Daume D et al. (2022), Patterns of tubb2b Promoter-Driven Fluorescence...

XB-ART-59235

Front Neuroanat 2022 Jan 01;16:914281. doi: 10.3389/fnana.2022.914281.

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Patterns of tubb2b Promoter-Driven Fluorescence in the Forebrain of Larval Xenopus laevis.

Daume D , Offner T , Hassenklöver T , Manzini I .

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Microtubules are essential components of the cytoskeleton of all eukaryotic cells and consist of α- and β-tubulin heterodimers. Several tissue-specific isotypes of α- and β-tubulins, encoded by distinct genes, have been described in vertebrates. In the African clawed frog (Xenopus laevis), class II β-tubulin (tubb2b) is expressed exclusively in neurons, and its promoter is used to establish different transgenic frog lines. However, a thorough investigation of the expression pattern of tubb2b has not been carried out yet. In this study, we describe the expression of tubb2b-dependent Katushka fluorescence in the forebrain of premetamorphic Xenopus laevis at cellular resolution. To determine the exact location of Katushka-positive neurons in the forebrain nuclei and to verify the extent of neuronal Katushka expression, we used a transgenic frog line and performed several additional antibody stainings. We found tubb2b-dependent fluorescence throughout the Xenopus forebrain, but not in all neurons. In the olfactory bulb, tubb2b-dependent fluorescence is present in axonal projections from the olfactory epithelium, cells in the mitral cell layer, and fibers of the extrabulbar system, but not in interneurons. We also detected tubb2b-dependent fluorescence in parts of the basal ganglia, the amygdaloid complex, the pallium, the optic nerve, the preoptic area, and the hypothalamus. In the diencephalon, tubb2b-dependent fluorescence occurred mainly in the prethalamus and thalamus. As in the olfactory system, not all neurons of these forebrain regions exhibited tubb2b-dependent fluorescence. Together, our results present a detailed overview of the distribution of tubb2b-dependent fluorescence in neurons of the forebrain of larval Xenopus laevis and clearly show that tubb2b-dependent fluorescence cannot be used as a pan-neuronal marker.

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Species referenced: Xenopus laevis
Genes referenced: calb2 crygdl.43 fh pax6 pax7 tubb2b
GO keywords: forebrain neuron differentiation [+]
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	FIGURE 1. tubb2b-dependent fluorescence within the MOE and OB of larval Xenopus laevis. (A) Dorsal view of a single focal plane of the left main olfactory epithelium (MOE). tubb2b-dependent fluorescence (magenta) is present in olfactory receptor neurons. (B) Close-up of tubb2b-positive olfactory receptor neurons of the MOE. (C) Schematic overview of the olfactory organs (magenta). Close-up of the left MOE. (D) Coronal slice of the OB (left and right) of a transgenic NBT-Katushka γ-cry-Venus tadpole. tubb2b-dependent fluorescence (magenta) is present in axons of ORNs terminating in all known axonal projection fields of the OB (projection fields 1–9 and projection fields α, β, γ, and δ; see Gaudin and Gascuel, 2005). (E) Coronal slice of the OB (one side only) of transgenic NBT-Katushka γ-cry-Venus tadpoles and tubb2b-dependent fluorescence in cells of the mitral cell layer of the main and accessory (projection field 8) olfactory bulb. (F–H) Coronal slices of the OB (one side only) of transgenic NBT-Katushka pax6-GFP tadpoles. (F) tubb2b- (magenta) and pax6-dependent fluorescence (green) in cells in the mitral cell layer. (G,H) Close-ups of the mitral cell layer of the main and accessory OB showing tubb2b-dependent fluorescence and pax6-dependent fluorescence. Examples of cells featuring tubb2b-dependent fluorescence only and pax6-dependent fluorescence only, and double-fluorescent cells (*) are encircled. (I–K) Maximum projection of the whole ventral OB of one brain hemisphere. (I) tubb2b-dependent fluorescence has not been detected in calretinin (CR; green)-positive cells. tubb2b-positive fibers that bypass the OB were found in the intermediate OB (white arrow). (J) tubb2b-dependent fluorescence has not been detected in tyrosine hydroxylase (TH; green)-positive cells. (K) tubb2b-dependent fluorescence and pax6-dependent fluorescence have been detected in cells of the mitral cell layer. tubb2b-dependent fluorescence was also found in the nucleus accumbens (Acc) and pax6-dependent fluorescence in the anterior part of the striatum (Str). (L) Scheme of the larval brain showing the approximate positions (lines) of the sections shown in (A–L). POA, preoptic area; Hyp, hypothalamus; NC, nasal cavity; ON, olfactory nerve; mcl, mitral cell layer; gcl, granule cell layer; V, ventricle; d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral.
	FIGURE 2. Schematic overview of regions featuring tubb2b-dependent fluorescence in the forebrain of premetamorphic Xenopus laevis. Ventral view of the larval forebrain and transverse sections of the olfactory bulb (OB; A–C), telencephalon (Tel; A–F), diencephalon (Di), and hypothalamus (G). The position and density of the cells shown for each region are presented based on observation and not via quantitative analysis. (A,B) tubb2b-dependent fluorescence is present in projection fields of axons arriving from the olfactory epithelium (1–9 + α, β, γ, δ; Gaudin and Gascuel, 2005). (C,D) tubb2b-dependent fluorescence in cells of the mitral cell layer of the main and accessory olfactory bulbs, the nucleus accumbens (Acc), and pallium. (E) tubb2b-dependent fluorescence in the medial pallium (Mp), lateral pallium (Lp), dorsal pallium (Dp), ventral pallium (Vp), striatum (Str), and septum (S). (F) tubb2b-dependent fluorescence in the anterior commissure (ac), medial amygdala (MeA), optic nerve (OpN), dorsal pallidum (DP), and preoptic area (POA). (G) tubb2b-dependent fluorescence in prosomeres 1–3 (p1–3). Hyp, Hypothalamus; lfb, lateral forebrain bundle; V, ventricle; MOE, main olfactory epithelium; ON, olfactory nerve; a, anterior; p, posterior; d, dorsal; v, ventral; l, lateral; m, medial.
	FIGURE 3. Cell density of tubb2b-positive neurons in the OB of premetamorphic Xenopus larvae. (A–C) Ventral view of a single focal plane of the OB of one brain hemisphere. (A) HuC/D-positive cells (green) are present throughout the whole OB. Few HuC/D-positive cells are also tubb2b-positive (magenta). (B) Pattern of HuC/D-positive cells within the OB. (C) tubb2b-positive cells are located in the mitral cell layer (mcl) of the OB. (D) Number of tubb2b-positive cells in comparison to HuC/D-positive cells of the whole OB. Average number of tubb2b-positive cells: 1,588 ± 191 (n = 10). Average number of HuC/D-positive cells: 6,517 ± 210 (n = 3). Cell density of tubb2b-positive cells compared to HuC/D-positive cells: 24,4%. (E) Scheme of the ventral OB showing the position of the images (A–C). ON, olfactory nerve; a, anterior; p, posterior; m, medial; l, lateral.
	FIGURE 4. tubb2b-dependent fluorescence in the telencephalon of larval Xenopus laevis. Coronal sections through the telencephalon of NBT-Katushka γ-cry-Venus (A,E,F) and NBT-Katushka pax6-GFP (B–D,G,H) transgenic tadpoles. (A) tubb2b-dependent fluorescence (magenta) in the anterior telencephalon. Stained cells could be detected in the nucleus accumbens (Acc), striatum (Str), accessory OB (projection field 8), and pallium. (B–D) Close-ups of various zones of the anterior telencephalon showing tubb2b-dependent fluorescence in cells of the dorsal pallium (Dp), ventral pallium (Vp), medial pallium (Mp), lateral pallium (Lp), septum (S), and medial amygdala (MeA). (E) Sections through the posterior telencephalon showing tubb2b-dependent fluorescence in cells of the Mp, Lp, Vp, MeA, dorsal pallidum (DP), preoptic area (POA), and optic nerve (OpN). (F–H) Close-ups of various zones of the ventral posterior telencephalon. (F) tubb2b-dependent fluorescence in cells of the POA. (G,H) No overlap was detected between tubb2b- and pax6-dependent fluorescence (green) in the POA, ac, and central amygdala (CeA). (I) Scheme of the larval brain showing the approximate positions (dashed rectangles) of the sections shown in (A–H). Hyp, hypothalamus; OB, olfactory bulb; BST, bed nucleus of the stria terminalis; V, ventricle; d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral.
	FIGURE 5. Location of tubb2b-dependent fluorescence in the diencephalon of larval Xenopus laevis. Coronal sections through the diencephalon of NBT-Katushka γ-cry-Venus transgenic tadpoles. (A) tubb2b-dependent fluorescence (magenta) in cells of prosomeres 1–3 (p1–3). (B) No tubb2b-dependent fluorescence could be detected in calretinin-positive cell bodies and fibers (CR; green) of the thalamus (Th). (C) tubb2b-dependent fluorescence-positive cell bodies in the area of the Th. CR-positive cells and fibers were not tubb2b-positive. (D) Cells in prosomere 3 (p3) did not show tubb2b-dependent fluorescence but could be stained with an antibody against Pax7 (green). (E) Approximate positions of the sliced regions are shown in a schematic image of the larval brain (lines). OB, olfactory bulb; Hyp, hypothalamus; POA, preoptic area; V, ventricle; d, dorsal; v, ventral; a, anterior; p, posterior; m, medial; l, lateral.

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