Domínguez L et al. (2013), Characterization of the hypothalamus of Xenopus...

XB-ART-45956

J Comp Neurol 2013 Mar 01;5214:725-59. doi: 10.1002/cne.23222.

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Characterization of the hypothalamus of Xenopus laevis during development. I. The alar regions.

Domínguez L , Morona R , González A , Moreno N .

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The patterns of expression of a set of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the alar hypothalamus of Xenopus laevis throughout development. Combined immunohistochemical and in situ hybridization techniques were used for the identification of subdivisions and their boundaries. The alar hypothalamus was located rostral to the diencephalon in the secondary prosencephalon and represents the rostral continuation of the alar territories of the diencephalon and brainstem, according to the prosomeric model. It is composed of the supraoptoparaventricular (dorsal) and the suprachiasmatic (ventral) regions, and limits dorsally with the preoptic region, caudally with the prethalamic eminence and the prethalamus, and ventrally with the basal hypothalamus. The supraoptoparaventricular area is defined by the orthopedia (Otp) expression and is subdivided into rostral and caudal portions, on the basis of the Nkx2.2 expression only in the rostral portion. This region is the source of many neuroendocrine cells, primarily located in the rostral subdivision. The suprachiasmatic region is characterized by Dll4/Isl1 expression, and was also subdivided into rostral and caudal portions, based on the expression of Nkx2.1/Nkx2.2 and Lhx1/7 exclusively in the rostral portion. Both alar regions are mainly connected with subpallial areas strongly implicated in the limbic system and show robust intrahypothalamic connections. Caudally, both regions project to brainstem centers and spinal cord. All these data support that in terms of topology, molecular specification, and connectivity the subdivisions of the anuran alar hypothalamus possess many features shared with their counterparts in amniotes, likely controlling similar reflexes, responses, and behaviors.

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???displayArticle.link??? J Comp Neurol

Species referenced: Xenopus laevis
Genes referenced: dll4 dlx2 isl1 lhx1 lhx5 lhx8 lhx9 nkx2-1 nkx2-2 otp shh sst.1
???displayArticle.antibodies??? GABA Ab2 Isl1/2 Ab1 Mst Ab1 Nkx2-1 Ab5 Nkx2-2 Ab1 Otp Ab1 Som Ab1 Tbr1 Ab1

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	Figure 2. Photomicrographs of Nissl-stained sagittal (a,e,i,j,k) and transverse (b–d,f–h,l–q) sections through the hypothalamus of a late embryo (a–d), a premetamorphic larva (e–h), and a prometamorphic larva (i–q), 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 roughly recognized in the hypothalamus during development. The dashed lines in (a,e) indicate the approximate boundaries between telencephalic, hypothalamic, and diencephalic regions. Scale bars 1⁄4 100 lm in d,h,k’ (applies to a–c,e– g,i’,j); 200 lm in k,q (applies to i,j,l–p).
	Figure 3. Photomicrographs of transverse sections through the main alar hypothalamic subdivisions of Xenopus at different developmental stages. The boundaries are depicted on the basis of the single expression for xDll4 (d) and the combined expressions of Isl1 and Otp (a), xShh and Otp (b,c), and xLhx9 and Otp (e). The boundaries identified are the PO-SPV (a,b), SPV-SC (c), SPV-PTh (a–c), SC-PTh (d), and SPV-PThE (e). Developmental stages are indicated in each photograph. A magenta-green version of this figure is provided as Supporting Information Figure 1. Scale bars = 50 μm in a–c; 100 μm in d,e
	Figure 4. Photomicrographs of sagittal sections through the main alar hypothalamic subdivisions of Xenopus at different developmental stages. The boundaries are depicted on the basis of the single expression for xShh and Nkx2.1 (a,b) and the combined expressions of xDll4 and Otp (c), xDll4 and Nkx2.2 (d), Nkx2.1 and Nkx2.1 (e), xLhx1 and Otp (f), Isl1 and Otp (g), and Trbr1 and Isl1 (h). The boundaries identified are the PO-SPV (a–c), SPV-SC (d,e), SPV-PTh (c,f), and SPV-PThE (h). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the boundaries between the different alar 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 Figure 2. Scale bars = 50 μm in a–f,h; 200 μm in g
	Figure 5. Photomicrographs of transverse sections through the developing Xenopus PO region showing the combined expressions of xShh and Nkx2.1 (a), Isl1 and Nkx2.1 (b–d), Nkx2.1 and TH (e), Isl1 and TH (f), Isl1 and GABA (g), Isl1 and Otp (h–j), xShh and Otp (k), and Nkx2.1 and Nkx2.2 (l). Developmental stages are indicated in each photograph. The arrowhead in g indicates double-labeled Isl1 and GABA cells. The photomicrograph j shows a higher magnification of the area shown in i by confocal microscropy to illustrate the limit between the Isl1+ PO and the Otp+ SPV. A magenta-green version of this figure is provided as Supporting Information Figure 3. Scale bars = 50 μm in a–i,k,l; 25 μm in j.
	Figure 6. Photomicrographs of sagittal sections through the developing Xenopus PO region showing the single expressions of Nkx2.1 (a) and xShh (b) and the combined expressions of Isl1 and GABA (c), Isl1 and Nkx2.1 (d,e), Isl1 and Otp (f), and xDll4 and Otp (g). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the combinatorial code of markers present in the PO and its adjacent domains in a lateral view (h) and a transverse section (i). A magenta-green version of this figure is provided as Supporting Information Figure 4. Scale bars = 50 μm in a–e,g; 200 μm in f.
	Figure 8. Photomicrographs of sagittal sections through the developing Xenopus SPV region showing the expressions of Nkx2.2 and Otp (a–c), Nkx2.2 and MST (d,e), Nkx2.2 and SOM (f,f'), xLhx5 (g), xDll4 and Otp (h), and Otp and Isl1 (i). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the combinatorial code of markers present in the SPV and its adjacent domains in a lateral view (j,j') and a transverse section (k). The yellow line in a indicates the level of the section shown in Figure 7b. The yellow box in f indicates the higher magnification shown in f' and the arrowhead in f' shows the coexpression of Nkx2.2 and SOM in SPVr. A magenta-green version of this figure is provided as Supporting Information Figure 6. Scale bars = 100 μm in a; 50 μm in b,d–h; 250 μm in g.
	Figure 9. Photomicrographs of transverse (a–d,h,m–o) and sagittal (e–g,i–l) sections through the developing Xenopus SC region showing the single expressions of xLhx7 (e), xLhx1 (f) and the combined expressions of xDll4 and Isl1 (a), Isl1 and GABA (b), Isl1 and Nkx2.1 (c), xShh and Nkx2.1 (d), xLhx7 and xDll4 (g), Nkx2.1 and Nkx2.2 (h–j), xLhx1 and Nkx2.2 (k), xShh and TH (l,m), Nkx2.1 and TH (n) and Nkx2.2 and TH (o). Developmental stages are indicated in each photograph. The arrowhead in b indicates a cell double-labeled for Isl1 and GABA. A magenta-green version of this figure is provided as Supporting Information Figure 7. Scale bars: 200 μm (j), 50 μm (a–i, k–o).
	Figure 10. Schematic representation summarizing the combinatorial code of markers present in the SC in a lateral view of the brain (a,a') and a transverse section (b).
	Figure 11. Photomicrographs of transverse (a,e–k) and sagittal (b–d) sections through the SC territory in late prometamorphic and juvenile Xenopus showing the single expressions of Nkx2.1 (b), Isl1 (b), xLhx1 (g), and xLhx7 (h) and the combined expressions of Isl1 and Nkx2.1 (a,d–f), Isl1 and Otp (i), Isl1 and GABA (j) and Isl1 and TH (k). Developmental stages are indicated in each photograph. The arrowhead in k indicates a cell double-labeled for Isl1 and TH. A magenta-green version of this figure is provided as Supporting Information Figure 8. Scale bars = 100 μm.
	Figure 12. Schematic sagittal representation of the alar hypothalamus of a premetamorphic Xenopus larva summarizing the combinatorial code of transcription factors used in the present study, following the color code indicated in the box.
	Figure 13. Photomicrographs of transverse sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the PO of juvenile Xenopus, combined with the marker MST (d,e,g) to corroborate that the tracer was delivered into this particular area. A magenta-green version of this figure is provided as Supporting Information Figure 9. Scale bars = 250 μm in a,e,i; 200 μm in b,d,f,g,h; 100 μm in c,j.
	Figure 14. Photomicrographs of sagittal sections showing anterograde and retrograde labeling after BDA injection in the SPV (a–h) and the SC (h'–n) of juvenile Xenopus, combined with the markers Otp (a–c,e–g,h'–j), TH (d,n), and Isl1 (h–j) to corroborate that the tracer was delivered into the selected area. The dashed box in h and j indicate the higher magnification shown in h' and j', respectively. The arrowheads point to retrogradely labeled cells, with the exception of the e,j',n, which indicate labeled fibers toward the hypophysis. In all photographs the olfactory bulb is to the right and the spinal cord to the left. A magenta-green version of this figure is provided as Supporting Information Figure 10. Scale bars = 200 μm in a–e,i,g,h'; 500 μm in f,h,i–n).
	Figure 15. Photomicrographs of transverse sections through the brain of Xenopus showing retrogradely labeled cells and anterogradely labeled fibers in the PO, SPV, and SC regions after BDA injections in the different brain centers indicated in each photograph. The arrowheads in a,d,e,q,v,w point to retrogradely labeled cells. Scale bars = 200 μm in b–d,g,j,l,q,s,u–w; 100 μm in a,e,f,h,I,k,m–p,r,t.
	Figure 16. Schemes in sagittal view summarizing the main afferent and efferent connections of the PO, SPV, and SC regions in Xenopus. A color version of this figure is provided as Supporting Information Figure 11.
	Supplementary Figure 1. Photomicrographs of transverse sections through the main alar hypothalamic subdivisions of Xenopus at different developmental stages. The boundaries are depicted on the basis of the single expression for xDll4 (d) and the combined expressions of Isl1 and Otp (a), xShh and Otp (b,c), and xLhx9 and Otp (e). The boundaries identified are the PO-SPV (a-b), SPV-SC (c), SPV-PTh (a-c), SC-PTh (d), and SPV-PThE (e). Developmental stages are indicated in each photograph. Scale bars: 50μm (a-c) and 100μm (d,e).This figure corresponds to the figure 3.
	Supplementary Figure 2. Photomicrographs of sagittal sections through the main alar hypothalamic subdivisions of Xenopus at different developmental stages. The boundaries are depicted on the basis of the single expression for xShh and Nkx2.1 (a,b) and the combined expressions of xDll4 and Otp (c), xDll4 and Nkx2.2 (d), Nkx2.1 and Nkx2.1 (e), xLhx1 and Otp (f), Isl1 and Otp (g), and Trbr1 and Isl1 (h). The boundaries identified are the PO-SPV (a-c), SPV-SC (d,e), SPV-PTh (c,f), and SPV-PThE (h). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the boundaries between the different alar 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. Scale bars: 50μm (a-f, h) and 200μm (g). This figure corresponds to the figure 4.
	Supplementary Figure 3. Photomicrographs of transverse sections through the developing Xenopus PO region showing the combined expressions of xShh and Nkx2.1 (a), Isl1 and Nkx2.1 (b-d), Nkx2.1 and TH (e), Isl1 and TH (f), Isl1 and GABA (g), Isl1 and Otp (h-j), xShh and Otp (k), and Nkx2.1 and Nkx2.2 (l). Developmental stages are indicated in each photograph. The arrowhead in g indicates double labeled Isl1 and GABA cells. The photomicrograph j shows a higher magnification of the area shown in i by confocal microscropy to illustrate the limit between the Isl1+ PO and the Otp+ SPV. Scale bars: 50μm (a-i, k, l) and 25μm (j). This figure corresponds to the figure 5.
	Supplementary Figure 4. Photomicrographs of sagittal sections through the developing Xenopus PO region showing the single expressions of Nkx2.1 (a) and xShh (b) and the combined expressions of Isl1 and GABA (c), Isl1 and Nkx2.1 (d,e), Isl1 and Otp (f), and xDll4 and Otp (g). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the combinatorial code of markers present in the PO and its adjacent domains in a lateral view (h) and a transverse section (i). Scale bars: 50μm (a-e,g) and 200μm (f). This figure corresponds to the figure 6.
	Supplementary Figure 5. Photomicrographs of transverse sections through the developing Xenopus SPV region showing the expressions of Otp (a), Nkx2.2 and Otp (b-f), Nkx2.2 and MST (e,f), Nkx2.2 and SOM (g), xShh and Otp (h), and Nkx2.2 and Nkx2.1 (i). Developmental stages are indicated in each photograph. The arrowhead in a indicates the Otp+ ventricular cells. The arrowheads in c' and d point to the double labeling for Nkx2.2 and Otp in the SPVr portion. Scale bars: 50μm (a-c, f-i), 100μm (d) and 200μm (e). This figure corresponds to the figure 7.
	Supplementary Figure 6. Photomicrographs of sagittal sections through the developing Xenopus SPV region showing the expressions of Nkx2.2 and Otp (a-c), Nkx2.2 and MST (d,e), Nkx2.2 and SOM (f,f'), xLhx5 (g), xDll4 and Otp (h), and Otp and Isl1 (i). Developmental stages are indicated in each photograph. The schematic representation at the bottom summarizes the combinatorial code of markers present in the SPV and its adjacent domains in a lateral view (j, j') and a transverse section (k). The yellow line in a indicates the level of the section shown in Fig.7b. The yellow box in f indicates the higher magnification shown in f' and the arrowhead in f' shows the coexpression of Nkx2.2 and SOM in SPVr. Scale bars: 100μm (a), 50μm (b,d-h), and 250μm (g). This figure corresponds to the figure 8.
	Supplementary Figure 7. Photomicrographs of transverse (a-d, h, m-o) and sagittal (e-i, i-n) sections through the developing Xenopus SC region showing the single expressions of xLhx7 (e), xLhx1 (f) and the combined expressions of xShh and Nkx2.1 (d), xDll4 and Isl1 (a), Isl1 and GABA (b), Isl1 and Nkx2.1 (c), xLhx7 and xDll4 (g), Nkx2.1 and Nkx2.2 (h-j), xLhx1 and Nkx2.2 (k), xShh and TH (l,m), Nkx2.1 and TH (n) and Nkx2.2 and TH (o). Developmental stages are indicated in each photograph. The arrowhead in b indicates a cell double-labeled for Isl1 and GABA. Scale bars: 200μm (j), 50μm (a-i, k-o). This figure corresponds to the figure 9.
	Supplementary Figure 8. Photomicrographs of transverse (a,e-k) and sagittal (b-d) sections through the SC territory in late prometamorphic and juvenile Xenopus showing the single expressions of Nkx2.1 (b), Isl1 (b), xLhx1 (g) and xLhx7 (h) and the combined expressions of Isl1 and Nkx2.1 (a,d-f), Isl1 and Otp (i), Isl1 and GABA (j) and Isl1 and TH (k). Developmental stages are indicated in each photograph. The arrowhead in k indicates a cell double-labeled for Isl1 and TH. Scale bars: 100 μm. This figure corresponds to the figure 11.
	Supplementary Figure 9. Photomicrographs of transverse sections showing the retrogradely labeled cells and anterogradely labeled fibers after BDA injection in the PO of juvenile Xenopus, combined with the marker MST (d,e,g) to corroborate that the tracer was delivered into this particular area. Scale bars: 250 μm (a,e,i), 200 μm (b,d,f,g,h), 100 μm (c,j). This figure corresponds to the figure 13.
	Supplementary Figure 10. Photomicrographs of sagittal sections showing anterograde and retrograde labeling after BDA injection in the SPV (a-h) and the SC (h-n) of juvenile Xenopus, combined with the markers Otp (a-c,e-g, h'-j), TH (d,n) and Isl1 (h-j) to corroborate to corroborate that the tracer was delivered into the selected area. The dashed box in h and j indicate the higher magnification shown in h' and j', respectively. The arrowheads point to retrogradely labeled cells, with the exception of the figures e, j' and n that indicate labeled fibers toward the hypophysis. In all photographs, the olfactory bulb is to the right and the spinal cord to the left. Scale bars: 200 μm (a-e, i, g, h'), 500μm (f, h, i-n). This figure corresponds to the figure 14.
	Supplementary Figure 11. Schemes in sagittal view summarizing the main afferent and efferent connections of the PO, SPV and SC regions in Xenopus. This figure corresponds to the figure 16.