<i>notch3</i> expression in the adult cerebellar niche.

<p>Cross-sections at the indicated level through the mesencephalon; cerebellar area shown in the micrographs is indicated in the cross-section schematic. <b>A</b>, Brightfield image shows <i>notch3</i> expressing cells in the cerebellum (black arrowheads). <b>B–D</b>, Confocal images showing localization of the glial marker S100 (red) and PCNA (green), with <i>notch3</i> by FISH (white). <b>B–C</b>, <i>notch3</i> is weakly expressed in a small subset of PCNA cells in the stem cell niche (unfilled arrowhead). <b>B–D</b>, Strong <i>notch3</i> expression is detected in S100 cells indicated by the white arrows; <i>notch3</i> expression is also detected in some scattered cells of the ML, IML and GL that do not localize with the analysed markers (unfilled arrows); <b>E</b>, Summary of the expression pattern and cellular characteristics of Notch receptor expressing cells in the adult cerebellum. Abbreviations: GL, granule cell layer; IML, intermediate layer; ML, molecular layer. Scale bars  = 50 in A and B; 20 in C and D.</p

<i>notch1a</i> and <i>notch1b</i> expression in the adult cerebellar niche.

<p>Cross-sections at the indicated level through the mesencephalon; cerebellar area shown in the micrographs is indicated in the cross-section schematic in A. Brightfield images show expression of <b>A</b>, <i>notch1a</i> and <b>D</b>, <i>notch1b</i> in the cerebellum (arrowheads). Confocal images showing localization of the glial marker S100 (red) and PCNA (green), with <b>B–C</b>, <i>notch1a</i> and <b>E–F</b>, <i>notch1b</i> by FISH (white). <b>B–C</b>, Expression of <i>notch1a</i> localizes with a large fraction of PCNA cells in the niche (unfilled arrowheads); weak (white arrow) or no expression of <i>notch1a</i> is detected in the Bergmann glia (S100). <b>E–F</b>, <i>notch1b</i> is expressed in a subpopulation of PCNA cells (unfilled arrowheads) and in a few S100 cells (white arrows). A few scattered <i>notch1a </i> and <i>notch1b </i> cells in the ML, IML and GL do not localize with the analysed markers (unfilled arrows). Abbreviations: GL, granule cell layer; IML, intermediate layer; ML, molecular layer. Scale bars  = 50 in A, B, D and E; 20 in C and F.</p

Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain

<div><p>The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 of proliferating radial glia express <i>notch1a</i>, <i>notch1b</i> and <i>notch3</i>. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express <i>notch3</i> and about half express <i>notch1a/1b</i>. In the non-proliferating radial glia <i>notch3</i> is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, <i>notch1a/1b/3</i> are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA cells. In this region <i>notch3</i> expression is mostly in Bergmann glia and at low levels in few PCNA cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches.</p></div

Overlapping and complementary <i>notch1a/3</i> expression in the adult zebrafish optic tectum.

<p>Confocal images showing localization of Notch receptor pairs by double FISH (white and red) and PCNA proliferating cells (green). Cross-sections at the indicated level through the mesencephalon; tectal area shown in the micrographs is indicated in the cross section schematics. <b>A</b>, <i>notch1a/3</i> and <b>B</b>, <i>notch1a/1b</i> expression domains in the dPGZ and mPGZ layers. Co-expression of these receptors both in PCNA (filled white arrowheads) and PCNA cells (unfilled white arrowheads); <i>notch3 </i> -only cells in A and <i>notch1b </i> -only cells in B are indicated by unfilled yellow arrowheads; white arrows indicate <i>notch1a </i> /<i>notch3 </i> /PCNA cells in A; a few PCNA cells are Notch receptor (filled yellow arrows); unfilled white arrows indicate cells positive for <i>notch1a</i> alone. <b>C</b>, Summary of Notch receptor expression pattern and cellular characteristics in the TeO. Abbreviations: Cce, corpus cerebelli; PGZ, periventricular gray zone of the optic tectum; dPGZ, deep layer of the PGZ; mPGZ, mitotic region of the PGZ; PML, posterior mesencephalic lamina; TeO, optic tectum. Scale bars  = 50 .</p

<i>notch1a</i>, <i>notch1b</i> and <i>notch3</i> expression in radial glia and proliferating cells of the dorso-medial ventricular zone of the telencephalon.

<p>Confocal images showing localization of Notch receptors by FISH (white), radial glia labelled with S100 (red), and PCNA proliferating cells (green); DAPI (blue) is used as nuclear counterstaining. Schematics in A indicate the cross-section levels through the telencephalon and the dorsal telencephalic area represented in the micrographs. <b>A–C</b>, <i>notch1a</i> and <b>D–E</b>, <i>notch1b</i> expressing cells are scattered throughout the dorso-medial ventricular zone of the dT and co-localize with both PCNA /S100 cells (filled arrowheads) and a subpopulation of PCNA /S100 cells (white arrows); yellow arrows indicate Notch receptor /PCNA /S100 cells; unfilled yellow arrowheads indicate <i>notch1b </i> /PCNA cells. <b>F–H</b>, <i>notch3</i> expressing cells localize to a great extent with the S100 marker, including both PCNA (white arrows) and PCNA (white arrowheads) cells; unfilled yellow arrowheads indicate <i>notch3 </i> /PCNA cells. <b>I</b>, Schematics indicating the cross-sections levels through the telencephalon, along the rostro-caudal axis, and examples of Dm areas used for marker co-localization analysis in J. <b>J</b>, Quantification on the co-localization of Notch receptor by FISH with PCNA and S100, for the Dm region; all ventricularly located cells of the indicated area were counted; cells were distinguished based on their glia character (S100), Notch receptor expression and proliferative status (PCNA ). n = 9 (fish), 4–6 tissue sections per fish, at the rostro-caudal levels indicated in I; total number of cells counted = 7245. Values represented as mean percentage SEM. Significance levels: , . Abbreviations: Dm, dorso-medial telencephalic area; dT, dorsal telencephalic area; vT, ventral telencephalic area; Scale bars  = 50 in A, in D and in F; 10 in C (applies to B), in E and in H (applies to G).</p

Notch receptor expression in radial glia and proliferating cells of the adult zebrafish optic tectum.

<p>Cross-sections at the indicated level through the mesencephalon; optic tectum area shown in the micrographs is indicated in the cross section schematic in A. Confocal images showing localization of the glial marker S100 (red) and the proliferation marker PCNA (green), with <b>A–D</b>, <i>notch1a</i>, <b>E–I</b>, <i>notch1b</i> and <b>J–N</b>,<i>notch3</i> by FISH (white). <b>A</b>, <i>notch1a</i>, <b>E</b>, <i>notch1b</i> and <b>J</b>, <i>notch3</i> are present in PCNA cells of the mPGZ and PML (unfilled white arrowheads) and in S100 glial cells of the dPGZ; cells in the superficial layer of the PGZ expressing <i>notch1a</i> and <i>notch1b</i> are also found (unfilled arrows), with relative more abundance for the <i>notch1b</i> receptor in this layer; <b>B, C</b>, <b>F,G</b> and <b>K,L</b> are higher magnifications of the respective framed areas in A, E and J, showing the overlap of <i>notch</i> expression with S100 (filled arrows) or PCNA (unfilled white arrowheads); unfilled yellow arrowheads in C, G and J indicate a few Notch receptor /PCNA cells; <b>D</b>, <b>H</b>, <b>I</b>, <b>M</b>,<b>N</b>, close ups of Notch receptor expressing cells in the above mentioned areas and orthogonal views of the indicated cells. Abbreviations: PGZ, periventricular gray zone of the optic tectum; dPGZ, deep layer of the PGZ; mPGZ, mitotic region of the PGZ; PML, posterior mesencephalic lamina. Scale bars  = 100 in A, E and J; 20 in B (applies to C), in F, G and K (applies to L); 5 in D, I (applies to H) and N (applies to M).</p

Overlapping and complementary Notch receptor expression in the dorsal telencephalon.

<p>Confocal images showing localization of Notch receptors pairs by double FISH (white and red) and PCNA proliferating cells (green); DAPI (blue) is used as nuclear counterstaining. Cross-sections at the indicated level through the telencephalon. Corresponding dorsal telencephalic areas represented in the micrographs are indicated in the cross section schematics of each panel. <b>A, B</b>, <i>notch1a/3</i> co-expression in Dm and Dl, respectively. <b>C</b>, <i>notch1a/b</i> co-expression in Dm. These receptors are co-expressed both in PCNA (filled white arrowheads) and PCNA (unfilled white arrowheads) cells; within the PCNA population, few cells are <i>notch1a </i> /<i>notch3 </i> (filled yellow arrowheads) while others are negative for both receptors (filled yellow arrows); within the PCNA population, some cells express either <i>notch3</i> or <i>notch1b</i> but not <i>notch1a</i> alone (unfilled yellow arrowheads). Scale bars  = 50 .</p

Summary of Notch receptor expression pattern and cellular characteristics in the adult zebrafish hypothalamus.

<p>Illustration shows the overall expression of the analysed Notch receptors at different rostro-caudal levels of the hypothalamus; they are mainly present in ventricular zone cells of Hv, Hd and Hc and localize with glial and proliferation markers. Abbreviations: DiV, diencephalic ventricle; Hc, periventricular caudal hypothalamus; Hd, periventricular dorsal hypothalamus; Hv, periventricular ventral hypothalamus; LR, lateral recess of the DiV; PR, posterior recess of the DiV; PTN, posterior tuberal nucleus.</p

<i>notch1a</i>, <i>notch1b</i> and <i>notch3</i> expression in radial glia and proliferating cells of the dorso-lateral telencephalic ventricular zone.

<p>Confocal images showing localization of Notch receptors by FISH (white), radial glia labelled with S100 (red), and PCNA proliferating cells (green); DAPI (blue) is used as nuclear counterstaining. Schematics in A indicate the cross-section levels through the telencephalon and the dorsal telencephalic area represented in the micrographs. <b>A–B</b>, <i>notch1a</i> and <b>C–D</b>, <i>notch1b</i> expressing cells are scattered throughout the dorso-lateral ventricular zone of the dT and localize with both PCNA /S100 cells (arrowheads) and a subpopulation of PCNA /S100 cells (white arrows); yellow arrows indicate Notch receptor /PCNA /S100 cells; unfilled yellow arrowheads indicate Notch receptor /PCNA /S100 cells. <b>E–F</b>, <i>notch3</i> expressing cells localize to a great extent with S100, including both PCNA (white arrows) and PCNA (white arrowheads) cells; unfilled yellow arrowheads indicate <i>notch3 </i> /PCNA cells. <b>G</b>, Quantification on the co-localization of Notch receptor by FISH with PCNA and S100, for the Dl region; schematics on the left indicate the cross-section levels through the telencephalon, along the rostro-caudal axis, and examples of Dl areas used for marker co-localization analysis; all ventricularly located cells of the indicated area were counted; cells were distinguished based on their glia character (S100), notch expression and proliferative status (PCNA ). n = 9 (fish), 4–6 tissue sections per fish, at different rostro-caudal levels; total number of cells counted = 6433. Values represented as mean percentage SEM. Significance levels: , , . Abbreviations: Dl, dorso-lateral telencephalic area; dT, dorsal telencephalic area; vT, ventral telencephalic area; Scale bars  = 50 in C (applies to A) and in E; 10 in B, D and F.</p

Comparison of Notch receptor expressing cells in the five analysed adult zebrafish neurogenic niches.

<p>Notch receptor expression is consistent in a subpopulation of proliferating progenitors or NSCs throughout the adult zebrafish brain. The existing heterogeneity and similarities observed in the combinatorial Notch receptor expression between neurogenic niches might be a reflection of their inherent cellular properties. This is depicted by the presented regional grouping, as indicated on the right side with some regions having similar characteristics. Approximate proportion of the cell populations found is represented by gradient triangle at the bottom, and box around the cells, with decreasing abundance from left (dark) to right (light). Cell populations regarding their Notch receptor expression in the proliferation zone ot the <b>A</b>, dorsal telencephalon and hypothalamus <b>B</b>, ventral telencephalon and optic tectum and <b>C</b> cerebellum. N – Notch receptor; N1 – <i>notch1a/1b</i>; N3 – <i>notch3</i>.</p