Embryonic origins of forebrain oligodendrocytes revisited by combinatorial genetic fate mapping

  1. Yuqi Cai
  2. Zhirong Zhao
  3. Mingyue Shi
  4. Mingfang Zheng
  5. Ling Gong
  6. Miao He  Is a corresponding author
  1. Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, China

eLife assessment

In this study the authors revisited the question of the embryonic origin of telencephalic oligodendrocytes using some new and powerful genetic tools. There is convincing evidence to support previous suggestions of a predominantly cortical origin of oligodendrocytes in the cerebral cortex, however the new studies suggest that LGE/CGE-derived oligodendrocytes make a modest contribution in some areas, while MGE/POA-derived oligodendrocytes make a small but enduring contribution. The findings are valuable and should be of interest to developmental and myelin biologists.

https://doi.org/10.7554/eLife.95406.3.sa0

Abstract

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 Nkx2.1Cre (Xu et al., 2008), Gsh2Cre (Kessaris et al., 2006), and Emx1Cre (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 (MPOLs) was 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 (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 Gsh2Cre.

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, OpalinP2A-Flpo-T2A-tTA2 (Figure 1), orthogonal to Cre drivers that label dorsal or ventral progenitors (ProgenitorCre). 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 OpalinP2A-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 OpalinFlp 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).

A new driver mouse for efficient and specific oligodendrocyte (OL) labeling.

(A) Scheme for generating the OpalinP2A-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.

Next, we established two types of genetic combinatorial fate-mapping strategies to directly visualize OLs from different embryonic origins (Figure 2): (1) combining OpalinFlp and ProgenitorCre with intersectional reporters Ai65 to label OLs derived from Cre+ progenitor domain by RFP (Figure 2A); (2) combining OpalinFlp and ProgenitorCre 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 MPOLs (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.

Figure 2 with 3 supplements see all
Combinatorial fate mapping of dOLs, MPOLs, and LCOLs.

(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 OpalinFlp::Emx1Cre::Ai65 (C), subtractional labeling of LCOLs in OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG (D), intersectional labeling of MPOLs in OpalinFlp::Nkx2.1Cre::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). MPOLs and LCOLs preferentially reside in the medial and lateral cc (cc-m and cc-l), respectively. Scale bar: 1 mm in low magnification images in (G–I), 250 μm in high magnification images of the boxed area in (G–I) and low magnification images in (K–M), 100 μm in high magnification images of the boxed area (cc-m and cc-l) in (K–M). n = 3 for dOLs and LCOLs; n = 4 for MPOLs. Dots represent data from individual mice. Error bar: standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001.

Deploying these strategies, we assessed the differential contributions of dOLs, LCOLs, and MPOLs by analyzing RFP+ cells in the following mice: OpalinFlp::Emx1Cre::Ai65 (Figure 2C), OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG (Figure 2D), and OpalinFlp::Nkx2.1Cre::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 LCOLs, the vast majority of neocortical OLs were dOLs but not LCOLs. 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 LCOLs. Considering the possibility of incomplete recombination in combinatorial reporters, and the relatively low Cre activity in the dorsal MGE of Nkx2.1Cre (Xu et al., 2008), 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 Gsh2Cre in previous study (Kessaris et al., 2006) or by GFP in OpalinFlp::Gsh2Cre::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 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 2I, J and Figure 2—figure supplement 2D–G). MPOLs display a clear rostrocaudal density decline (Figure 2—figure supplement 2E, F), a higher density in somatosensory cortex (SS) than motor cortex (Mo) (Figure 2I), and a laminar preference toward layer 4 (L4) in SS (Figure 2—figure supplement 2G). In contrast, the distribution of dOLs and LCOLs do not vary significantly across the rostrocaudal axis (Figure 2—figure supplement 2E, 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 MPOLs 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 LCOLs than dOLs. MPOLs make the lowest contribution (Figure 2J) at a density similar to SS and higher than Mo (Figure 2I).

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 high density of OLs (Figure 2K–M). We found that, similar to the neocortex, cc is mainly populated by dOLs and supplemented by very low proportions of LCOLs and MPOLs (Figure 2K–N). Interestingly, LCOLs and MPOLs seem to show preferential distribution in the lateral and medial regions of cc (cc-l and cc-m), respectively (Figure 2L–N). Different from cc, ac is mainly populated by LCOLs and MPOLs and supplemented by very low proportion of dOLs (Figure 2K–N).

To substantiate the above results, we further breed OpalinFlp::Emx1Cre::Nkx2.1Cre::Ai65 to label dOLs together with MPOLs 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 LCOL 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 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 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. LCOLs are the major contributor to both regions, while dOLs and MPOLs mainly contribute to Pir and ac, respectively (Figure 2).

The classical and revised model of forebrain oligodendrocyte (OL) origins.

(A) In the classical model (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 LCOLs (blue dots) and dOLs (purple dots) and are devoid of MPOLs (orange dots). (B) In the new model, NCx and cc mainly contain dOLs with very low contribution from the ventral origins. LCOLs mainly contribute to piriform cortex (Pir) and anterior commissure (ac). MPOLs makes a small but sustained contribution to NCx, with a strong laminar preference toward layer 4 in somatosensory cortex (SS). In addition, dOLs and MPOLs also make substantial contributions to Pir and ac, respectively. Gray dots indicate OLs in unanalyzed regions.

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 12). 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.

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)Nkx2.1CreThe Jackson LaboratoryStrain#: 008661; RRID: IMSR_JAX:008661
Genetic reagent (Mus musculus)Gsh2CreThe Jackson LaboratoryStrain#: 025806; RRID: IMSR_JAX:025806
Genetic reagent (Mus musculus)Emx1CreThe Jackson LaboratoryStrain#: 005628; RRID: IMSR_JAX:005628
Genetic reagent (Mus musculus)Ai65The Jackson LaboratoryStrain#: 021875; RRID: IMSR_JAX:021875
Genetic reagent (Mus musculus)RC::FLTGThe Jackson LaboratoryStrain#: 026932; RRID: IMSR_JAX:026932
Genetic reagent (Mus musculus)Ai62The Jackson LaboratoryStrain#: 022731; RRID: IMSR_JAX:022731
Genetic reagent (Mus musculus)HG-FRTThe Jackson LaboratoryStrain#: 028581; RRID: IMSR_JAX:028581
Genetic reagent (Mus musculus)OpalinP2A-Flpo-T2A-tTA2This paperSee Materials and methods, Mice
Antibodyanti-RFP (goat polyclonal)SICGENCat# AB0081-200; RRID: AB_2333095IF (1:2000)
Antibodyanti-RFP (rabbit polyclonal)RocklandCat# 600-401-379; RRID: AB_2209751IF (1:2000)
Antibodyanti-GFP (chicken polyclonal)Aves LabsCat# GFP-1020; RRID: AB_10000240IF (1:1000)
Antibodyanti-MBP (rat polyclonal)AbD SerotecCat# MCA409S; RRID: AB_325004IF (1:500)
Antibodyanti-CC1 (rabbit polyclonal)Oasis BiofarmCat# OB-PRB070; RRID: AB_2934254IF (1:500)
Antibodyanti-CC1 (mouse polyclonal)MilliporeCat# OP80; RRID: AB_2057371IF (1:300)
Antibodyanti-ASPA (rat polyclonal)Oasis BiofarmCat# OB-PRT005; RRID: AB_2938679IF (1:200)
Antibodyanti-Sox9 (rabbit polyclonal)ChemiconCat# AB5535; RRID: AB_2239761IF (1:2000)
Antibodyanti-NeuN (mouse monoclonal)MilliporeCat# MAB377; RRID: AB_2298772IF (1:500)
Sequence-based reagentOpalin-FThis paperPCR primersGGCCTATGTTTGATTTCCAGCACTG
Sequence-based reagentOpalin-RThis paperPCR primersAGCACTTATGACTGCTGAGCCGTTC
Chemical compound, drugTail lysis bufferViagenCat# 102-T
Chemical compound, drugProteinase KBeyotimeCat# ST535
Chemical compound, drugSodium pentobarbitalSigma-AldrichCat# P3761
Chemical compound, drugNormal Donkey SerumAbcamCat# ab7475
Chemical compound, drugTriton X-100Sigma-AldrichCat# X100PC
Chemical compound, drugCitrate bufferOasis-BiofarmCat# BR-AB001
OtherAqua-mountSouthern BiotechCat# 0100-01
OtherDAPI stainInvitrogenCat# D1306(10 mg/ml)
Software, algorithmImageJNational Institutes of HealthRRID: SCR_003070
Software, algorithmQuPathQueen’s University BelfastRRID: SCR_018257
Software, algorithmAdobe PhotoshopAdobe SystemsRRID: SCR_014199
Software, algorithmGraphPad Prism v8.0.1GraphPad SoftwareRRID: SCR_002798

Mice

All mouse studies were carried out in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of School of Basic Medical Sciences, Fudan University. All husbandry and experimental procedures were reviewed and approved by the same committee (Permit Number: 20210302-137). 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: Nkx2.1Cre (Jax 008661) (Xu et al., 2008), Gsh2Cre (Jax 025806) (Kessaris et al., 2006), Emx1Cre (Jax 005628) (Gorski et al., 2002), Ai65 (Jax 021875) (Madisen et al., 2015), and RC::FLTG (Jax 026932) (Plummer et al., 2015). 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 OpalinP2A-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

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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

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Mice were anesthetized 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 phosphate buffer. Following post fixation at 4°C for 24 hr, brain samples were sectioned at 30 μm using a vibratome (Leica VT1000S), or transferred into 30% sucrose in 0.1 M PB for cryoprotection, embedded in optimal cutting temperature (OCT) compound, 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 (pH 6.0). Sections were blocked in phosphate buffered saline (PBS) containing 0.05% Triton and 5% normal donkey serum and then incubated with the following primary antibodies in the blocking solution at 4°C 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), and NeuN (mouse monoclonal antibody, 1:500, Millipore, MAB377). Sections were then incubated with appropriate Alexa fluor dye-conjugated IgG secondary antibodies (1:500, Thermo Fisher 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 (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 PaxinosThe Mouse BrainAtlas 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

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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.

Data availability

All data generated or analyzed during this study are included in the manuscript. Source data have been provided for Figures 1 and 2.

References

Article and author information

Author details

  1. Yuqi Cai

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Zhirong Zhao and Mingyue Shi
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0009-0001-5831-4980
  2. Zhirong Zhao

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Validation, Investigation, Writing - review and editing
    Contributed equally with
    Yuqi Cai and Mingyue Shi
    Competing interests
    No competing interests declared
  3. Mingyue Shi

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Data curation, Validation, Investigation, Writing - review and editing
    Contributed equally with
    Yuqi Cai and Zhirong Zhao
    Competing interests
    No competing interests declared
  4. Mingfang Zheng

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
  5. Ling Gong

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Formal analysis, Funding acquisition, Visualization, Methodology, Writing - original draft, Project administration
    Competing interests
    No competing interests declared
  6. Miao He

    Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Department of Neurobiology, Zhongshan Hospital, Fudan University, Shanghai, China
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    hem@fudan.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0731-6801

Funding

National Science and Technology Innovation 2030 Major Projects of China (STI2030-Major Projects-2022ZD0206500)

  • Miao He

National Natural Science Foundation of China (32171087)

  • Miao He

National Natural Science Foundation of China (32371145)

  • Ling Gong

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

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, 32371145).

Ethics

All mouse studies were carried out in strict accordance with the guidelines of the Institutional Animal Care and Use Committee of School of Basic Medical Sciences, Fudan University. All husbandry and experimental procedures were reviewed and approved by the same committee (Permit Number: 20210302-137). All applicable institutional and/or national guidelines for the care and use of animals were followed.

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  1. Yuqi Cai
  2. Zhirong Zhao
  3. Mingyue Shi
  4. Mingfang Zheng
  5. Ling Gong
  6. Miao He
(2024)
Embryonic origins of forebrain oligodendrocytes revisited by combinatorial genetic fate mapping
eLife 13:RP95406.
https://doi.org/10.7554/eLife.95406.3

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    Maiko Kawasaki, Katsushige Kawasaki ... Atsushi Ohazama
    Research Article

    Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma, and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested tongue swelling formation. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations to disturb cellular processes which were disrupted in mutant mice. Our results provide hints for possible future treatment in ciliopathies.

    1. Developmental Biology
    2. Genetics and Genomics
    Morgane Djebar, Isabelle Anselme ... Christine Vesque
    Research Article

    Cilia defects lead to scoliosis in zebrafish, but the underlying pathogenic mechanisms are poorly understood and may diverge depending on the mutated gene. Here, we dissected the mechanisms of scoliosis onset in a zebrafish mutant for the rpgrip1l gene encoding a ciliary transition zone protein. rpgrip1l mutant fish developed scoliosis with near-total penetrance but asynchronous onset in juveniles. Taking advantage of this asynchrony, we found that curvature onset was preceded by ventricle dilations and was concomitant to the perturbation of Reissner fiber polymerization and to the loss of multiciliated tufts around the subcommissural organ. Rescue experiments showed that Rpgrip1l was exclusively required in foxj1a-expressing cells to prevent axis curvature. Genetic interactions investigations ruled out Urp1/2 levels as a main driver of scoliosis in rpgrip1 mutants. Transcriptomic and proteomic studies identified neuroinflammation associated with increased Annexin levels as a potential mechanism of scoliosis development in rpgrip1l juveniles. Investigating the cell types associated with annexin2 over-expression, we uncovered astrogliosis, arising in glial cells surrounding the diencephalic and rhombencephalic ventricles just before scoliosis onset and increasing with time in severity. Anti-inflammatory drug treatment reduced scoliosis penetrance and severity and this correlated with reduced astrogliosis and macrophage/microglia enrichment around the diencephalic ventricle. Mutation of the cep290 gene encoding another transition zone protein also associated astrogliosis with scoliosis. Thus, we propose astrogliosis induced by perturbed ventricular homeostasis and associated with immune cell activation as a novel pathogenic mechanism of zebrafish scoliosis caused by cilia dysfunction.