Bandín S et al. (2015), Prepatterning and patterning of the thalamus al...

XB-ART-51177

Front Neuroanat 2015 Feb 03;9:107. doi: 10.3389/fnana.2015.00107.

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Prepatterning and patterning of the thalamus along embryonic development of Xenopus laevis.

Bandín S , Morona R , González A .

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Previous developmental studies of the thalamus (alar part of the diencephalic prosomere p2) have defined the molecular basis for the acquisition of the thalamic competence (preparttening), the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains (rostral and caudal progenitor domains) in response to inducing activities and that are shared in birds and mammals. In the present study we have analyzed the embryonic development of the thalamus in the anuran Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined In situ hybridization (ISH) and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain vs. Gbx2 and Ngn2 (among others) in the caudal domain strongly suggest the role of Shh as morphogen in the amphibian thalamus. All these data showed that the molecular characteristics observed during preparttening and patterning in the thalamus of the anuran Xenopus (anamniote) share many features with those described during thalamic development in amniotes (common patterns in tetrapods) but also with zebrafish, strengthening the idea of a basic organization of this diencephalic region across vertebrates.

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Species referenced: Xenopus laevis
Genes referenced: dbx2 dll4 emx1l fezf1 gad1.1 gbx2.1 gbx2.2 gli1 gli2 gli3 irx1 lhx2 lhx9 neurog2 nkx2-2 pax6 pax7 ptbp1 shh slc17a6 tcf7l2 trhd wnt3a
???displayArticle.antibodies??? GABA Ab2 Isl1 Ab1 Nkx2-2 Ab1 Pax7 Ab1

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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.
	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.
	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.
	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.
	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.
	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.
	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.




	lhx9 (LIM homeobox 9) gene expression in Xenopus laevis embryonic brain, NF stage 40/41, transverse section, dorsal up,
	lhx2 (LIM homeobox 2) gene expression in Xenopus laevis embryonic brain, NF stage 40/41, transverse section, dorsal up,


	nkx2-2
	dll4/isl1 abs

	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.

References [+] :

Abbasi, Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development. 2010, Pubmed