Illustration of neurogenic niches. (A–E) Whole mount (WM) overview of Edu+ cells in 7-week-old Nothobranchius furzeri visualized 4 h after intraperitoneal injection. Dorsal view of the entire brain (A) and telencephalic region (B). (C–E) Ventral view of the entire brain, low magnification (C), and two different orientations at higher magnification (D, E). Neurogenic niches are identified by Latin numerals: I = telencephalic proliferative niche corresponding to the subpallial region visible in panel A – II, III = telencephalic proliferative niches corresponding to the pallial regions visible in panel B, D, E – IV = preoptic proliferative niche visible in panel E were the optic nerves were removed – V = rostro-dorsal part of the proliferative niche in the optic tectum (OT), visible in panel A, B (partially) – VI = caudal part of the proliferative niche in the OT, visible in panel A, C – VII = medial proliferative niche of the cerebellum, visible in panel A – VIII = caudal proliferative niche of the cerebellum, visible in panel A – IX = caudal proliferative niche along the roof of the IV ventricle, visible in panel A. *The region of the telencephalic surface devoid of proliferating cells, possibly of pial origin. Arrowheads in panels B and D indicate areas of higher concentration of proliferating cells in the caudal margin of niche III. F-N: horizontal and coronal brain sections illustrating PCNA+ (red) and Edu+ (green) cells in 6-week-old N. furzeri visualized 4 h after intraperitoneal injection. The approximate level corresponding to coronal sections represented in panels L, M, N is shown as dotted lines on a dorsal view of the brain in the inset of panel F. (F) Emi-section of brain in horizontal view: all telencephalic proliferative niches described in (A–E) are visible (I, II, III), as well as the preoptic niche (IV) and the niche of the caudal margin of the OT (VI). (G–I) Magnification of areas corresponding to the boxed regions in panel F. (L) Coronal section of the brain at median telencephalic level: the three proliferative niches are visible (I, II, III) and magnified in panels L^I, L^II, L^III. (M) Coronal section of the brain at rostral level of the OT: the dorso-medial tectal niche bordering with the torus longitudinalis (tl) is visible (V) and three different areas of the proliferative niches distributed along the ventricular wall are magnified in panels M^I, M^II, M^III. (N) Coronal section of the brain at median level of the OT: the medial tectal niche bordering with the cerebellum (crb) is visible (VII), as well as the niche of the tectal ventro-caudal margin (VI); regions VII is magnified in panels N′; in N″, a partial magnification of region VI shows few double-labeled cells scattered within the subventricular gray zone of the OT (arrowheads). DAPI nuclear counterstaining is visualized in blue.

Comparison of double-staining for S-100 (green) and glial fibrillary acid protein (GFAP) (red) in the central area of the optic tectum (OT) of young (5-week-old) vs. old (25-week-old) Nothobranchius furzeri brain; confocal images (projection of seven optical sections for 6 μm thickness). (A, B, C) Representation of single GFAP (A), S-100B (B), and merged staining GFAP+S-100B (C) in the tectal central region of a 5-week-old fish. (D, E, F) Representation of single GFAP (D), S-100B (E), and merged staining GFAP+S-100B (F) in the tectal central region of a 25-week-old fish; S-100B shows intense staining in both young and old subjects, whereas GFAP staining is weak in the young tissue but undergoes a strong age-dependent up-regulation. Scale bar reported in panel A is representative for all the other panels (B–F). (G) Quantification of labeling intensity of GFAP. Intensity is expressed as pixels over threshold and is normalized on the number of pixels positive for S100B. Error bars are standard errors of means. **P < 0.01, Student’s t-test. Four animals for each age were used. (H) Quantification of mRNAs per real time. Fold changes are relative to 5-week-old controls. Values are normalized with respect to TBP. Error bars are standard errors of means. *P < 0.05, permutation-based test, REST software. Number of animal: 5-week-old N = 8, 27-week-old N = 8, 32-week-old N = 6. (I–M) Double-labeling for 5-ethynil-2′deoxyuridin (EdU) (green) and S100B (red). Single confocal planes, double-labeled cells are indicated by arrowheads and cells labeled by EdU only by arrows. (I) Subpallial neurogenic niche, note lack of co-localization. (L) Pallial neurogenic niche. (M) Germinal layer of the OT. (N–Q) Double-labeling for EdU (green) and Msi-1 (red). Single confocal planes. Note complete co-localization in all areas. (N) subpallial, (O) pallial, (P) germinal layer of the OT, (Q) radial glia of the OT. See Fig. S6 (Supporting Information) for single channels.

Coronal sections of 11-week-old Nothobranchius furzeri stained for PCNA (red) and 5-ethynil-2′deoxyuridin (EdU) (green), 5 weeks after intraperitoneal EdU injection. Approximate position of the sections in panels A, B, H, I along the rostro-caudal axis of the brain is shown on top view of a whole-mount brain in the inset of panel A. (A) Coronal section of the telencephalon in median position: after 5 weeks, EdU+ cells migrate centrifugally from the ventricular and dorso-lateral margins into the parenchyma, outside of the PCNA+ proliferative regions. (B) Coronal section of a rostral portion of the optic tectum (OT): in the antero-dorsal margin (magnification in panel F) EdU+ cells form a strip into the tectal parenchyma well distinct from the PCNA+ cells area. (C–E) Magnifications of the dorsal, lateral, and ventricular margin of the telencephalic section, respectively, represented in pictures A. (F, G) Magnifications of the antero-dorsal tectal margin and the III ventricle, respectively: in both cases, EdU+ cells migrate after 5 weeks into the parenchyma. (H, I) Coronal sections of the central and caudal portions of the OT: the antero-dorsal and postero-ventral strips of EdU+ cells into the parenchyma can be appreciated as well (red arrows); centripetally migrating EdU+ cells can be observed also in the torus longitudinalis (picture B, magnified panel F). A very large number of EdU+ cells can be found in the granule cell layer of the valvula cerebelli (picture H, magnified panel M) and corpus cerebelli (picture I, magnified panel N). Scale bar for magnification panels C, D, E, F, G, L, and M are the same as indicated in panel N.

In situ hybridization for miR-124 and miR-9 (A–H) in the telencephalic and tectal structures of 7-week-old Nothobranchius furzeri (young adult), and double-staining for doublecortin (DCX)/PCNA andDCX/5-ethynil-2′deoxyuridin (EdU) (I-T). (A) Overview of the right telencephalon in horizontal section where EdU+ cells (green) and miR-124+ cells (red) are visualized. (B) Magnification of the pallial neurogenic niche boxed in A is shown in panel B; the exclusion of miR-124 labeling from the EdU+ cells area is apparent. (C) Overview of the right telencephalon in horizontal section where EdU+ cells (green) and miR9+ cells (red) are visualized. (D) Magnification of the telencephalic subpallial neurogenic area boxed in C. (E) Confocal image of a single optical plane, horizontal section of the pallial neurogenic area. Magnification of the region boxed in E is shown in panel F. (G, H) In situ hybridization labeling in the optic tectum for miR-9 (G) and miR-124 (H). Note the exclusion of miR-124 and miR-9 as well from the germinal layer (arrow) and the complementary labeling pattern of miR-124 and miR-9 in the radial glia (arrowheads). (N–T) Horizontal sections of 7-week-old N. furzeri (young adult) double-labeled for DCX and PCNA (I–Q) or DCX and EdU, 1-2 weeks after intraperitoneal injection (R–T). (I) Horizontal overview of the right telencephalon stained for PCNA (red), DCX (green). (M, N) Magnification of the telencephalic pallial and subpallial neurogenic regions, respectively. Note that in the pallial neurogenic region (L), a strip of DCX+ cells is present immediately adjacent to the PCNA+ cells marginal stratum. In contrast, in the subpallial niche (M) only DCX+ fibers can be detected. (N, O) Representation at low (N) and high (M) magnification of the ventro-posterio tectal margin: a strip of DCX+ neuronal processes (green) is present a few cell diameter medial to the PCNA+ cells (red) in the germinal layer. (P, Q) Representation at low (P) and high (Q) magnification of the retinal ciliary marginal zone: a concentration of DCX+ neuronal processes (red) is present medial from the PCNA+ cells (green) proliferative margin. (R) Representation of the ventro-posterio tectal margin stained for DCX (red) and EdU (green, 1 week postinjection): the strip of EdU+ cells is aligned with the strip of DCX+ processes. (S, T) Representation at low (S) and high (T) magnification of the latero-posterior telencephalic margin stained for DCX (red) and EdU (green, 2 weeks postinjection): the majority of EdU+ cells after 2 weeks correspond with the strip of DCX+ cells. The nuclear DAPI is visualized as blue staining in panel B, D, G, H. (T) Scale bars are directly indicated in the panels: bar in the same for pictures L and M. DCX, doublecortin.

Horizontal sections double-labeled for PCNA+ (red) and Edu+ (green) in the ventro-posterior margin (niche VI) of the optic tectum (OT): 5- vs. 25-week-old Nothobranchius furzeri, visualized 4 h after intraperitoneal 5-ethynil-2′deoxyuridin (EdU) injection. (A, B) Hemisection of young (5-week-old, (A) vs. aged (25-week-old, (B) brain in horizontal view; regions undergoing evident age-dependent reduction in PCNA+ and EdU+ labeling are better appreciated in the correspondent magnification in the lower panel strip. (A′) the OT posterior margin niche of a young animal (5 weeks old) is represented, showing an intense double-labeled proliferative cells in the germinal layer. By comparison, in the old subject represented in B′), few double-labeled PCNA+/EdU+ cells can be detected in the PCNA+ germinal layer. (C) Histogram reporting unbiased estimation of EdU+ cells into the tectal germinal layer at three different age-steps. Estimates for left and right tecta are reported separately. Five animals per time point were analyzed, and error bars represent standard deviations. (D) The panel on the right represents the growth curve of N. furzeri males. The analyzed age-steps for the quantification are marked by red arrows. Forty-five animals were measured to produce this curve. Error bars represent standard errors of means. Scale bar for magnification panels (A′, A″, B″) is the same, as indicated in panel B″. (E–N) Comparison of doublecortin (DCX) expression (green) in telencephalic and tectal areas of young (5-week-old) vs. old (25-week-old) N. furzeri brain; confocal images (projection of seven optical sections for 6 μm thickness) A. (D) Representation of the telencephalic subpallial (E) and pallial (F) neurogenic niches, and the germinal layer (G) and central region (H) of the OT of a 5-week-old brain: DCX+ processes are present in all telencephalic and tectal margins, as well as in the central tectum. Representation of the subpallial (I) and pallial (L) neurogenic niches of the telencephalon, and the germinal layer (M) and central region (N) of the OT of a 25-week-old brain: DCX+ processes are clearly reduced in the neurogenic areas (I–M) when compared to the corresponding regions (E–G) in the 5-week-old brain. On the other hand, DCX staining in the central tectum shows similar pattern and intensity at both ages and can be regarded as a control for labeling quality.