Morona R and González A (2013), Pattern of calbindin-D28k and calretinin immuno...

XB-ART-45411

J Comp Neurol 2013 Jan 01;5211:79-108. doi: 10.1002/cne.23163.

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Pattern of calbindin-D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development.

Morona R , González A .

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The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions.

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Species referenced: Xenopus laevis
Genes referenced: calb1 calb2 chat nos1 nos3 tff3.7 th

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	Figure 1. Schematic representation of the general distribution of calbindin (empty dots) and calretinin (filled dots) immunoreactive cells in a sagittal views of the brain at representative early embryonic (a) late embryonic (b) premetamorphic (c) and prometamorphic (d) stages. In all schemes dorsal is oriented upward and rostral to the left. The main subdivisions with regard to the neuromeric models are indicated. In each scheme the dashed lines represent the levels of the sections shown in the microphotographs in subsequent figures. Levels indicated in a and b correspond to photographs in Figure 2, levels indicated in c correspond to photographs in Figure 3, and levels indicated in d correspond to photographs in Figure 4. For abbreviations, see list.
	Figure 2. Photomicrographs of double-labeled CB/CR sagittal (a, dorsal is upwards and rostral to the left) and transverse (b) sections through representative levels of the brain of Xenopus at embryonic stages (indicated on each photomicrograph). The levels of transverse sections are indicated in Figure 1a,b. In all photographs green is CB and red is CR, whereas yellow represents double-labeled cells. Dashed lines mark the approximate boundary between neuromeres and telencephalic subdivisions. Arrowheads in f point to the lateral CRir fiber tract in p2, in hours, and in s point to the lateral CRir fiber tract in the basal plate, and in q to double-labeled cells in the LDT. The asterisks in r and t mark the CBir fibers in the lateral rhombencephalon. For abbreviations, see list. A magenta-green version of this figure is provided as Supporting Information Figure 1. Scale bars = 50 μm in a; 100 μm in i.
	Figure 3. Photomicrographs of double-labeled CB/CR sagittal (a,k,l; dorsal is upwards and rostral to the left) and transverse (b) sections through representative levels of the brain of Xenopus at premetamorphic stages (indicated on each photomicrograph). The levels of transverse sections for stage 50, and the area shown in a, are indicated in Figure 1c. In all photographs green is CB and red is CR, whereas yellow represents double-labeled cells. Dashed lines represent the approximate boundary between telencephalic (e,f) or diencephalic (a,b,h,k,l) subdivisions, the mesencephalic longitudinal bands (c,g,k), and rhombencephalic regions (d,i,j). For abbreviations, see list. A magenta-green version of this figure is provided as Supporting Information Figure 2. Scales bar = 100 μm in a,l; 200 μm in k.
	Figure 4. Photomicrographs of double-labeled CB/CR transverse (a) and sagittal (i; dorsal is upwards and rostral to the left) sections through representative levels of the brain of Xenopus at prometamorphic stages (indicated on each photomicrograph). The levels of transverse sections are indicated in Figure 1d. In all photographs green is CB and red is CR, whereas yellow represents double-labeled cells. Dashed lines represent the approximate boundary between telencephalic (a,i), diencephalic (e,i), mesencephalic (g,h), and rhombencephalic (i) subdivisions. i: Low magnification image of a lateral sagittal section showing the relationship between CB and CR labeled structures throughout the brain of a prometamorphic larva. j,k: Details of sagittal sections through the hypothalamic (j) and pretectal (k) region showing the relative localization of CB- and CR-labeled structures in each subdivision. Inset in g′ shows trigeminal mesencephalic neurons (arrowheads) in the rostral optic tectum. For abbreviations, see list. A magenta-green version of this figure is provided as Supporting Information Figure 3. Scale bars = 100 μm in a,k; 200 μm in i,j.
	Figure 5. Photomicrographs of double-labeled sagittal (a; dorsal is upwards and rostral to the left) and horizontal (j; rostral is to the left) sections through the brain of Xenopus at different developmental stages showing the distribution of CB or CR immunoreactive cells with nitric oxide synthase (NOS), tyrosine hydroxylase (TH), or choline acetyltransferase (ChAT). The markers shown in each panel are indicated in appropriate color code, and the developmental stage is indicated in the upper right corner. a: Relative localization of nitrergic (a) catecholaminergic (b) and cholinergic (c) populations compared to CR at stage 45. d: Colocalization of CR in the large nitrergic cells of the central amygdala (double-labeled cells in yellow). e: Distribution of CR in relation to the catecholaminergic cells in the preoptic and suprachiasmatic regions. f: Segmental distribution of the alar CRir cell groups in relation to the basal catecholaminergic cell groups, g: CRir and NOSir cells in the distinct dorsal and lateral bands in the mesencephalon and along the rhombencephalon. h: High magnification of double CR/NOS-labeled cells in the inferior reticular nucleus. i: CBir cells in the alar region of r0 and r1 in relation to the cholinergic nuclei. Arrowhead points to the fibers of the trochlear nucleus in the basal plate (not shown). j: Horizontal section showing the relative position and the segmental arrangement of CR groups in relation to the ChATir motor and nonmotor cholinergic groups in the rhombencephalon. k: Relative localization CBir cells in relation to the oculomotor, isthmic, rhombencephalic, and spinal cholinergic cell groups. Double-labeled cells always appear in yellow. n: CB and NOS-stained structures in a premetamorphic larva in the mesencephalon and rostral rhombencephalon (n) and in prometamorphic larvae in the caudal telencephalon (o) and mesencephalon and rostral rhombencephalon (p). Double-labeled CB/NOSir cells (in yellow) in the basal band of the mesencephalon and in the LDT at stage 50 were not observed at stage 60. For abbreviations, see list. Arrows point to double-labeled cells. A magenta-green version of this figure is provided as Supporting Information Figure 4. Scale bar = 50 μm in a,b,d,h,k; 100 μm in c,g,i,j.
	Supporting Figure 1 (Magentahyphen;green version of Figure 2 for the assistance of colorhyphen;blind readers): Photomicrographs of doublehyphen;labeled CB/CR sagittal (a, dorsal is upwards and rostral to the left) and transverse (bhyphen;t) sections through representative levels of the brain of Xenopus at embryonic stages (indicated on each photomicrograph).The levels of transverse sections are indicated in Figure 1a and b. In all photographs green is CB and red is CR, whereas yellow represents double labeled cells. Dashed lines mark the approximate boundary between neuromeres and telencephalic subdivisions. Arrowheads in f point to the lateral CRir fiber tract in p2, in h and s point to the lateral CRir fiber tract in the basal plate, and in q to doble labeled cells in the LDT. The asterisks in r and t mark the CBir fibers in the lateral rhombencephalon. For abbreviations, see list. A magentahyphen;green version of this figure is provided as Supporting Information Figure 1. Scale bar = 50 μm in ahyphen;h; 100 μm in ihyphen;t.
	Supporting Figure 2 (Magentahyphen;green version of Figure 3 for the assistance of colorhyphen;blind readers): Photomicrographs of doublehyphen;labeled CB/CR sagittal (a,k,l; dorsal is upwards and rostral to the left) and transverse (bhyphen;j) sections through representative levels of the brain of Xenopus at premetamorphic stages (indicated on each photomicrograph). The levels of transverse sections for stage 50, and the area shown in a, are indicated in Figure 1c. In all photographs green is CB and red is CR, whereas yellow represents double labeled cells. Dashed lines represent the approximate boundary between telencephalic (e,f) or diencephalic (a,b,h,k,l) subdivisions, the mesencephalic longitudinal bands (c,g,k) and rhombencephalic regions (d,i,j). For abbreviations, see list. A magentahyphen;green version of this figure is provided as Supporting Information Figure 2. Scale bar =100 μm in ahyphen;j,l; 200 μm in k.
	Supporting Figure 3 (Magentahyphen;green version of Figure 4 for the assistance of colorhyphen;blind readers): Photomicrographs of doublehyphen;labeled CB/CR transverse (ahyphen;h) and sagittal (ihyphen;k; dorsal is upwards and rostral to the left) sections through representative levels of the brain of Xenopus at prometamorphic stages (indicated on each photomicrograph). The levels of transverse sections are indicated in Figure 1d. In all photographs green is CB and red is CR, whereas yellow represents double labeled cells. Dashed lines represent the approximate boundary between telencephalic (ahyphen;d, i), diencephalic (ehyphen;g, ihyphen;k), mesencephalic (g,h) and rhombencephalic (i) subdivisions. i: Low magnification image of a lateral sagittal section showing the relationship between CB and CR labeled structures throughout the brain of a prometamorphic larva. j,k: Details of sagittal sections through the hypothalamic (j) and pretectal (k) region showing the relative localization of CBhyphen; and CRhyphen;labeled structures in each subdivision.. Inset in panel g' shows trigeminal mesencephalic neurons (arrowheads) in the rostral optic tectum. For abbreviations, see list. A magentahyphen;green version of this figure is provided as Supporting Information Figure 3. Scale bar =100 μm in ahyphen;h,k; 200 μm in i,j.
	Supporting Figure 4 (Magentahyphen;green version of Figure 5 for the assistance of colorhyphen;blind readers): Photomicrographs of doublehyphen;labeled sagittal (ahyphen;i; dorsal is upwards and rostral to the left) and horizontal (j; rostral is to the left) sections through the brain of Xenopus at different developmental stages showing the distribution of CB or CR immunoreactive cells with nitric oxide synthase (NOS), tyrosine hydroxylase (TH) or choline acelyltransferase (ChAT). The markers shown in each panel are indicated in apriate color code, and the developmental stage is indicated in the upper rigth corner. ahyphen;c: relative localization of nitrergic (a) catecholaminergic (b) and cholinergic (c) populations compared to CR at stage 45. d: Colocalization of CR in the large niterergic cells of the central amygdala (double labeled cells in yellow). e: Distribution of CR in relation to the catecholaminergic cells in the preoptic and suprachiasmatic regions. f: Segmental distribution of the alar CRir cell groups in relation to the basal catecholaminergic cell groups, g: CRir and NOSir cells in the distinct dorsal and lateral bands in the mesencephalon, and along the rhombencephalon. h: High magnification of double CR/NOS labeled cells in the inferior reticular nucleus. i: CBir cells in the alar region of r0 and r1 in relation to the cholinergic nuclei. Arrowhead points to the fibers of the troclear nucleus in the basal plate (not shown). j: Horizontal section showing the relative position and the segmental arrangement of CR groups in relation to the ChATir motor and nonhyphen;motor cholinergic groups in the rhombencephalon. khyphen;m: Relative localization CBir cells in relation to the oculomotor, isthmic, rhombencephalic and spinal cholinergic cell groups. Doublehyphen;labeled cells always appear in yellow. nhyphen;p: CB and NOShyphen;stained structures in a premetamorphic larva in the mesencephalon and rostral rhombencephalon (n) and in prometamorphic larvae in the caudal telencephalon (o) and mesencephalon and rostral rhombencephalon (p). Double labeled CB/NOSir cells (in yellow) in the basal band of the mesencephalon and in the LDT at stage 50 were not observed at stage 60. For abbreviations, see list. Arrows point to doble labeled cells. A magentahyphen;green version of this figure is provided as Supporting Information Figure 4. Scale bar = 50 μm in a,b,dhyphen;f,h, khyphen;p; 100 μm in c,g, i,j.