mCherry expression in 4- and 6-dpf Tg(mbpa:mCherry-T2A-CreER^T2) larvae. A: Scheme of the Tg(mbpa:mCherry-T2A-CreERT^T2) construct, which expresses a single open reading frame coding for mCherry (red) and CreER^T2 (dark gray) separated by a viral T2A peptide sequence (white) under the control of the Zebrafish mbpa promoter (light gray). B–I: Native mCherry fluorescence in 4-dpf (B–E) and 6-dpf (F–I) transgenic Zebrafish larvae. B,F: Lateral view of the larvae, showing mCherry expression in presumptive myelinating cells. Scale bar, 200 µm. C,D,G,H: Higher magnification of the areas indicated by white boxes in B,F. C,G: mCherry expressing oligodendrocytes in the hindbrain and (D,H) the spinal cord. E,I: Dorsal view of the hindbrain with mCherry fluorescence in presumptive oligodendrocyte cell bodies (arrowhead) and processes (arrow). Asterisks mark auto fluorescence from yolk and pigmentation. Scale bar, 100 µm.

The mbpa promoter recapitulates expression in mature oligodendrocytes in the larval and adult Zebrafish CNS. A: 6-dpf larvae from the Tg(mbpa:mCherry-T2A-CreER^T2) line stained with antibodies against mCherry and MBP show coexistence of mCherry + cells (red) in MBP + regions (green), suggesting mature oligodendrocytes. Scale bar, 100 µm. B: Higher magnification of the area depicted (white rectangle) in A. Yellow arrowheads mark mCherry-positive oligodendrocytes located in MBP-expressing regions. Scale bar, 50 µm. C: Double-transgenic Tg(mbpa:mCherry-T2A-CreER^T2); Tg(mbpa:EGFP) at 7 dpf show co-localization of EGFP and mCherry expression in oligodendrocytes in the spinal cord and hindbrain. D: Higher magnification of the area encircled in C showing EGFP/mCherry-positive oligodendrocytes. E: Scheme of cross-sections of the diencephalon with optic nerve/tract in adult Zebrafish brain as shown in F and G. F: Expression of mCherry in oligodendrocytes of the optic nerve/tract shown by IHC against mCherry (red) and MBP (green) in Tg(mbpa:mCherry-T2A-CreER^T2) adult fish. Scale bar, 100 µm. G: Higher magnification of the area depicted (white rectangle) in F showing mCherry-expressing cells (yellow arrowheads) located within the MBP-expressing optic tract. Yellow arrowheads point to oligodendrocytes. Scale bar, 50 µm. H: Cross-section of the retina with the exit point of the optic nerve immunostained for mCherry (red) and MBP (green), showing mCherry-expressing cells within the MBP-expressing processes in the optic nerve as shown in individual panels. Asterisks mark unspecific auto fluorescence of the retinal pigment epithelium, photoreceptors, and extra-retinal tissue. Scale bar, 75 µm. I: Higher magnification of the area depicted (white rectangle) in H showing presumptive oligodendrocytes expressing mCherry (yellow arrowheads) and located within the MBP staining along axons of the optic nerve. DAPI stains all nuclei. Scale bar, 25 µm.

Conditional CreER^T2-mediated recombination in Tg(mbpa:mCherry-T2A-CreER^T2) in 5-dpf larvae. A: Schematic representation of treatments applied to achieve conditional CreER^T2-mediated recombination with the temperature-inducible, Cre-dependent reporter line Tg(hsp70l:loxP-DsRed-loxP-EGFP). B: Scheme of the ligand-dependent recombination event in double-transgenic Tg(mbpa:mCherry-T2A-CreER^T2), Tg(hsp70:loxP-DsRed-stop-loxP-EGFP) larvae. Application of TAM results in Tg(hsp70: loxP-EGFP) in mbpa-expressing cells. C: Lateral view of a 7-dpf larva after 4-OHT treatment and heat shock. Recombination is indicated by a switch from red to green fluorescence in cells in the hindbrain and spinal cord. Scale bar, 200 µm. D,E: Higher magnification of the areas indicated by white boxes in C showing EGFP-expressing oligodendrocytes. F: Dorsal view of the hindbrain with EGFP fluorescence in presumptive oligodendrocyte cell bodies (arrowhead) and processes (arrow). Scale bar, 100 µm. G,H: Recombination can also be observed in Schwann cells myelinating the anterior and posterior lateral lines (arrowheads) Scale bar, 200 µm. I: Recombination only ever occurred in larvae treated with 4-OHT and HS, but never in control siblings treated with ethanol and HS. Scale bar, 200 µm. J: Asterisks mark auto fluorescence from yolk and pigmentation. Scale bar, 200 µm.

Conditional CreER^T2-mediated recombination in Tg(mbpa:mCherry-T2A-CreER^T2) in the adult Zebrafish CNS. A: Schematic representation of treatments applied to achieve conditional CreER^T2-mediated recombination with the temperature-inducible, Cre-dependent reporter line Tg(hsp70l:loxP-DsRed-loxP-EGFP). B: Cross-section of the diencephalon with optic tracts and the rostral optic tectum immunostained for MBP (red) and EGFP (green). Scale bar, 100 µm. C: Higher magnification of the area depicted (white rectangle) in B showing presumptive oligodendrocytes (marked by arrowheads) after recombination expressing EGFP and co-stained with MBP (red). Scale bar, 50 µm. D: Cross-section of the retina with the exit point of the optic nerve immunostained for EGFP (green) and MBP (red), showing recombined cells (EGFP) within the MBP-expressing optic nerve. Scale bar, 50 µm. E: Higher magnification of the area depicted (white rectangle) in C showing EGFP-expressing cells (yellow arrowheads) co-expressing MBP. Scale bar, 25 µm. F: Cross-section of the dorsal telencephalon showing presumptive mature oligodendrocytes after recombination expressing EGFP (yellow arrowheads), co-immunostained with MBP (red) and located along the MBP-positive lateral olfactory tract (yellow dotted line) and the dorsal part of the entopeduncular nucleus. DAPI stains nuclei. Scale bar, 25 µm.