Introduction

Oligodendrocytes (OLs) are an important class of macroglia responsible for producing the myelin sheaths that insulate and protect neuronal axons. Forebrain OLs arise from multiple embryonic origins. Previous fate mapping study using Nkx2.1^Cre[1], Gsh2^Cre[2] and Emx1^Cre[3] reported consecutive and competing waves of OLs derived from medial ganglionic eminence/preoptic area (MGE/POA), lateral/caudal ganglionic eminences (LGE/CGE) and dorsal pallium[2]. The first-wave of OLs generated by MGE/POA (_MPOLs) were believed to be eliminated postnatally, while those from the second and third waves (_LCOLs and dOLs) survive and populate the cortex and corpus callosum at comparable proportions. Several other studies provided both supporting and contradicting evidence to this model[4–9]. Moreover, Gsh2 was recently found to be expressed in dorsal progenitors[10], casting doubt on the interpretation of lineage tracing data from Gsh2^Cre.

In this study, we generated new genetic tools and combinatorial fate mapping strategies which allow direct visualization and comparison among OLs derived from different origins. We found that neocortical OLs are primarily composed of dOLs, rather than similar proportions of _LCOLs and dOLs. In contrast, _LCOLs and dOLs made comparable contributions to piriform cortex. We also found that although _MPOLs only make a small contribution, they do persist in the cortex beyond adulthood with a unique spatial pattern distinct from that of the dOLs. In the two major white matter commissure tracts, dOLs are the vast majority in corpus callosum but make little contribution to anterior commissure, while _LCOLs behaved the opposite. These findings significantly revised the classical view and provided a new and more comprehensive picture of cortical and white matter OL origins.

Results and Discussion

To unambiguously track OLs from different embryonic origins, we first generated a knock-in driver, Opalin^{P2A-Flpo-T2A-tTA2} (Figure 1), orthogonal to Cre drivers that label dorsal or ventral progenitors (Progenitor^Cre). Opalin (also known as Tmem10) encodes oligodendrocytic myelin paranodal and inner loop protein that are specifically expressed in differentiated oligodendrocytes[11–14]. In Opalin^{P2A-Flpo-T2A-tTA2}, Flpo and tTA2 were inserted before the STOP codon and linked by self-cleavage peptide P2A and T2A (Figure 1A-C), allowing co-transcription and translation with Opalin. Flp-mediated recombination by this driver (hereinafter referred to as Opalin^Flp for simplicity) enables highly specific, efficient and irreversible OL labeling, while the tTA2 component offers the flexibility for labeling in tunable densities (Figure 1D-F).

Figure 1.

Next, we established two types of genetic combinatorial fate mapping strategies to directly visualize OLs from different embryonic origins (Figure 2): (1) combining Opalin^Flp and Progenitor^Cre with intersectional reporters Ai65 to label OLs derived from Cre+ progenitor domain by RFP (Figure 2A); (2) combining Opalin^Flp and Progenitor^Cre with RC::FLTG[15] to simultaneously label OLs derived from Cre+ progenitors by GFP and OLs derived from the complementing Cre-progenitors by RFP (Figure 2B). The first approach allowed us to track dOLs and _MPOLs (Figure 2C, E). The second approach empowered us to observe and compare OLs generated from dorsal and ventral origins, or those from Gsh2+ and Gsh2-progenitors, in the same brain (Supplementary Figure 1). Importantly, the subtraction power enabled us to target OLs derived from LGE/CGE progenitors that express neither Emx1 nor Nkx2.1 (Figure 2D). In addition, these strategies greatly facilitated the identification of OLs derived from specific origin which exist at relatively low density in certain regions.

Figure 2.

Deploying these strategies, we assessed the differential contributions of dOLs, _LCOLs and _MPOLs by analyzing RFP+ cells in the following mice: Opalin^Flp::Emx1^Cre::Ai65 (Figure 2C), Opalin^Flp::Emx1^Cre::Nkx2.1^Cre::RC::FLTG (Figure 2D) and Opalin^Flp::Nkx2.1^Cre::Ai65 (Figure 2E). We observed two significant differences from the traditional model in the neocortex. The first major deviation is that, instead of comparable contributions by dOLs and _LCOLs, the vast majority of neocortical OLs were dOLs but not _LCOLs. The density of dOLs is significantly higher than that of _LCOLs in the motor cortex (Mo) (504.5±25.1 vs. 5.3±0.7; P<0.001; n=3) and somatosensory cortex (SS) (518.0±22.6 vs.21.6±2.3; P<0.001; n=3) (Figure 2C, D, F). Co-staining with aspartoacylase (ASPA)[16], a marker for mature OL, also revealed a very high neocortical OL labeling rate in Opalin^Flp::Emx1^Cre::Ai65 (RPF+ASPA+/ASPA+: 94.6±2.0% in Mo and 90.5±2.2% in SS; n=3).Considering the possibility of incomplete recombination in combinatorial reporters, and the relatively low Cre activity in the dorsal MGE of Nkx2.1^Cre[1], the genuine contribution of _LCOLs to the neocortex could be even lesser than our current observation. Therefore, the large quantity of neocortical OLs labeled by Gsh2^Cre in previous study[2] or by GFP in Opalin^Flp::Gsh2^Cre:: RC::FLTG (Supplementary Figure 1B) most likely were predominantly dOLs generated by Gsh2+ dorsal progenitors[10], rather than bona fide _LCOLs.

The second major deviation is that cortical _MPOLs are not completely depleted postnatally, instead, they make a small but continued contribution with a unique spatial distribution pattern (Figure 2D and Supplementary Figure 2). _MPOLs display a clear rostrocaudal density decline (Supplementary Figure 2A-B) and a laminar preference towards layer 4 (L4) in SS (Supplementary Figure 2C-E). In contrast, the distribution of dOLs does not vary significantly across the rostrocaudal axis but exhibits increased density towards deeper layers (Supplementary Figure 2A-E). Importantly, we have observed cortical _MPOLs in mice as old as one year (Supplementary Figure 2F), well beyond the age analyzed in previous reports[2, 7, 8], suggesting a persisted contribution.

We then turned our attention to the lateral three-layer archicortex, piriform cortex (Pir). Different from the neocortex, Pir contains comparable density of dOLs and _LCOLs (114.1±12.5 vs. 171.7±23.6; P=0.058; n=3) (Figure 2F). _MPOLs make the lowest contribution at a density similar to SS(16.4±2.4 vs.15.8±1.4; P=0.966; n=4) (Figure 2E).

These combinatorial models also grant us the opportunity to revisit the differential contributions of dOLs, _LCOLs and _MPOLs to the two commissural white matter tracts, corpus callosum (cc) and anterior commissure (ac), which contain higher density of OLs than the grey matter (Figure 3). We found that, similar to the neocortex, cc is mainly populated by dOLs and supplemented by very low density of _LCOLs and _MPOLs(Figure 3A-D). Interestingly, _LCOLs and _MPOLs seem to show preferential distribution in the lateral and medial regions of cc (cc-l and cc-m), respectively (Figure 3B-D, boxed regions in right panels). Different from cc, ac is mainly populated by _LCOLs (2454.6±254.1, n=3) and _MPOLs (1146.3±101.6; n=4) and supplemented by very low density of dOLs (85.2±34.5; n=3) (Figure 3D).

Figure 3.

To substantiate the above results, we further breed Opalin^Flp::Emx1^Cre::Nkx2.1^Cre::Ai65 to label dOLs together with _MPOLs by RFP and co-stained them with ASPA (Supplementary Figure 3). RFP-ASPA+ cells were difficult to find in Mo, SS and cc, but were more easily observed in Pir and ac, consistent with the respective low and high _LCOL densities in these regions.

In summary, our findings significantly revised the canonical model of forebrain OL origins (Figure 4A), and provided a new and more comprehensive view (Figure 4B). We demonstrated that neocortical OLs are mainly derived from dorsal origin with small but lasting contribution from the ventral origin (Figure 2, Supplementary Figure 1A). Our data showed that LGE/CGE makes little contribution to neocortex and cc, but makes major contribution to piriform cortex and ac. This finding is supported by another report[17] in which in utero electroporation failed to label LGE-derived cortical OLs. Taken together, these results suggest that the lack of _LCOL in neocortex is less likely caused by competitive postnatal elimination, but more likely due to limited production and/or allocation. We further discovered that MGE/POA makes a small but persistent contribution to the neocortex with a distinct distribution pattern featured by a rostral-high to caudal-low gradient and a preference towards L4 in SS. Whether their enduring existence and highly biased localization has functional implications awaits future exploration. In addition, we found that the cc showed a similar OL composition as the neocortex, but the Pir and the ac each exhibited distinct OL compositions in term of their embryonic origins. _LCOLs are the major contributor to both regions, while dOLs and _MPOLs mainly contribute to Pir and ac, respectively.

Figure 4.

In addition to the new framework of forebrain OL origins (Figure 4), we also generated a new driver (Figure 1) and established multiple combinatorial genetic models (Figure 2) for efficient tracking and direct visualization of OLs from different embryonic origins without interference from other cells types. These tools set up a firm foundation and will provide reliable experimental access for future inquiries on the development and function of diverse OLs in healthy and disease brains[18], especially to uncover the relationship between developmental origins and the functional and molecular heterogeneity of OLs.

Material and Methods

Mice

All mouse studies were carried out in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of Fudan University. All husbandry and experimental procedures were reviewed and approved by the same committee. All applicable institutional and/or national guidelines for the care and use of animals were followed. The following transgenic mouse lines were used in this study: Nkx2.1^Cre (Jax 008661)[1],Gsh2^Cre(Jax 025806)[2], Emx1^Cre (Jax 005628)[3], Ai65 (Jax 021875)[19], RC::FLTG (Jax 026932)[15]. The tTA2-dependent tdTomato reporter (TRE-RFP) was derived from Ai62 (Jax 022731) [19], by removing LoxP-STOP-LoxP with E2a-Cre (Jax 003724). The Flp dependent H2B-GFP reporter (HG-FRT) was derived from HG-dual (Jax 028581) via removal of loxP flanking STOP cassette by CMV-Cre[20]. The Opalin^{P2A-Flpo-T2A-tTA2}allele was generated by targeted insertion of the T2A-Flpo-P2A-tTA2 sequence immediately before the STOP codon of the endogenous Opalin gene using homologous recombination. Gene targeting vector was generated using PCR-based cloning approach as described before[20]. More specifically, a 4.7 kb 5’ homology arm, a loxP flanking Neo positive selection cassette, a T2A-FlpO-P2A-tTA2 cassette and a 2.7 kb 3’ homology arm were cloned into a building vector containing the DTA negative selection cassette to generate the targeting vector. Targeting vector was linearized and transfected into a C57/black6 ES cell line. ES clones that survived through negative and positive selections were first screened by genomic PCR, then confirmed by Southern blotting using appropriate DIG-dUTP labelled probes. One positive ES cell clone was used for blastocyst injection to obtain male chimera mice carrying the modified allele following standard procedures. Chimera males were bred with C57BL/6J females to confirm germline transmission by genomic PCR. The Neo selection cassette was self-excised during spermatogenesis of F0 chimeras. Heterozygous F1 siblings were bred with one another to establish the colony. Targeting vector construction, ES cell transfections and screening, blastocyst injections and chimera breeding were performed by Biocytogen Co, China.

Genomic PCR

Genomic DNA was prepared from mouse tails. Tissue was lysed by incubation in tail lysis buffer (Viagen, 102-T) with 0.1 mg/ml proteinase K (Diamond, A100706) overnight at 55°C followed by 45 min at 90°C in an air bath to inactivate proteinase K. The lysate was cleared by centrifugation at maximum speed (21,130 G) for 15 min in a table-top centrifuge. Supernatant containing genomic DNA was used as the PCR template for amplifying DNA products. The following primers were used:

Opalin-F: 5’-GGCCTATGTTTGATTTCCAGCACTG-3’

Opalin-R: 5’-AGCACTTATGACTGCTGAGCCGTTC-3’

Immunohistochemistry and microscopy

Mice were anaesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (0.09 mg/g body weight) and then intracardially perfused with saline followed by 4% paraformaldehyde in 0.1 M PB. Following post fixation at 4°C for 24 hours, brain samples were sectioned at 30 μm using a vibratome (Leica VT1000S), or transferred into 30% sucrose in 0.1M PB for cryoprotection, OCT embedded, and sectioned using a cryostat (Leica CM1950). For CC1 immunostaining, antigen retrieval was performed prior to blocking by boiling for 3 min in 10 mM citrate buffer (pH6.0). Sections were blocked in PBS containing 0.05% Triton and 5% normal donkey serum and then incubated with the following primary antibodies in the blocking solution at 4 ℃ overnight: RFP (goat polyclonal antibody, 1:2000, SICGEN AB0081-200; rabbit polyclonal antibody, 1:2000, Rockland 600-401-379), GFP (chicken polyclonal antibody, 1:1000, Aves Labs, GFP-1020), MBP (rat polyclonal antibody, 1:500, AbD Serotec, MCA409S), CC1 (rabbit polyclonal antibody, 1:500, Oasis Biofarm, OB-PRB070, mouse polyclonal antibody, 1:300, Millipore, OP80), ASPA (rat polyclonal antibody, 1:200, Oasis Biofarm, OB-PRT005), Sox9(rabbit polyclonal antibody, 1:2000, Chemicon, AB5535), NeuN (mouse monoclonal antibody, 1:500, Millipore, MAB377). Sections were then incubated with appropriate Alexa fluor dye-conjugated IgG secondary antibodies (1:500, Thermofisher Scientific) or CF dye-conjugated IgG secondary antibodies (1:250, Sigma) in blocking solution and mounted in Aqua-mount (Southern Biotech, 0100-01). Sections were counterstained with DAPI. Sections were imaged with confocal microscopy (Olympus FV3000), fluorescence microscopy (Nikon Eclipse Ni; Olympus VS120; Olympus VS200) and fluorescent stereoscope (Nikon SMZ25). Anatomical regions in the postnatal brain were identified according to the Paxinos “The Mouse Brain” Atlas and the Allen Reference Atlas, and their areas were measured in ImageJ, whenever applicable. One out of every four sections within the range of selection from each brain and at least three brains per genotype were analyzed. To quantify density and co-localization, cells were identified and counted in Image J or QuPath.

Statistical analysis

GraphPad Prism version 8.0.1 was used for statistical calculations. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications. Data collection and analysis were performed blind to the conditions of the experiments whenever possible. No animals or data points were excluded from the analysis. Normalcy was assessed using Shapiro-Wilk test. Equal variances were assessed using F test or Bartlett’s test. Statistical significance was tested using two-tailed unpaired t-test, Welch’s t-test, one-way ANOVA and two-way ANOVA followed by Tukey’s or Bonferroni post hoc test, wherever appropriate. Data are presented as mean ± standard error of the mean (S.E.M.). P < 0.05 was considered significant. Significance is marked as *P < 0.05; **P < 0.01 and ***P < 0.001.

Acknowledgements

We thank Dr. Yilin Tai for helpful discussion and Dr. Min Jiang from core facility of IOBS, Fudan University, for technical support on imaging experiments. This study was supported by funds from the National Science and Technology Innovation 2030 Major Projects of China (STI2030-Major Projects-2022ZD0206500), National Natural Science Foundation of China (32171087, 32200918, 32371145).

Author contributions

Conceptualization: MH

Methodology: YC, MH

Investigation: YC, ZZ, MS

Visualization: YC, MH, LG

Supervision: MH, LG

Writing—original draft: MH, YC, LG

Writing—Review and editing: MH, YC, ZZ, MZ

Declaration of interests

The authors declare no competing interests.

Supplementary information

Supplementary Figure 1.

Supplementary Figure 2.

Supplementary Figure 3.