Figures and data
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A new driver mouse for efficient and specific OL labeling
(A) Scheme for generating the OpalinP2A-Flp-T2A-tTA2allele. (B) Southern blot confirmation of correctly targeted ES clone. (C) Genomic 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 RFP with MBP staining, which further demonstrated the myelination ability of labeled OLs. (F) Quantification of marker staining. Left panel quantified the labeling specificity. Both systems are highly specific, as shown by the complete co-localization with OL marker (CC1) and lack of co-staining by the neuronal marker (NeuN) or astrocyte marker (Sox9). Right panel quantified the labeling efficiency. Close to complete OL labeling was achieved by Flp-dependent H2B-GFP reporter in all analyzed regions, while sparser labeling with variable regional density was achieved by tTA2-dependent tdTomato reporter driven by TRE promoter. Scale bar: 50 μm in low magnification images, 5μm in high magnification images. Quantification: n=3. Dots represent data from individual mice.
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Combinatorial fate mapping of dOLs, MPOLs and LCOls
(A) Strategy for intersectional labeling. Flp-AND-Cre labels OLs from Cre-expressing progenitors with RFP. (B) Strategy for simultaneous intersectional and subtractional labeling of OLs derived from complementary origins. Flp-NOT-Cre labels OLs from non-Cre-expressing progenitors with RFP, while Flp-AND-Cre labels OLs from Cre-expressing progenitors with GFP. (C-E) Intersectional labeling of dOLs in OpalinFlp::Emx1Cre::Ai65, subtractional labeling of LCOLs in OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG, and intersectional labeling of MPOLs in OpalinFlp::Nkx2.1Cre::Ai65 and their cortical distribution. Schematics, representative images and quantification of RFP+ cell density in motor cortex (Mo), somatosensory cortex (SS) and piriform cortex (Pir) were displayed. (F) Differential contribution of OLs from the three embryonic origins to Mo, SS and Pir.
Scale bar: 1mm in low magnification images, 500 μm in high magnification images of the boxed area. Quantification: n=3 for dOLs and LCOLs; n=4 for MPOLs. Dots represent data from individual mice. **P < 0.01, ***P < 0.001. Error bar: S.E.M.
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dOLs, MPOLs and LCOLs differentially contribute to the two major white matter tracts
(A-C) Schematics and representative images of dOLs, MPOLs and LCOLs 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. (D) Quantification of dOL, MPOL and LCOL densities in cc-m, cc-l and ac. Scale bar: 1mm in low magnification images, 500μm in cc and ac, 100 μm in high magnification images of the boxed area (cc-m and cc-l) in cc. Quantification: n=3 for dOLs and LCOLs; n=4 for MPOLs. Dots represent data from individual mice. **P < 0.01, ***P < 0.001. Error bar: S.E.M.
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The classical and revised model of forebrain OL origins
(A) In the classical model[2], OLs derived from MGE/POA (orange) were largely eliminated postnatally (thin dashed line), while those from 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 contains dOLs with very low contribution from the ventral origins. LCOLs mainly contribute to Pir and ac. MPOLs makes a small but sustained contribution to NCx, with a strong laminar preference towards layer 4 in somatosensory cortex (SS). In addition, dOLs and MPOLs also make substantial contributions to Pir and ac, respectively. Grey dots indicate OLs in unanalyzed regions.
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Simultaneous differential labeling of OLs derived from complementary embryonic origins
(A) Coronal sections showing GFP+ OLs from dorsal origin (dOLs) and RFP+ OLs from ventral origin (vOLs) in OpalinFlp::Emx1Cre::RC::FLTG. (B) Coronal sections showing GFP+ OLs derived from Gsh2+ progenitors (Gsh2+OL) and RFP+ OLs derived from Gsh2-progenitors (Gsh2-OL) in OpalinFlp::Gsh2Cre::RC::FLTG. Scale bar: 1mm.
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The distribution pattern of neocortical dOLs and MPOLs
(A-B) Rostrocaudal distribution of neocortical dOLs and MPOLs. (A) Within the range of analysis, RFP+ OLs in neocortex of every fourth section were quantified. (B) Slices were grouped into seven bins numbered rostrocaudally. Density of dOLs showed no significant change across bins, while MPOLs exhibited lower density in more caudal regions. (C-E) Laminar distribution of dOLs and MPOLs in somatosensory cortex (SS). (C) Representative images of SS from P56 mice. (D) Distribution of dOLs and MPOLs across 6 layers. More dOLs reside in deeper layers, while MPOLs are highly enriched in L4. (E) dOLs and MPOLs exhibited significant differences in their relative laminar distributions. (F) Representative image of SS from 1 year old OpalinFlp::Nkx2.1Cre::Ai65 mouse. Scale bar: 200 μm. Dots represent data from individual mice. Error bar: S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.
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Intersectional labeling of OLs derived from both dorsal origin and MGE/POA
(A) Coronal sections showing both dOLs and MPOLs labeled by RFP in OpalinFlp::Emx1Cre::Nkx2.1Cre::Ai65. (B) Higher magnification images showing ASPA+ RFP+ dOLs/MPOLs (closed arrow heads) and ASPA+RFP-putative LCOLs (open arrow heads).The latter cells were difficult to find in neocortical regions such as motor cortex (Mo) and somatosensory cortex (SS), and corpus callosum (cc), but were frequently encountered in piriform cortex (Pir) and anterior commissure (ac). Scale bar: 1mm in low magnification images, 200μm in Mo, SS, cc and ac, 10 μm in high magnification images of boxed area (all shared the same scale).