Domínguez L et al. (2014), Characterization of the hypothalamus of Xenopus...

XB-ART-47852

J Comp Neurol 2014 Apr 01;5225:1102-31. doi: 10.1002/cne.23471.

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Characterization of the hypothalamus of Xenopus laevis during development. II. The basal regions.

Domínguez L , González A , Moreno N .

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The expression patterns of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development by means of combined immunohistochemical and in situ hybridization techniques. The connectivity of the main subdivisions was investigated by in vitro tracing techniques with dextran amines. The basal hypothalamic region is topologically rostral to the basal diencephalon and is composed of the tuberal (rostral) and mammillary (caudal) subdivisions, according to the prosomeric model. It is dorsally bounded by the optic chiasm and the alar hypothalamus, and caudally by the diencephalic prosomere p3. The tuberal hypothalamus is defined by the expression of Nkx2.1, xShh, and Isl1, and rostral and caudal portions can be distinguished by the distinct expression of Otp rostrally and Nkx2.2 caudally. In the mammillary region the xShh/Nkx2.1 combination defined the rostral mammillary area, expressing Nkx2.1, and the caudal retromammillary area, expressing xShh. The expression of xLhx1, xDll4, and Otp in the mammillary area and Isl1 in the tuberal region highlights the boundary between the two basal hypothalamic territories. Both regions are strongly connected with subpallial regions, especially those conveying olfactory/vomeronasal information, and also possess abundant intrahypothalamic connections. They show reciprocal connections with the diencephalon (mainly the thalamus), project to the midbrain tectum, and are bidirectionally related to the rhombencephalon. These results illustrate that the basal hypothalamus of anurans shares many features of specification, regionalization, and hodology with amniotes, reinforcing the idea of a basic bauplan in the organization of this prosencephalic region in all tetrapods.

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Species referenced: Xenopus laevis
Genes referenced: acta4 dlx2 isl1 nkx2-1 nkx2-2 otp pax7 shh sst.1 tub
???displayArticle.antibodies??? GABA Ab2 Isl1/2 Ab1 Nkx2-1 Ab5 Nkx2-2 Ab1 Otp Ab1 Pax7 Ab1 Som Ab1 Th Ab1

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	Figure 1. Photomicrographs of Nissl-stained sagittal (A,D,G) and transverse (B,C,E,F,H,I) sections through the basal hypothalamus of a late embryo (A–C), a premetamorphic larva (D–F), and a prometamorphic larva (G–I) at the approximate levels indicated on the respective schemes of the left column. Note that the coordinate system for the hypothalamus rotates 90° because the longitudinal axis of the brain bends in the diencephalon, and this is also the case for all photomicrographs of sagittal sections in all figures. These photomicrographs illustrate the poorly segregated regions in the basal hypothalamus and the scarce cell migration from the periventricular lining during development. The dashed lines in (A,D,G) indicate the approximate boundaries between hypothalamic and diencephalic regions, whereas in (H,I) they also indicate the approximate boundaries of the thalamus, prethalamus, and pretectal diencephalic regions. Scale bars = 200 μm (in C,F,I are valid for B,E,H, respectively)
	Figure 2. Photomicrographs of sagittal (A,E,G) and transverse (B–D,F) sections through the mammillary and tuberal hypothalamus of Xenopus, showing the expressions of xShh (A), Nkx2.1 (B), and Isl1 (C) and the combined expressions of Isl1 and Nkx2.1 (D,E), Isl1 and Otp (F), Nkx2.1 and Nkx2.2 (G), and Pax7 and Nkx2.1 (H). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the boundaries between the different basal hypothalamic subdivisions and the combinatorial code of markers of each region. The yellow lines indicate the limit between two adjacent regions revealed by the different markers. A magenta-green version of this figure is provided as Supporting Information Fig. 1. Scales bars = 100 μm in A,F–H, B (valid for C,D); 50 μm in E.
	Figure 3. Photomicrographs of transverse (A–E,G,H) and sagittal (F,I–K) sections through the hypothalamus of Xenopus showing in the tuberal region and adjacent areas the expressions of Nkx2.1 (A) and Isl1 (B), and the combined expressions of Isl1 and Nkx2.1 (C), xShh and Isl1 (D), Isl1 and Otp (E,F), Nkx2.2 and Nkx2.1 (G), Nkx2.2 and otp (H,I), Nkx2.2 and SOM (J), and xLhx1 and Nkx2.2 (K). Developmental stages are indicated in each photograph. The schematic representations at the bottom summarize the combinatorial codes of the markers in the tuberal hypothalamus in a lateral view of the brain and a transverse section. A magenta-green version of this figure is provided as Supporting Information Fig. 2. Scale bars = 100 μm (in A is valid for B,C).
	Figure 4. Photomicrographs of transverse (A,C–F,H,J) and sagittal (B,G,I) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expression of Isl1 and Nkx2.1 (A,B), xShh and Nkx2.1 (C), Isl1 and Otp (D,E,F), Nkx2.2 and Otp (G,I,J), Nkx2.2 and Nkx2.1 (H). The level of the transverse section shown in (J) is indicated by the vertical yellow line drawn in (I). Developmental stages are indicated in each photograph. A magenta-green version of this figure is provided as Supporting Information Fig. 3. Scale bars = 100 μm.
	Figure 5. Photomicrographs of sagittal (A,E–I) and transverse (B–D) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expression of Nkx2.2 and Nkx2.1 (A–C), xDll4 and Isl1 (D), xDll4 and Otp (E), xLhx1 and Otp (F). Single expression for xLhx1 (G) and Nkx2.2 (H) are shown together with the merged image (I). Developmental stages are indicated in each photograph. A magenta-green version of this figure is provided as Supporting Information Fig. 4. Scale bars = 100 μm (in G is valid for H,I).
	Figure 6. Photomicrographs of transverse (A,B,D–K) and sagittal (C,L) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expressions of Pax7 and Nkx2.1 (A,D) and Pax7 and Otp (E). The arrowheads in (A,D,E,F) show double-labeled cells. In addition, images are shown of the combined labeling for TH with Nkx2.1 (F), xShh (G), Isl1 (H), Otp (I), xLhx1 (J), and Nkx2.2 (K,L). The schematic representations at the bottom summarize the combinatorial codes of the markers in the mammillary hypothalamus in a lateral view of the brain and a transverse section. A magenta-green version of this figure is provided as Supporting Information Fig. 5. Scale bars = 100 μm in A,B,E–L (in E is valid for D); 50 μm in C
	Figure 7. Schematic sagittal (A) and transverse (B,C) representations of the basal hypothalamus of Xenopus summarizing the combinatorial code of expression for the transcription factors and markers used in the present study, following the color code indicated in the box.
	Figure 8. Photomicrographs of sagittal (A) and transverse (B–N) sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the Tub of juvenile Xenopus, combined with the marker Isl1 (A,C,E,H–J,L) to corroborate that the tracer was delivered into this paricular area. The dashed box in (C,E,K) indicate the higher magnification shown in (C',E',K'), respectively. The arrowheads in (A,B,C',E',J,M) and N point to retrogradely labeled cells or fibers. A magenta-green version of this figure is provided as Supporting Information Fig. 6. Scale bars = 500 μm in A; 100 μm in C–N; 50 μm in B,K'; 25 μm in C',E'.
	Figure 9. Photomicrographs of transverse sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the mammillar area of juvenile Xenopus, combined with the markers Isl1 (B-I) and Otp (D) to corroborate that the tracer was delivered into this particular area. The arrowheads in B–J indicate retrogradely labeled cells. A magenta-green version of this figure is provided as Supporting Information Fig. 7. Scale bars = 200 μm in G–I,J'; 100 μm in A–F,K.
	Figure 10. Schematic drawings of transverse sections through the brain of X. laevis illustrating the representative injection sites used in the confirmation experiments. The levels of the selected transverse sections (A–G) are indicated in the upper right scheme of the brain. H–J: Photomicrographs showing the examples of injection sites in the bed nucleus of the stria terminalis (H), central amygdala (I), and lateral amygdala (J). Scale bars = 100 μm.
	Figure 11. Photomicrographs of transverse sections through the brain of X. laevis showing retrogradely labeled cells and anterogradely labeled fibers in the tuberal and mammillary regions after BDA injections carried out in the different brain centers indicated in each photograph. The arrowheads in (B,C,E–G,I,J,M,N,S) point to labeled cells and fibers. Scale bars = 200 μm in B,C,E–J,L–N,P,R,U; 100 μm in A,D,O,Q,S,T; 50 μm in K.
	Figure 12. Schemes in sagittal view summarizing the main afferent and efferent connections of the tuberal and mammillary regions in Xenopus.
	Supplementary Figure 1. Photomicrographs of sagittal (A,E,G) and transverse (B-D, F) sections through the mammillary and tuberal hypothalamus of Xenopus, showing the expressions of xShh (A), Nkx2.1 (B), and Isl1 (C) and the combined expressions of Isl1 and Nkx2.1 (D,E), Isl1 and Otp (F), Nkx2.1 and Nkx2.2 (G) and Pax7 and Nkx2.1 (H). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the boundaries between the different basal hypothalamic subdivisions and the combinatorial code of markers of each region. The yellow lines indicate the limit between two adjacent regions revealed by the different markers. Scales bars: 100μm (A, F-H), 50μm (E). The scale bar in b is valid for C and D = 100μm. This figure corresponds to the figure 2.
	Supplementary Figure 2. Photomicrographs of transverse (A-E,G, H) and sagittal (F, I-K) sections through the hypothalamus of Xenopus showing in the tuberal region and adjacent areas the expressions of Nkx2.1 (A) and Isl1 (B), and the combined expressions of Isl1 and Nkx2.1 (C), xShh and Isl1 (D), Isl1 and Otp (E,F), Nkx2.2 and Nkx2.1 (G), Nkx2.2 and otp (H,I), Nkx2.2 and SOM (J), and xLhx1 and Nkx2.2 (K). Developmental stages are indicated in each photograph. The schematic representations at the bottom summarize the combinatorial codes of the markers in the tuberal hypothalamus in a lateral view of the brain and a transverse section. Scale bars: 100μm. The scale bar in A is valid for B and C. This figure corresponds to the figure 3.
	Supplementary Figure 3. Photomicrographs of transverse (A, C-F, H, J) and sagittal (B, G, I) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expression of Isl1 and Nkx2.1 (A,B), xShh and Nkx2.1 (C), Isl1 and Otp (D, E, F), Nkx2.2 and Otp (G I, J), Nkx2.2 and Nkx2.1 (H). The level of the transverse section shown in J is indicated by the vertical yellow line drawn in I. Developmental stages are indicated in each photograph. Scale bars: 100μm. This figure corresponds to the figure 4.
	Supplementary Figure 4. Photomicrographs of sagittal (A, E-I) and transverse (B-D) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expression of Nkx2.2 and Nkx2.1 (A-C), xDll4 and Isl1 (D), xDll4 and Otp (E), xLhx1 and Otp (F). Single expression for xLhx1 (G) and Nkx2.2 (H) are shown together with the merged image (I). Developmental stages are indicated in each photograph. Scale bars: 100μm. The scale bar in G is valid for H and I. This figure corresponds to the figure 5.
	Supplementary Figure 5. Photomicrographs of transverse (A, B, D-K) and sagittal (C,L) sections through the developing hypothalamus of Xenopus showing in the mammillary region and adjacent areas the combined expressions of Pax7 and Nkx2.1 (A, D) and Pax7 and Otp (E). The arrowheads in A, D, E and F show double labeled cells. In addition, imagines are shown of the combined labelling for TH with Nkx2.1 (F), xShh (G), Isl1 (H), Otp (I), xLhx1 (J) and Nkx2.2 (K,L). The schematic representations at the bottom summarize the combinatorial codes of the markers in the mammillary hypothalamus in a lateral view of the brain and a transverse section. Scale bars: 100μm (A, B, E-L), 50μm (C). The scale bar in E is valid for D. This figure corresponds to the figure 6.
	Supplementary Figure 6. Photomicrographs of sagittal (A) and transverse (B-N) sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the tuberal region of juvenile Xenopus, combined with the marker Isl1 (A,C,E,H-J, L) to corroborate that the tracer was delivered into this paricular area. The dashed box in C,E and K indicate the higher magnification shown in C',E' and K', respectively. The arrowheads in A,B,C',E',J,M and n point to retrogradely labeled cells or fibers. Scale bars: 500μm (A), 100 μm (C-N), 50 μm (B, K'), 25 μm (C', E'). This figure corresponds to the figure 8.
	Supplementary Figure 7. Photomicrographs of transverse sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the Ma of juvenile Xenopus, combined with the markers Isl1 (B-I) and Otp (D) to corroborate that the tracer was delivered into this particular area. The arrowheads in b-j indicate retrogradely labeled cells. Sclae bars: 200 μm (G-I, J'), 100 μm (A-F, K). This figure corresponds to the figure 9.