLIG2. The inner subventricular zone (ISVZ) is enriched with neuronally committed progenitor cells that express ASCL1 and DLX2. The outer SVZ (OSVZ) is a heterogeneous mixture of both undifferentiated and lineage-committed progenitor cells, together with newborn neurons that have downregulated SOX2, OLIG2, ASCL1 and Ki67 but continue to express DLX2. Early formation of type II clusters is observed in the OSVZ, featured in magnified regions 3 and 4. D, V, R, C: dorsal, ventral, rostral, caudal.

Supplementary Figure 3 Progenitor cell compartments in the embryonic rat MGE.

SVZ in rat GE can be divided into two progenitor cell compartments. SVZ1 is adjacent to the VZ, is enriched with VZ-derived intermediate progenitor cells that express Ascl1 and/or Dlx2 (not shown), and is equivalent to what we have termed inner SVZ in human and macaque GE. SVZ2 is further from the ventricle and includes progenitor cells that express Sox2 (shown above) and Olig2 (not shown), raising the possibility that SVZ2 contains at least some undifferentiated progenitor cells. Rodent SVZ2 likely bears some evolutionary relationship to the OSVZ in human GE, with key differences being scale of expansion, lack of pronounced clustering in SVZ2, and possibly degree of differentiation. Top: Entire LGE and MGE germinal regions outlined in red using Ki67 to mark proliferating cells. Area outlined in green (lower left) is magnified (lower right) to show MGE progenitor cell compartments. D, V, M, L: dorsal, ventral, medial, lateral.

Supplementary Figure 4 Instances of ganglionic eminences progenitor cells with M phase fibers.

a) In contrast to OSVZ progenitors, dividing cells in the VZ often showed retention of fibers during M-phase and radial orientation. Nestin and DAPI are shown only in LGE images. Scale bars, 10 μm. b) Occasional examples of progenitor cell association with the developing neurovasculature were observed in the MGE OSVZ. Left: This unipolar 4A4+ cell displayed a contorted fiber that appeared to terminate on the endothelial basement membrane (marked with collagen IV). Middle: Example of a 4A4+ fiber without a clearly associated cell body, terminating on endothelial basement membrane. Right: Example of 4A4+ cell body directly associated with a blood vessel marked by dashed white lines (discernible by eye when focusing up and down). Scale bars, 10 μm. c-e) Rare examples MGE and LGE progenitors in the OSVZ with clear oRG cell morphology and radial orientation. The MGE oRG cell (c) is associated with a bundle of radial glial fibers in a type I progenitor cell cluster. Boxed or numbered areas are magnified to show cell morphology by phospho-vimentin stain and coexpression with nestin and SOX2. R, C, M, L, D, V: rostral, caudal, medial, lateral, dorsal, ventral. Cd, caudate; IC, internal capsule; Cx, cortex.

Supplementary Figure 5 Type I clustering of MGE progenitor cells.

a) Type I clustering in PCW12 MGE, observed as streaks of progenitor cells extending from the inner SVZ into the OSVZ. The streaks are most apparent with markers of undifferentiated cells such as OLIG2 (left and center images), but are also discernible by the absence of signal for LHX6 (right image, orange arrows), which is expressed mainly in postmitotic neurons. b) Type I clusters are very pronounced in PCW13 horizontal sections, with narrow streaks of SOX2+ OLIG2+ progenitor cells around pockets of SOX2– OLIG2– NKX2-1+ neurons. c) The early formation of nestin+ radial fiber bundles, the core of type I clusters, can be seen in the dorsal MGE at PCW10, before the clusters are apparent using nuclear markers.

Supplementary Figure 6 Type II clustering of MGE progenitor cells

a) Type I and type II progenitor cell clusters are seen in PCW12 MGE OSVZ, with OLIG2+ and DLX2high progenitor cells segregated from OLIG2– DLX2+ neurons. The type II clusters of neurons appear as streaks toward the ventrolateral MGE border, where NKX2-1+ DLX2+ neurons migrate into the basal ganglia. GP, globus pallidus; Th, thalamus. b) Type II clusters in PCW14 MGE, showing clumps of SOX2+ and ASCL1+ progenitor cells amid streaks of NKX2-1+ neurons extending toward the dorsolateral MGE border and the LGE en route to the cortex. c) LHX6 mRNA expression in PCW14 MGE shows densely labeled streaks in the OSVZ (orange arrowheads), suggestive of neuronal migratory streams. The absence of signal in the periventricular region (magenta arrowheads) indicates that LHX6 is expressed mainly in post-mitotic cells. (This image from the same section used for main Fig. 5b.)

Supplementary Figure 7 MGE-derived cells migrating into the LGE and striatum.

a) In the MGE, many NKX2-1+ cells are progenitor cells that coexpress SOX2. A front of NKX2-1+ cells with downregulated SOX2 can be seen exiting the MGE toward the LGE. Further into the LGE, the frequency of NKX2-1 expression dissipates as MGE-derived interneurons migrating to the cortex downregulate NKX2-1. (Asterisk, nonspecific staining of blood vessels.) b) The relative lack of Ki67 signal reveals a hypoproliferative region of OSVZ (arrows) between the MGE and LGE. In these sections, the width of the hypoproliferative region is roughly 0.4 mm at PCW10, 0.6 mm at PCW12, and 1.3 mm at PCW14. (Asterisk, microhemorrhage.) c) Striatal interneurons exiting the MGE into the caudate (Cd) appear as a front of NKX2-1+ cells with downregulated progenitor cell markers.

Supplementary Figure 8 MGE-derived cells populate the globus pallidus.

a) NKX2-1+ cells that have downregulated progenitor cell markers migrate ventrocaudally out of the rostral MGE toward the globus pallidus (GP). The region boxed in red from this PCW10 sagittal section is magnified and shown with NKX2-1 alone and in combination with SOX2 and ASCL1. The region boxed in white is further magnified, showing MGE-derived cells streaming from the germinal region toward the GP (dashed arrows). b) In situ hybridization for LHX6 mRNA in PCW10 frontal section shows apparent stream of MGE-derived cells migrating ventrally along the border of the thalamus (Th) toward pallidal structures. c) In these PCW12 sections, the external (GPe) and internal (GPi) globus pallidi are seen as distinct nuclei using NKX2-1 staining. Pu, putamen; IC, internal capsule.

Supplementary Figure 9 Transcription factor patterning in the MGE ventricular zone.

a) At the interganglionic sulcus (asterisk), COUP-TFII is expressed in the same region of VZ where MGE fate determinants are downregulated, similar to the pattern in mouse (Nery et al., 2002). Sections from rostral-intermediate region of PCW12 GE. b) Going from ventral MGE to dorsal MGE, the VZ changes from containing mostly NKX2-1high progenitor cells (box 1) to mostly NKX2-1low cells (box 3), with the region between being a mixture of NKX2-1high and NKX2-1low cells (box 2). COUP-TFII is expressed infrequently in the MGE and in distinct cells from those that express NKX2-1. In the dorsalmost VZ near the interganglionic sulcus (asterisk), the regions of NKX2-1 and COUP-TFII expression overlap but the two factors are expressed in mutually exclusive cells (box 4). Thus, these COUP-TFII+ cells may actually be CGE-type progenitors. The few OSVZ cells that appear to be co-labeled are juxtaposed cells with overlapping signal. Section from intermediate rostral-caudal location of PCW12 MGE. c) The high-to-low ventral-dorsal gradient of NKX2-1 expression in the MGE VZ (outlined in right panel) is also observed in PCW10 tissue. The expression level of NKX2-1 in MGE progenitors may be related to the preferential production of parvalbumin+ or somatostatin+ cortical interneurons in the ventral or dorsal MGE, respectively. Section from intermediate rostral-caudal location. Cd, caudate; IC, internal capsule; Pu, putamen; Cx, cortex.

Supplementary Figure 10 NKX2-1+ COUP-TFII+ progenitors in the caudalmost MGE.

a) At caudal MGE locations, spotty COUP-TFII expression is seen in the VZ and SVZ (box 2). A long stretch of VZ with solid COUP-TFII expression connects the MGE and LGE (box 3), suggesting the expansion of CGE-type progenitor domains as the GE progresses caudally. Scale bars, 1 mm (orange), 0.2 mm (white). b-c) At its caudalmost extremity, the MGE thins into a narrow belt of progenitor cells that extends caudoventrally along the telencephalic-thalamic interface. At this location, NKX2-1 and COUP-TFII are widely coexpressed in subventricular progenitor cells near the interganglionic sulcus (asterisk) and in VZ/SVZ cells within the caudoventral MGE extension. These progenitors could give rise to Martinotti cells, cortical interneurons in layer V that express both somatostatin and calretinin—markers associated with MGE-derived and CGE-derived interneurons, respectively. d) The caudalmost MGE extending between the striatum and thalamus is marked by transcription factors typical of MGE progenitor cells: NKX2-1, SOX2, and OLIG2, as well as ASCL1 and Ki67 (not shown). (These sections from a different PCW12 tissue than that featured in panels a/b/c/e, and cut in a slightly different orientation. The tissue fragment did not include certain caudoventral structures such as the inferior GE and the ventral cortex.) e) This frontal section of PCW12 tissue is from the same tissue featured in panels b-c, but from a slightly more caudal location. The NKX2-1+ cells represent MGE-derived cells (but not the MGE itself since progenitor cell markers are downregulated) in a caudal migratory stream. Cx, cortex; Str, striatum; Th, thalamus.

Supplementary Figure 11 Distinguishing CGE and LGE-type germinal regions.

The CGE ventricular zone can be subdivided into at least two regions based on COUP-TFII expression. Immunostaining tissues from postconception week 10 (frontal section) and week 13 (horizontal section) showed COUP-TFII+ VZ in the medial CGE (arrows), and COUP-TFII– VZ in the lateral CGE (arrowheads). The COUP-TFII– region and its adjacent SVZ is likely a caudal extension of the LGE. D, V, M, L, R, C: dorsal, ventral, medial, lateral, rostral, caudal. Th, thalamus; Cx, cortex.

Supplementary Figure 12 Non-proliferative COUP-TFII+ DLX2+ cells in the LGE and cortex.

a) PCW10 sagittal section stained for COUP-TFII, DLX2, and Ki67.The boxed area where rostralmost LGE meets cortex is magnified, with a line marking the boundary between LGE and cortex. b) PCW12 frontal section stained for COUP-TFII and Ki67 (same section as the COUP-TFII stain shown in main Fig. 2a.) Magnified images of OSVZ are shown to the right, with panels 1,2 from the dorsal LGE (dLGE) and panel 3 from the CGE. Cells that coexpress COUP-TFII and Ki67 (appearing yellow) are frequent in the CGE but nearly absent in the LGE, including in the dLGE, reported to be a source of COUP-TFII+ cortical interneurons in mouse. Our data suggests that COUP-TFII+ interneurons are not produced within but simply migrate through the dLGE before entering the cortex. D, V, R, C, M, L: dorsal, ventral, rostral, caudal, medial, lateral. Str, striatum; Th, thalamus; Cx, cortex. c) In the dorsal cortex (dCx), the fraction of DLX2+ interneurons that coexpress COUP-TFII increases from ∼35% at PCW10 to more than 50% by PCW14, within the SVZ/IZ where the majority of interneurons are found at these ages. Additionally, the ventral cortex (vCx) shows a greater proportion of CGE-derived interneurons than the dorsal cortex at PCW12. IZ, intermediate zone; CP, cortical plate.

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Supplementary Figure 13 Markers of LGE-cortical boundary in the OSVZ.

The dorsal border of the LGE is neatly marked in the OSVZ by the boundary within which a high level of DLX2 is frequently coexpressed with Ki67 (a), OLIG2 (b), ASCL1 (b), and SOX2 (c) in progenitor cells. In the cortex, DLX2 is expressed at lower levels in post-mitotic inhibitory neurons, in which OLIG2 and Ki67 are completely repressed and SOX2 and ASCL1 are severely downregulated. The OLIG2+ cells in the cortex (b) are DLX2– oligodendrocyte progenitor cells. Note that essentially all COUP-TFII+ cells in both the LGE and in the dorsal cortex (a) are DLX2+ Ki67– postmitotic interneurons, the main exception being occasional COUP-TFII+ DLX2– pericytes (see Supplementary Fig. 15e). Note that certain cortical areas outside the dorsal cortex have COUP-TFII+ DLX2– progenitor cells and pyramidal neurons (see Supplementary Fig. 14a).

Supplementary Figure 14 Locating DLX2+ Ki67+ cortical cells near the LGE or CGE.

a) Unbiased searches of diverse cortical areas revealed only one Ki67+ cell among thousands of DLX2+ cells examined, indicating that proliferation of interneuron precursors within the cortex is exceptionally rare during postconception weeks 10 through 14. The DLX2+ Ki67+ cell was a short distance from the CGE boundary and coexpressed COUP-TFII (see arrowhead in magnified image of boxed area), suggesting that it was a CGE-derived progenitor cell. b, c) Targeted searches of cortical areas adjacent to the LGE and CGE identified isolated examples of DLX2+ progenitor cells that coexpressed Ki67, ASCL1 (b), SOX2 (c), or OLIG2 (not shown). The boxed area of dorsal cortical OSVZ in panel b contains three DLX2+ ASCL1+ cells—two Ki67+ (arrowheads) and one Ki67– (arrow)—within 0.2 mm of the LGE boundary. Within the boxed area in panel c, the ventral cortex (vCx) portion contained well over a thousand DLX2+ cells. Among these, we identified several within 0.2 mm of the CGE that coexpressed SOX2 and Ki67, and five (arrowheads) between 0.2 and 1 mm away. None were found more than 1 mm outside the CGE. For each of the cortical DLX2+ progenitor cells we identified in these and other tissue sections, their proximity to the corticostriatal boundary suggested that they originated in the LGE or CGE. Images in this figure from PCW12.

Supplementary Figure 15 Calretinin+ cells in the cortex are almost exclusively non-proliferative.

a) Human PCW12 frontal section stained for calretinin (CALB2), DLX2, and Ki67 to analyze the proliferative state of CALB2+ cells. While most were non-proliferative, DLX2+ CALB2+ cells in the CGE occasionally expressed the proliferation marker Ki67 (not shown). Many newborn/migrating neurons in the CGE and LGE expressed CALB2 (not shown). In the cortex, CALB2 labeled many interneuron precursors migrating in the germinal region or in the cortical plate, certain neurons of layer I, and thalamocortical fibers. Most CALB2+ cells in the cortex coexpressed DLX2 and COUP-TFII. Th, thalamus; dCx, dorsal cortex; vCx, ventral cortex. B-d) We searched cortical germinal regions for proliferating CALB2+ cells. Many examples of apparent overlap for CALB2 and Ki67 signals were observed, but upon closer examination it was clear that essentially all of these were false double positives from distinct cells lying one atop the other. Such an example is shown in c, with optical z sections showing the separate layering of Ki67 and CALB2/DLX2 signals (arrows). Only one out of 766 DLX2+ CALB2+ cells examined expressed Ki67 (arrowhead in d, single optical section). Similar to other isolated examples of DLX2+ Ki67+ cells that we observed in cortex, this cell was less than 0.5 mm outside the CGE, suggesting it was a CGE-derived interneuron precursor that proliferated after entering the cortex, and it also expressed COUP-TFII (not shown). We also examined cortical CALB2+ cells outside the germinal region, but did not find any that expressed Ki67. Panel e shows CALB2+ cells in cortical layer I, with the nearby neurovasculature containing two Ki67+ cells and three COUP-TFII+ pericytes.

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Hansen, D., Lui, J., Flandin, P. et al. Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences. Nat Neurosci 16, 1576–1587 (2013). https://doi.org/10.1038/nn.3541

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