Embryonic origins of forebrain oligodendrocytes revisited by combinatorial genetic fate mapping

Multiple embryonic origins give rise to forebrain oligodendrocytes (OLs), yet controversies and uncertainty exist regarding their differential contributions. We established intersectional and subtractional strategies to genetically fate map OLs produced by medial ganglionic eminence/preoptic area (MGE/POA), lateral/caudal ganglionic eminences (LGE/CGE), and dorsal pallium in the mouse brain. We found that, contrary to the canonical view, LGE/CGE-derived OLs make minimum contributions to the neocortex and corpus callosum, but dominate piriform cortex and anterior commissure. Additionally, MGE/POA-derived OLs, instead of being entirely eliminated, make small but sustained contribution to cortex with a distribution pattern distinctive from those derived from the dorsal origin. Our study provides a revised and more comprehensive view of cortical and white matter OL origins, and established valuable new tools and strategies for future OL studies.


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 (Xu et al., 2008), Gsh2 Cre (Kessaris et al., 2006), and Emx1 Cre (Gorski et al., 2002) 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 (Kessaris et al., 2006).The first wave of OLs generated by MGE/POA ( MP OLs) was believed to be eliminated postnatally, while those from the second and third waves ( LC OLs 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 (Nakahira et al., 2006;Tsoa et al., 2014;Naruse et al., 2016;Orduz et al., 2019;Liu et al., 2021;Shen et al., 2021;Tripathi et al., 2011;Winkler et al., 2018).Moreover, Gsh2 was recently found to be expressed in dorsal progenitors (Zhang et al., 2020), 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 LC OLs and dOLs.In contrast, LC OLs and dOLs made comparable contributions to piriform cortex.We also found that although MP OLs 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 LC OLs 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 OLs (Kippert et al., 2008;Yoshikawa et al., 2008;Jiang et al., 2013;Marques et al., 2016).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 OL-specific labeling in tunable densities (Figure 1D-F).
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 (Plummer et al., 2015) to simultaneously label OLs derived from Cre+ progenitors by green fluorescent protein (GFP) and OLs derived from the complementing Cre− progenitors by RFP (Figure 2B, Figure 2-figure supplement 1A).The first approach allowed us to track dOLs and MP OLs (Figure 2C, E).The second approach empowered us to observe and compare OLs generated from dorsal and ventral origins (Figure 2-figure supplement 1B), or those from Gsh2+ and Gsh2− progenitors, in the same brain (Figure 2-figure supplement 1C).Importantly, the subtraction power enabled us to target OLs derived from LGE/CGE progenitors that express neither Emx1 nor Nkx2.1 (Figure 2D and Figure 2-figure supplement 1D).In addition, these strategies greatly facilitated the identification of OLs derived from specific origin which exist at relatively low density in certain regions.
Deploying these strategies, we assessed the differential contributions of dOLs, LC OLs, and MP OLs by analyzing RFP+ cells in the following mice: Opalin Flp ::Emx1 Cre ::Ai65 (Figure 2C), Opalin Flp ::Emx-1 Cre ::Nkx2.1 Cre ::RC::FLTG (Figure 2D), and Opalin Flp ::Nkx2.1 Cre ::Ai65 (Figure 2E).To better assess their contributions to the total OL population (Figure 2F), we co-stained RFP with the mature OL marker aspartoacylase (ASPA) (Huang et al., 2023;Figure 2G-I) and quantified the ratio of co-localization (Figure 2J).Notably, all RFP+ cells are ASPA+, reassured the specificity of our label strategies.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 LC OLs, the vast majority of neocortical OLs were dOLs but not LC OLs.The densities (Figure 2G-I and Figure 2-figure supplement 2A-F) and ASPA ratios (Figure 2J) of dOLs are much higher than those of LC OLs.Considering the possibility of incomplete recombination in combinatorial reporters, and the relatively low Cre activity in the dorsal MGE of Nkx2.1 Cre (Xu et al., 2008), the genuine contribution of LC OLs 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 (Kessaris et al., 2006) or by GFP in Opalin Flp ::Gsh2 Cre ::RC::FLTG (Figure 2-figure supplement 1C) most likely were predominantly dOLs generated by Gsh2+ dorsal progenitors (Zhang et al., 2020), rather than bona fide LC OLs.
The second major deviation is that cortical MP OLs are not completely depleted postnatally.Instead, they make a small but continued contribution with a unique spatial distribution pattern (Figure 2I In contrast, the distribution of dOLs and LC OLs do not vary significantly across the rostrocaudal axis F) or between Mo and SS (Figure 2G, H), but exhibits increased density toward deeper layers (Figure 2-figure supplement 2G).Importantly, we have observed cortical MP OLs in mice as old as 1 year (Figure 2-figure supplement 2H), well beyond the age analyzed in previous reports (Kessaris et al., 2006;Orduz et al., 2019;Liu et al., 2021), suggesting a persisted contribution.
We then turned our attention to the lateral three-layer archicortex, piriform cortex (Pir).Different from the neocortex, Pir contains higher proportions (Figure 2J) of LC OLs than dOLs.MP OLs make the lowest contribution (Figure 2J) at a density similar to SS and higher than Mo (Figure 2I).(Kessaris et al., 2006), OLs derived from medial ganglionic eminence/preoptic area (MGE/POA) (orange) were largely eliminated postnatally (thin dashed line), while those from lateral/caudal ganglionic eminences (LGE/CGE) (blue) and dorsal origin (purple) survive at similar proportions (thick solid line).Therefore, neocortex (NCx) and corpus callosum (cc) contain comparable density of LC OLs (blue dots) and dOLs (purple dots) and are devoid of MP OLs (orange dots).(B) In the new model, NCx and cc mainly contain dOLs with very low contribution from the ventral origins.LC OLs mainly contribute to piriform cortex (Pir) and anterior commissure (ac).MP OLs makes a small but sustained contribution to NCx, with a strong laminar preference toward layer 4 in somatosensory cortex (SS).In addition, dOLs and MP OLs also make substantial contributions to Pir and ac, respectively.Gray dots indicate OLs in unanalyzed regions.cc, ac is mainly populated by LC OLs and MP OLs and supplemented by very low proportion of dOLs (Figure 2K-N).
To substantiate the above results, we further breed Opalin Flp ::Emx1 Cre ::Nkx2.1 Cre ::Ai65 to label dOLs together with MP OLs by RFP and co-stained them with ASPA (Figure 2-figure supplement 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 LC OL contributions in these regions.
In summary, our findings significantly revised the canonical model of forebrain OL origins (Figure 3A), and provided a new and more comprehensive view (Figure 3B).We demonstrated that neocortical OLs are mainly derived from dorsal origin with small but lasting contribution from the ventral origin (Figure 2, Figure 2-figure supplements 1B and 2).Our data showed that LGE/CGE makes little contribution to neocortex and cc, but makes major contribution to piriform cortex and ac (Figure 2 and Figure 2-figure supplement 3).This finding is supported by another report in which in utero electroporation failed to label LGE-derived cortical OLs in both embryonic and early postnatal brains, and an exclusion strategy revealed very low percentage of LGE/CGE-derived cortical OLs in neonatal brains (Li et al., 2023).The lack of adult labeling in our study together with the lack of developmental labeling in the other study suggests that the lack of LC OL 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 toward L4 in SS (Figure 2-figure supplement 2).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.LC OLs are the major contributor to both regions, while dOLs and MP OLs mainly contribute to Pir and ac, respectively (Figure 2).
In addition to the new framework of forebrain OL origins (Figure 3), 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 sharing the same progenitor domains such as OL precursors, astrocytes, and neurons (Figures 1-2).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 (Gong et al., 2022), especially to uncover the relationship between their developmental origins and the functional and molecular heterogeneity.Opalin P2A-Flpo-T2A-tTA2  This paper

See Materials and methods, Mice
The tTA2-dependent tdTomato reporter (TRE-RFP) was derived from Ai62 (Jax 022731) (Madisen et al., 2015), 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 (He et al., 2016).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 (He et al., 2016).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-labeled 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 Cyagen.

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:
Sections were counterstained with DAPI (4',6-diamidino-2-phenylindole).Sections were imaged with confocal microscopy (Olympus FV3000), fluorescence microscopy (Nikon Eclipse Ni; Olympus VS120; Olympus VS200), and fluorescent stereoscope (Nikon SMZ25).All quantifications were performed in 2-month-old adult mice from coronal sections between Bregma +1.94 and −2.80 mm.Anatomical regions were identified according to the Paxinos 'The Mouse Brain' Atlas and the Allen Reference Atlas, and their areas were measured in ImageJ for density calculations, whenever applicable.For cortical regions, every fourth section within the range of selection was analyzed.For whiter matter tracts, three consecutive sections at Bregma 0.14 were analyzed.At least three brains were analyzed for each genotype.To quantify density and co-localization, cells were identified and counted in Adobe Photoshop or ImageJ in conjugation with 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 analysis of variance (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.p < 0.05 was considered significant.Significance is marked as *p < 0.05, **p < 0.01, and ***p <0.001.

Figure 1 .
Figure 1.A new driver mouse for efficient and specific oligodendrocyte (OL) labeling.(A) Scheme for generating the Opalin P2A-Flpo-T2A-tTA2 allele.(B) Southern blot confirmation of correctly targeted embryonic stem cell clone.(C) Genomic polymerase chain reaction (PCR) to genotype F1 offspring.(D) OL labeling by Flp.(E) OL labeling by tTA2.High magnification images of the boxed region showing co-localization of red fluorescent protein (RFP) with myelin basic protein (MBP) staining, which further demonstrated the myelination ability of labeled OLs.(F) Quantification of labeling specificity (left panel) and efficiency (right panel) by colacalization with OL marker CC1.Both reporting systems are highly specific, as shown by the complete co-localization of fluorescent protein (XFP) with OL marker (CC1) and lack of co-staining with neuronal marker (NeuN) or astrocyte marker (Sox9).Quantification bar graph was not presented for NeuN and Sox9 as zero co-localizations were observed in all analyzed regions.Close to complete OL labeling was achieved by Flp-dependent H2B-GFP reporter in all analyzed regions (green dots), while sparser labeling with variable regional density was achieved by tTA2-dependent tdTomato reporter driven by TRE promoter (red dots).NCx: neocortex.Pir: piriform cortex.cc: corpus callosum.ac: anterior commissure.Scale bar: 50 μm in low magnification images, 5 μm in high magnification images.Quantification: n = 3. Dots represent data from individual mice.The online version of this article includes the following source data for figure 1: Source data 1.Raw unedited blot for Figure 1B.Source data 2. Uncropped and labeled blot for Figure 1B.Source data 3. Raw unedited gel for Figure 1C.Source data 4. Uncropped and labeled gel for Figure 1C.Source data 5.The raw data for the visualization of data presented in Figure 1F.

Figure 2 .
Figure 2. Combinatorial fate mapping of dOLs, MP OLs, and LC OLs.(A) Strategy for intersectional labeling.Flp-AND-Cre labels oligodendrocytes (OLs) from Cre-expressing progenitors with RFP.(B) Strategy for subtractional labeling of OLs derived from non-Cre-expressing progenitors with RFP.The eGFP expressing OLs derived from Cre-expressing progenitors were not used for analysis in this scenario and thereby were not highlighted by color.Schematics showing intersectional labeling of dOLs in Opalin Flp ::Emx1 Cre ::Ai65 (C), subtractional labeling of LC OLs in Opalin Flp ::Emx1 Cre ::Nkx2.1 Cre ::RC::FLTG (D), intersectional labeling of MP OLs in Opalin Flp ::Nkx2.1 Cre ::Ai65 (E), and cortical OLs derived from all three origins (F).(G-I) Representative images (left panels) and quantifications (right panels) of RFP+ cell density in motor cortex (Mo), somatosensory cortex (SS), and piriform cortex (Pir).(J) Quantification of differential contribution to ASPA+ OLs by three embryonic origins to Mo, SS, and Pir.Representative images (K-M) and quantifications (N) of differential contribution to ASPA+ OLs by three embryonic origins in the two major commissure white matter tracts: corpus callosum (cc) and anterior commissure (ac).MP OLs and LC OLs preferentially reside in the medial and lateral cc (cc-m and cc-l), respectively.

Figure 2
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Figure supplement 2 .
Figure supplement 2. The distribution pattern of cortical dOLs, MP OLs, and LC OLs.

Figure supplement 2
Figure supplement 2-source data 1.The raw data for the visualization of data presented in Figure 2-figure supplement 2F, G.