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FIGURE 1 | Expression of early markers of the diencephalic
prepatterning and formation of the Zli. Microphotographs of lateral
views of the forebrain in whole mounts labeled for double in situ
hybridization (ISH) to reveal Shh (purple) and Gbx2 (orange) at the
embryonic stages indicated (A–D); the orientation is indicated in each
panel to highlight the dorsoventral and rostrocaudal change of the axis
through the thalamus at each stage. (E–O): Microphotographs of whole
mounts (I,J,M) and sagittal (E,F,H,K,L,N) or transverse (G,O) sections of
embryos at stages 33/34. The photographs correspond to single ISH
(E,I,J), single immunohistochemistry (IHC; K), and combinations of
double ISH (F,G,M–O) or combined ISH and IHC (H,L). The markers
labeled are indicated in the upper left of each photograph. All images
are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. The levels of the transverse sections (G,O) are indicated in photographs (F,N), respectively. Scale bars = 100 µm (E,F,L–N), 50 µm (A–D,G–K,O). See list for abbreviations.
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FIGURE 2 | Expression of thalamic markers at early embryonic
stages 37/38. Microphotographs of whole mounts (A,N) and sagittal
(B,C,E,J,L,O,R) or transverse (D,F–I,K,M,P,Q) sections of embryos at
stages 37/38. Photographs correspond to single ISH (purple; A,I), double
ISH (purple/orange; G,N–Q) and combination of ISH (purple) with IHC
(brown) (B–E, F,H,J,K,L,M,R). The markers labeled are indicated in the
upper left of each photograph. All images are oriented following the
same standard: dorsal is upwards in transverse and sagittal sections, and
rostral is to the left in sagittal sections. The neuromeric boundaries and
main brain subdivisions are indicated to assist in the precise localization
of the labeling. At these stage c-Th and r-Th subdivision of the thalamus
were distinguished (M). The levels of the transverse sections (D,F,P) are
indicated in photographs (C,E,O), respectively. Scale bars = 100 µm
(A,B,L,N,O), 50 µm (C–K,M,P–R). See list for abbreviations.
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FIGURE 3 | Expression of thalamic markers at embryonic stages 40/41.
Microphotographs of transverse (A,B,C,E,F,H,J,L,M) or sagittal (D,G,I,K)
sections of embryos at stages 40/41. Photographs correspond to single ISH (purple; A,B), single IHC (C) double ISH (purple/orange; D,E,G,H,K) and
combination of ISH (purple) with IHC (brown) (F,I,J,L,M). The markers labeled
are indicated in the upper left of each photograph. All images are oriented
following the same standard: dorsal is upwards in transverse and sagittal
sections, and rostral is to the left in sagittal sections. The neuromeric
boundaries and main brain subdivisions are indicated to assist in the precise
localization of the labeling. Note the mutually exclusive expression between
Gbx2 in the thalamus and the Xiro1 in the habenular region, (compare E and H;
D and G). The levels of the transverse sections (H,J) are indicated in
photographs (G,I), respectively. Scale bars = 100 µm (G,K), 50 µm
(A–F,H,I,K–M). See list for abbreviations.
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FIGURE 4 Expression of thalamic markers at embryonic stages 40/41.
Microphotographs of transverse (A–E,G–I,K–M) or sagittal (F,J,N,O) sections of embryos at stages 40/41. In all cases, photographs correspond to combination of ISH (purple) with IHC (brown), except for a double fluorescent ISH (red) and IHC (green) panel (E) and two single ISH (purple; H,I). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. The levels of the transverse sections (A,K,L) are indicated in photograph (J), the level of (G) is indicated in (F), and the level of (M) is indicated in (N). Scale bars = 50 µm. See list for abbreviations.
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FIGURE 5 | Expression of thalamic markers at embryonic stage 45.
Microphotographs of sagittal (A,E,G–I,K,O,R,T), transverse
(B,F,J,L–N,P,Q,S,U), and horizontal (C,D) sections of embryos at stage
45. In all cases, photographs correspond to combination of ISH (purple)
with IHC (brown), except for the double fluorescent ISH (red) and IHC
(green) panels (K–O) and a double fluorescent IHC (P). The markers
labeled are indicated in the upper left of each photograph. All images are
oriented following the same standard: dorsal is upwards in transverse
and sagittal sections, and rostral is to the left in sagittal sections; in the
horizontal sections rostral is to the left. The neuromeric boundaries and
main brain subdivisions are indicated to assist in the precise localization
of the labeling. The levels of the transverse section (B) is indicated in
photograph (A), and the levels of (L,M) are indicated in (K). Scale bars
= 100 µm (K–M,O), 50 µm (A–J,N,P–U). See list for abbreviations.
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FIGURE 6 | Main markers involved in the early thalamic
prepatterning. Schemes of lateral views of the forebrain at early and
late embryonic stages representing the main expressed factors that
lead in Xenopus to a first induction of the thalamic region and the
position of the Zli (A), and the subsequent prepatterning of the
thalamus and formation of the Zli (B). The prethalamo-thalamic
boundary (PTB) can be inferred in the zone where the rostral and
caudal expressions abut.
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FIGURE 7 | Expression patterns of the main thalamic markers.
Summary diagrams representing in lateral view of the forebrain the extent of the expressions of the main markers analyzed in Xenopus with respect to the thalamus and neighboring regions at early (A,B) and late (C,D) embryonic
stages.
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lhx9 (LIM homeobox 9) gene expression in Xenopus laevis embryonic brain, NF stage 40/41, transverse section, dorsal up,
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lhx2 (LIM homeobox 2) gene expression in Xenopus laevis embryonic brain, NF stage 40/41, transverse section, dorsal up,
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nkx2-2
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dll4/isl1 abs
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Figure 1. Expression of early markers of the diencephalic prepatterning and formation of the Zli. Microphotographs of lateral views of the forebrain in whole mounts labeled for double in situ hybridization (ISH) to reveal Shh (purple) and Gbx2 (orange) at the embryonic stages indicated (A–D); the orientation is indicated in each panel to highlight the dorsoventral and rostrocaudal change of the axis through the thalamus at each stage. (E–O): Microphotographs of whole mounts (I,J,M) and sagittal (E,F,H,K,L,N) or transverse (G,O) sections of embryos at stages 33/34. The photographs correspond to single ISH (E,I,J), single immunohistochemistry (IHC; K), and combinations of double ISH (F,G,M–O) or combined ISH and IHC (H,L). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. The levels of the transverse sections (G,O) are indicated in photographs (F,N), respectively. Scale bars = 100 μm (E,F,L–N), 50 μm (A–D,G–K,O). See list for abbreviations.
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Figure 2. Expression of thalamic markers at early embryonic stages 37/38. Microphotographs of whole mounts (A,N) and sagittal (B,C,E,J,L,O,R) or transverse (D,F–I,K,M,P,Q) sections of embryos at stages 37/38. Photographs correspond to single ISH (purple; A,I), double ISH (purple/orange; G,N–Q) and combination of ISH (purple) with IHC (brown) (B–E, F,H,J,K,L,M,R). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. At these stage c-Th and r-Th subdivision of the thalamus were distinguished (M). The levels of the transverse sections (D,F,P) are indicated in photographs (C,E,O), respectively. Scale bars = 100 μm (A,B,L,N,O), 50 μm (C–K,M,P–R). See list for abbreviations.
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Figure 3. Expression of thalamic markers at embryonic stages 40/41. Microphotographs of transverse (A,B,C,E,F,H,J,L,M) or sagittal (D,G,I,K) sections of embryos at stages 40/41. Photographs correspond to single ISH (purple; A,B), single IHC (C) double ISH (purple/orange; D,E,G,H,K) and combination of ISH (purple) with IHC (brown) (F,I,J,L,M). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. Note the mutually exclusive expression between Gbx2 in the thalamus and the Xiro1 in the habenular region, (compare E and H; D and G). The levels of the transverse sections (H,J) are indicated in photographs (G,I), respectively. Scale bars = 100 μm (G,K), 50 μm (A–F,H,I,K–M). See list for abbreviations.
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Figure 4. Expression of thalamic markers at embryonic stages 40/41. Microphotographs of transverse (A–E,G–I,K–M) or sagittal (F,J,N,O) sections of embryos at stages 40/41. In all cases, photographs correspond to combination of ISH (purple) with IHC (brown), except for a double fluorescent ISH (red) and IHC (green) panel (E) and two single ISH (purple; H,I). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. The levels of the transverse sections (A,K,L) are indicated in photograph (J), the level of (G) is indicated in (F), and the level of (M) is indicated in (N). Scale bars = 50 μm. See list for abbreviations.
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Figure 5. Expression of thalamic markers at embryonic stage 45. Microphotographs of sagittal (A,E,G–I,K,O,R,T), transverse (B,F,J,L–N,P,Q,S,U), and horizontal (C,D) sections of embryos at stage 45. In all cases, photographs correspond to combination of ISH (purple) with IHC (brown), except for the double fluorescent ISH (red) and IHC (green) panels (K–O) and a double fluorescent IHC (P). The markers labeled are indicated in the upper left of each photograph. All images are oriented following the same standard: dorsal is upwards in transverse and sagittal sections, and rostral is to the left in sagittal sections; in the horizontal sections rostral is to the left. The neuromeric boundaries and main brain subdivisions are indicated to assist in the precise localization of the labeling. The levels of the transverse section (B) is indicated in photograph (A), and the levels of (L,M) are indicated in (K). Scale bars = 100 μm (K–M,O), 50 μm (A–J,N,P–U). See list for abbreviations.
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Figure 6. Main markers involved in the early thalamic prepatterning. Schemes of lateral views of the forebrain at early and late embryonic stages representing the main expressed factors that lead in Xenopus to a first induction of the thalamic region and the position of the Zli (A), and the subsequent prepatterning of the thalamus and formation of the Zli (B). The prethalamo-thalamic boundary (PTB) can be inferred in the zone where the rostral and caudal expressions abut.
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Figure 7. Expression patterns of the main thalamic markers. Summary diagrams representing in lateral view of the forebrain the extent of the expressions of the main markers analyzed in Xenopus with respect to the thalamus and neighboring regions at early (A,B) and late (C,D) embryonic stages.
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Figure 8. Diagram of the main expression patterns for markers of the thalamic subdivisions. Schematic representation of the early progenitor domains in the thalamus of Xenopus, indicating the main markers found in each domain in the present study. (A) is a drawing of a lateral view of the thalamus in which a color-code for the distinct expression patterns for the markers used is depicted in (B). (C) corresponds to the representation of a transverse section at the level indicated in (A) in which the extent of the thalamic progenitor domains is indicated in relation to the Zli.
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