A new driver mouse for efficient and specific OL labeling

(A) Scheme for generating the OpalinP2A-Flpo-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 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, 5μm in high magnification images. Quantification: n=3. Dots represent data from individual mice.

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 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. (C-F) 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. (K-N) 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: 1mm 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: S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

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 piriform cortex (Pir) and anterior commissure (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.

Simultaneous differential labeling of OLs derived from complementary embryonic origins

(A) Strategy for simultaneous 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 eGFP. (B) Coronal sections showing GFP+ OLs from dorsal origin (dOLs) and RFP+ OLs from ventral origin (vOLs) in OpalinFlp::Emx1Cre::RC::FLTG. (C) 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. (D) Coronal sections showing GFP+ OLs from dorsal and MGE/POA origin (dOLs+MPOLs) and RFP+ OLs from LGE/CGE origin (LCOLs) in OpalinFlp:: Emx1Cre:: Nkx2.1Cre::RC::FLTG. Scale bar: 1mm.

The distribution pattern of cortical dOLs, MPOLs and LCOLs

(A) Schematics of cortical regions chosen for quantifications and for showing representative images (boxed regions). Every fourth coronal section between Bregma +1.94 and –2.80 mm was analyzed. Position (P) 1 to 5 correspond to sections from which representative images were taken from. P2 corresponds to the sections shown in Figure 2G-I. (B-D) For each cortical region, two representative images at the rostral (r) and caudal(c) ends were presented for each combination. (E-F) Quantification of rostrocaudal distribution of neocortical (grey shaded region in E) dOLs, LCOLs and MPOLs. Slices were grouped into seven bins numbered rostrocaudally. Densities of dOLs and LCOLs showed no significant change across bins, while MPOLs exhibited lower density in more caudal regions. (G) Distributions of dOLs, LCOLs and MPOLs across 6 layers in SS. Similar to the total OL distribution quantified based on ASPA staining, more dOLs and LCOLs reside in deeper layers. In contrast, MPOLs are highly enriched in L4 at the cost of L6 with significant deviation from the total OLs. (H) Representative image of SS from 1 year old OpalinFlp::Nkx2.1Cre::Ai65 mouse. Scale bar: 200 μm. n=3 for dOLs and LCOLs; n=4 for MPOLs and ASPA. Dots represent data from individual mice. Error bar: S.E.M. *P < 0.05, ***P < 0.001.

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.