Spatiotemporally different origins of NG2 progenitors produce cortical interneurons versus glia in the mammalian forebrain Rosemarie W. Tsoaa,1, Volkan Coskuna,1, Chi K. Hoa, Jean de Vellisa,2, and Yi E. Suna,b,2 aDepartment of Psychiatry and Biobehavioral Sciences, Intellectual and Developmental Disabilities Research Center, University of California, Los Angeles, CA 90095; and bTranslational Stem Cell Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai 200092, China Edited* by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved March 25, 2014 (received for review January 8, 2014) + The studies on the exact lineage composition of NG2 expressing the differentiation potential of heterogeneous NG2 progenitors. progenitors in the forebrain have been controversial. A number of Our genetic lineage tracing, together with BrdU pulse-labeling, studies have revealed the heterogeneous nature of postnatal NG2 identified two independent origins of NG2 progenitors in the + cells. However, NG2 cells found in embryonic dates are far less forebrain. The first group is MGE-derived NG2 progenitor cells that are born at early embryonic dates and give rise to deep-layer understood. Our study indicates that early NG2 progenitors from + a ventral origin (i.e., before embryonic day 16.5) tangentially mi- interneurons in dorsal cortex (CTX). The second NG2 progenitor pools are found in the postnatal subventricular zone (SVZ), de- grate out of the medial ganglionic eminence and give rise to inter- + neurons in deep layers of the dorsal cerebral cortex. The majority rived from GFAP neural stem/progenitor cells (NPCs). This of myelinating oligodendrocytes found in both cortical gray and particular progenitor pool includes the classical OPCs, responsible white matters are, in contrast, derived from NG2 progenitors with for generating mature OLs found in corpus callosum (CC) and a neonatal subventricular zone origin. Our lineage tracing data dorsal CTX. reflect the heterogeneous nature of NG2 progenitor populations Results and define the relationship between lineage divergence and spa- + α + tiotemporal origins. Beyond the typical lineage tracing studies of NG2-Cre BAC Transgenic Mice as a Model System. The NG2 ;PDGF R + NG2 cells, by costaining with lineage-specific markers, our study cells in the CNS are characterized as OPCs that give rise to mature OLs in both white and gray matter. Recent evidence addresses the origins of heterogeneity and its implications in the + + + α differentiation potentials of NG2 progenitors. suggests that CNS NG2 ;PDGF R OPCs are more diverse in terms of their electrophysiological responses and possible lineage potentials (12–14). In general, studies on the lineage lineage differentiation + + potential of NG2 ;PDGFαR cells have focused on postnatal dates. Several findings have indicated that the peak of oligoden- he relationship between progenitor origins and their possible drogliogenesis occurs during early postnatal dates and then Tterminal cell fates in the central nervous system (CNS) de- slows down but remains continuous at low levels in both white velopment is a complex question that remains to be fully and gray matter in the adult CNS. All these studies provided addressed. Depending on the origin from which a progenitor cell strong evidence for the important biological function of postnatal + + arises, physical and molecular regulatory mechanisms define NG2 /PDGFαR progenitors in the mammalian CNS. However, + + both cell identity and direct specific lineage potentials during NG2 /PDGFαR progenitors can also be found within early differentiation and migration. For example, cortical pyramidal embryonic CNS [embryonic day (E)14–E18] at basal ganglia + + neurons are generated in the ventricular zone (VZ) of the pal- (5, 15). These embryonic NG2 ;PDGFαR progenitors are as- lium and are guided by radial glia to their final position in the sumed to be the progenitors that eventually differentiate into ma- cortical plate (1, 2). However, cortical interneurons born in ture OLs in the adult CNS. This raised the question of whether subpallium germinal zones during early embryonic dates tan- gentially migrate to the cortical plate up to the neonatal period. Significance Not only neurons but also the differentiation of glial cells follow a specific spatial and temporal patterning (3, 4). The devel- The CRE/LoxP cell lineage tracing strategy has been applied opmental origin of oligodendrocytes (OLs) is a longstanding effectively to label progenies derived from specific progenitors controversial issue with many valid hypotheses (5). One hypothesis in different model organisms. Although this approach effica- suggests that OLs are developed throughout all regions of the ciously labels cells expressing a specific marker, it often dis- CNS, with multiple and diverse developmental origins that pro- counts the heterogeneity of the cell populations sharing the vide the progenitor sources of all OLs (6, 7). This hypothesis was same cellular marker. In this study, we combined the CRE/LoxP challenged in the early 1990s as a series of observations suggested tracing strategy with BrdU birth-dating analysis to separate the that commitment to the OL lineage occurs in a specialized domain NG2 expression progenitor populations and identified the de- of the ventral VZ in development of the spinal cord and forebrain fined interneuronal versus oligodendroglial lineages on the (8). Both strategies provide mature OLs for myelination, but basis of special and temporal specific origins. separate and distinct developmental regulatory strategies that di- rect the OL development are required for each scenario. Author contributions: R.W.T., J.d.V., and Y.E.S. designed research; R.W.T. performed The subpallium germinal zones are divided into three areas: research; R.W.T., V.C., C.K.H., and Y.E.S. analyzed data; and R.W.T., V.C., J.d.V., and Y.E.S. the medial ganglionic eminence (MGE), the lateral ganglionic wrote the paper. eminence, and the caudal ganglionic eminence. The distribution The authors declare no conflict of interest. of cortical interneurons correlates with the origin of their pro- *This Direct Submission article had a prearranged editor. genitors (9, 10). Additional genetic studies have demonstrated 1R.W.T. and V.C. contributed equally to this work. that NG2 cells in the subpallium give rise to cortical interneurons 2To whom correspondence may be addressed. E-mail: [email protected] or (11). However, it remains unresolved whether cortical inter- [email protected]. neurons and OLs share the same progenitor pool. Collectively, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the aforementioned findings underscore the need for reconciling 1073/pnas.1400422111/-/DCSupplemental. 7444–7449 | PNAS | May 20, 2014 | vol. 111 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1400422111 Downloaded by guest on September 27, 2021 + + these diverse NG2 cell populations give rise exclusively to OLs in D and E), and 93.3% ± 2.08% of all β-gal cells were GST-π the CNS or whether different terminal cell fates are possible. To terminally differentiated OLs (Fig. 1F). These percentages re- + + determine whether all NG2 precursors found in CC and dorsal mained the same in 1-y-old mice (91.3% ± 3.025% of GST-π + + CTX are single-lineage, we traced cells using genetic fate cells were β-gal ;Fig.1E), suggesting that NG2 progenitors in this region mainly undergo oligodendrogliogenesis. In the dorsal mapping by crossing the Ng2-Cre mice that we generated with + CTX at 1 y, ∼79.9% ± 4.97% of all GST-π cells were β-gal-la- the Cre-LoxP reporter mice, Rosa26-LacZ (Fig. 1A). The Cre + recombinase activity under the control of CSPG4 (the gene beled (n = 3) (Fig. 1 D and E). At P30, 76.5% of GST-π OLs encoding the NG2 protein) promoter regulation would induce were already colabeled with β-gal in dorsal CTX (Fig. 1E). These permanent and heritable expression of the reporter gene observations suggest that the majority of differentiated OLs lo- β-galactosidase (β-gal) through mitosis. To confirm the cor- cated in the cortical gray matter are derived from NG2 progeni- + relation of Cre-expressing cells and NG2 cells, we focused on tors, leaving only ∼20% of all myelinating OLs in the dorsal CTX the costaining analysis throughout developmental stages, derived from a non-NG2 progenitor source (Fig. 1E). In contrast, ± β + starting at embryonic dates to postnatal day 30 (P30), and only 3.52% 1.38% of all -gal cells (progeny derived from + + + π+ showed that close to 100% of NG2 cells were also Cre and NG2 cells) in the dorsal CTX became GST- OLs, a stark vice versa in both CC and CTX (Fig. 1A). From the costaining difference from CC, where most (93.3%; Fig. 1F) NG2-derived of Cre and β-gal, the recombination efficiency of Cre regu- progeny became OLs. Therefore, the vast majority of OLs in the lated by CSPG4 promoter was found to be about 98% (Fig. CC and dorsal CTX are derived from NG2 progenitors, whereas 1 B and C). only a very small proportion of NG2 progenitors in dorsal CTX were designated to an OL lineage, leaving it possible for cortical NG2 Progenitors Give Rise to OLs in Both CC and Dorsal CTX. To in- NG2 progenitors to take on other neural cell fates. In summary, our lineage tracing in CC is consistent with previous models sug- spect NG2 lineage development, we first examined whether NG2 + progenitor cells that came from different developmental ages and gesting that NG2 cells are limited to an OL lineage. However, origins all gave rise equally and exclusively to mature OLs in data from the dorsal CTX suggested that these models may not be postnatal mice, as NG2 progenitors are historically considered to true in all cases: Not all NG2 progenitors must become OLs. Therefore, NG2 progenitors may be able to differentiate into non- be exclusively OPCs (16).