Figure 1.
Notch receptor expression in the adult zebrafish telencephalon.
Notch receptors are differentially expressed in the ventricular zone of the adult zebrafish telencephalon. Brightfield micrographs of cross-sections at the indicated levels through the telencephalon; the schematic at the bottom-left illustrates the corresponding areas in the cross-section shown at a higher magnification. A, B and C, expression of zebrafish notch1a, notch1b and notch3 in the rostral telencephalon, respectively; dashed line indicates the division between ventral and dorsal telencephalon; A –C
, Higher magnification of the ventral expression domain; A
–C
, Higher magnification of the dorsal expression domain; dashed line indicates the dorso-lateral most region. Arrowheads and unfilled arrowheads indicate, respectively, strongly and weakly expressing cells located at the ventricular surface; arrows indicate expressing cells in the parenchyme. Abbreviations: Dll, lateral part of the dorso-lateral telencephalic area; Dil, intermediate part of the dorso-lateral telencephalic area; Dm, dorso-medial telencephalic area; dT, dorsal telencephalic area; vT, ventral telencephalic area; Vd, dorsal nucleus of the ventral telencephalon; Vv, ventral nucleus of the ventral telencephalon. Scale bars = 200
in A (applies to B, C); 25
in A
(applies to B
,C
, A
–C
and A
–C
).
Figure 2.
notch1a, notch1b and notch3 expression in radial glia and proliferating cells of the dorso-medial ventricular zone of the telencephalon.
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. A–C, notch1a and D–E, notch1b 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 notch1b
/PCNA
cells. F–H, notch3 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 notch3
/PCNA
cells. I, 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. J, 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).
Figure 3.
notch1a, notch1b and notch3 expression in radial glia and proliferating cells of the dorso-lateral telencephalic ventricular zone.
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. A–B, notch1a and C–D, notch1b 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. E–F, notch3 expressing cells localize to a great extent with S100
, including both PCNA
(white arrows) and PCNA
(white arrowheads) cells; unfilled yellow arrowheads indicate notch3
/PCNA
cells. G, 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.
Figure 4.
Overlapping and complementary Notch receptor expression in the dorsal telencephalon.
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. A, B, notch1a/3 co-expression in Dm and Dl, respectively. C, notch1a/b 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 notch1a
/notch3
(filled yellow arrowheads) while others are negative for both receptors (filled yellow arrows); within the PCNA
population, some cells express either notch3 or notch1b but not notch1a alone (unfilled yellow arrowheads). Scale bars = 50
.
Figure 5.
Notch receptor expression in the ventral telencephalic proliferation zone.
Confocal images showing the localization of Notch receptors by FISH (white) in PCNA proliferating cells (green) and in the S100
glial cells of Vd (red). A–A
, notch1a, B–B
, notch1b and C–C
, notch3 expression in the ventral telencephalon. All three receptors are express in S100
cells of Vd (white arrows). A
-A
, B
-B
and C
-C
show the individual channels for Notch receptor expression and PCNA at a higher magnification of the respective boxed areas. Notch receptor
/PCNA
cells with apical nucleus (unfilled white arrowheads) display a stronger expression intensity than PCNA
cells with basal nucleus (unfilled red arrowheads); unfilled yellow arrowhead indicate Notch receptor
/PCNA
cells. Scale bars = 50
in A (applies to B and C) and 25
in A
(applies to B
, C
and A
-C
). D, The notched boxplot represents the relative mean grey values of notch expression in proliferating cells with apical or basal nucleus, as illustrated in the schematics and by the respective dashed lines indicated in the higher magnification pictures shown above; the middle line of the box represents the median; the bottom and top of the box represents the 25
and 75
percentiles, respectively; whiskers indicate the minimum and maximum; circle indicates an outlier; total n = 13. Significance levels:
,
.
Figure 6.
Overlapping and complementary notch1a/3 expression in the ventral telencephalic proliferation zone, along the rostro-caudal axis.
Confocal images showing localization of notch1a (white) and notch3 (red) by double FISH (white and red) and PCNA proliferating cells (green); DAPI (blue) is used as nuclear counterstaining. Areas represented in the micrographs are indicated in the cross-section schematics on the right as well as the different levels through the telencephalon. A–D, notch1a and notch3 expression domains along the rostro-caudal vT proliferation zone. Co-expression of these receptors both in PCNA
(white filled arrowheads) and PCNA
cells (unfilled white arrowheads); a few PCNA
cells are Notch receptor
(yellow arrows) while others are notch1a
/notch3
(white filled arrows); unfilled yellow arrowheads indicate notch1a
/notch3
/PCNA
cells. Abbreviations: dT, dorsal telencephalic area; vT, ventral telencephalic area; Vd, dorsal nucleus of the ventral telencephalon; Vv, ventral nucleus of the ventral telencephalon. Scale bars = 50
in A, B (applies to C) and D.
Figure 7.
Summary of Notch receptor expression pattern and cellular characteristics in the adult zebrafish telencephalon.
A, Illustration shows the overall expression of the analysed notch receptors; they are mainly present in ventricular zone cells, with a few positive cells in the parenchyma, overlapping with oligodendrocytic or endothelial markers. B, Cellular characteristics of Notch expressing cells in the Dm ventricular zone (vz) and C, in the vT proliferation zone. Abbreviations: Dil, intermediate part of the dorso-lateral telencephalic area; Dll, lateral part of the dorso-lateral telencephalic area; Dm, dorso-medial telencephalic area; dT, dorsal telencephalic area; vT, ventral telencephalic area; Vd, dorsal nucleus of the ventral telencephalon; Vv, ventral nucleus of the ventral telencephalon.
Figure 8.
Notch receptor expression in the adult zebrafish hypothalamus.
Notch receptors are differentially expressed throughout the hypothalamus. Cross-sections at the indicated level through the diencephalon. Brightfield images show the expression of A–A , notch1a, B–B
, notch1b and C–C
, notch3 at different rostro-caudal levels of the hypothalamus. Strong (filled arrowheads) and weak (unfilled arrowheads) expressing cells are detected. Asterisks (
) indicate areas of no or very weak expression in the Hd. 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. Scale bar = 100
in A (applies to all).
Figure 9.
Summary of Notch receptor expression pattern and cellular characteristics in the adult zebrafish hypothalamus.
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.
Figure 10.
Notch receptor expression in the adult zebrafish optic tectum.
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. Brighfield images show the expression (by ISH) of A, notch1a, B, notch1b and C, notch3 along the PML and mPGZ (filled arrowheads) and in the dPGZ (filled arrows). A few notch1a and notch1b
cells are also found in more superficial layers of the PGZ and TeO (unfilled arrows). 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; TeO, optic tectum. Scale bars = 100
in A (applies to all).
Figure 11.
Notch receptor expression in radial glia and proliferating cells of the adult zebrafish optic tectum.
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 A–D, notch1a, E–I, notch1b and J–N,notch3 by FISH (white). A, notch1a, E, notch1b and J, notch3 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 notch1a and notch1b are also found (unfilled arrows), with relative more abundance for the notch1b receptor in this layer; B, C, F,G and K,L are higher magnifications of the respective framed areas in A, E and J, showing the overlap of notch 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; D, H, I, M,N, 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).
Figure 12.
Overlapping and complementary notch1a/3 expression in the adult zebrafish optic tectum.
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. A, notch1a/3 and B, notch1a/1b 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); notch3
-only cells in A and notch1b
-only cells in B are indicated by unfilled yellow arrowheads; white arrows indicate notch1a
/notch3
/PCNA
cells in A; a few PCNA
cells are Notch receptor
(filled yellow arrows); unfilled white arrows indicate cells positive for notch1a alone. C, 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
.
Figure 13.
notch1a and notch1b expression in the adult cerebellar niche.
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 A, notch1a and D, notch1b in the cerebellum (arrowheads). Confocal images showing localization of the glial marker S100 (red) and PCNA (green), with B–C, notch1a and E–F, notch1b by FISH (white). B–C, Expression of notch1a localizes with a large fraction of PCNA
cells in the niche (unfilled arrowheads); weak (white arrow) or no expression of notch1a is detected in the Bergmann glia (S100
). E–F, notch1b is expressed in a subpopulation of PCNA
cells (unfilled arrowheads) and in a few S100
cells (white arrows). A few scattered notch1a
and notch1b
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.
Figure 14.
notch3 expression in the adult cerebellar niche.
Cross-sections at the indicated level through the mesencephalon; cerebellar area shown in the micrographs is indicated in the cross-section schematic. A, Brightfield image shows notch3 expressing cells in the cerebellum (black arrowheads). B–D, Confocal images showing localization of the glial marker S100 (red) and PCNA (green), with notch3 by FISH (white). B–C, notch3 is weakly expressed in a small subset of PCNA
cells in the stem cell niche (unfilled arrowhead). B–D, Strong notch3 expression is detected in S100
cells indicated by the white arrows; notch3 expression is also detected in some scattered cells of the ML, IML and GL that do not localize with the analysed markers (unfilled arrows); E, 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.
Figure 15.
Comparison of Notch receptor expressing cells in the five analysed adult zebrafish neurogenic niches.
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 A, dorsal telencephalon and hypothalamus B, ventral telencephalon and optic tectum and C cerebellum. N – Notch receptor; N1 – notch1a/1b; N3 – notch3.