Cre-mediated recombination in the red-to-green reporter line.

<p>(a) Scheme of the recombination event. In the absence of Cre the <i>EF1</i>α promoter drives the expression of DsRed2 but changes to EGFP after successful Cre-mediated recombination. (b) Embryos of the red-to-green reporter line show strong DsRed2 and no EGFP fluorescence. (c) Maternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele results in complete loss of DsRed2 and ubiquitous EGFP expression in double transgenic embryos. (d) Paternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele leads to strong DsRed2 and mosaic EGFP expression in double transgenic embryos. (e) Paternal contribution of the <i>Tg(hsp70:EGFP-Cre)</i> allele and brief heat induction at mid-gastrulation stages results in reduced DsRed2 and strong ubiquitous EGFP expression in double transgenic embryos. (f) Embryos of the <i>Tg(hsp70:EGFP-Cre)</i> line show only weak EGFP fluorescence after brief heat induction at mid-gastrulation stages. b–f Lateral views of live 24 hpf embryos bearing different transgenes. Scale bar, 125 µm.</p

Ligand-dependent Cre-mediated recombination.

<p>(a) Scheme of the ligand-dependent recombination event in cells of the red-to-green reporter line. The chimeric CreER^T2 recombinase is retained in the cytoplasm in the absence of the ligand. After administration of TAM which is converted to the active ligand 4-OHT, CreER^T2 translocates to the nucleus, where it catalyzes the recombination event. (b) Expression of CreER^T2 in the diencephalon of the <i>Tg(pax2a:CreER^T2)^#19</i> line at early segmentation stages revealed by <i>in situ</i> hybridization. (c) EGFP expression in the diencephalon of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#19</i> alleles after TAM treatment at mid-gastrulation stages. (d) Expression of CreER^T2 in rhombomere 3 and 5 of the <i>Tg(pax2a:CreER^T2)^#45</i> line at early segmentation stages revealed by <i>in situ</i> hybridization. (e) EGFP expression in rhombomere 3 and 5 of double transgenic embryos at 24 hpf bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#45</i> alleles after TAM treatment at mid-gastrulation stages. Abbreviations: f, forebrain; h, hindbrain; m, midbrain. Scale bar, 50 µm.</p

Dose-dependent recombination in the <i>Tg(pax2a:CreER^T2)^#19</i> and <i>Tg(pax2a:CreER^T2)^#45</i> lines by TAM.

<p>(a) Double transgenic embryos bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#19</i> alleles show strong EGFP expression in the diencephalon after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM or 0.05 µM TAM at the same stage, results in reduced EGFP expression or single EGFP-positive cells, respectively. (b) Double transgenic embryos bearing the red-to-green reporter and the <i>Tg(pax2a:CreER^T2)^#45</i> alleles show strong EGFP expression in rhombomere 3 and 5 after application of 5 µM TAM at mid-gastrulation stages. Application of 0.5 µM TAM at the same stage, results in single EGFP-positive cells. a, b Dorsal views of double transgenic embryos at 24 hpf. Scale bar, 30 µm.</p

Kinetics of ligand-dependent Cre-mediated recombination.

<p>(a) Expression of CreER^T2 in the <i>Tg(pax2a:CreER^T2)^#19</i> line at the 12-somite stage revealed by <i>in situ</i> hybridization. (b) Control embryos treated with DMSO never show any EGFP. (c) Immunofluorescence staining with antibodies to EGFP is detectable 4 hours after application of TAM and expanded further after 6 hours. (d) Onset of EGFP expression by immunofluorescence staining is detected after 2 hours and expanded further after 4 and 6 hours after application of 4-OHT. a–d Dorsal views of double transgenic embryos at 12-, 16-, 20 and 24-somite stage (15, 17, 19 and 21 hpf). Abbreviations: f, forebrain; e, eye anlage; h, hours; m, midbrain. Scale bar, 30 µm.</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

<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

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

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