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???displayArticle.abstract??? Thyroid hormone (T(3)) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T(3) receptors (TR) of which there are two subtypes, TRalpha and TRbeta. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRalpha and TRbeta in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpolebrain increased dramatically during metamorphosis. This increase was dependent on T(3) until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T(3). Using double labeling fluorescent histochemistry with confocal microscopy we found TRalpha expressed throughout the tadpolebrain, with strongest expression in proliferating cells. By contrast, TRbeta was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpolebrain with a Xenopus TRbeta promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRalpha selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRbeta-selective agonists GC1 or GC24 did not. Our findings support the view that T(3) acts to induce cell proliferation in the tadpolebrain predominantly, if not exclusively, via TRalpha.
Fig. 2. Thyroid hormone induces cell proliferation in the tadpolebrain by shortening cell cycle time. (A) T3 induces cell proliferation in the premetamorphic tadpole (NF stage 52) brain. Tadpoles were treated as described for Experiment 1 (see Fig. 1) and representative sagittal brain sections are shown stained for BrdU. Abbreviations: LV — lateralventricle, IIIv — third ventricle, tect — optic tectum, Cb — cerebellum. Scale bar = 200 μm. (B) T3 increases cell proliferation as evidenced by an increased number of phosphorylated histone 3 (pH3) positive cells. Tadpoles were treated as described for Experiment 1 and representative sagittal brain sections in the region of the optic tectum are shown stained for pH3. Scale bar = 50 μm. (C) Quantitation of the number of pH3 positive cells throughout the brain of tadpoles treated with or without T3 (50 nM) for 24 or 48 h (n = 5/treatment). Transverse sections were prepared and the number of pH3-positive nuclei were counted for each brain. The asterisk indicates statistically significant difference from controls (P < 0.001; unpaired t-test). (D) T3 shortens cell cycle time in early prometamorphic tadpolebrain. Tadpoles were treated as described for Experiment 2. Transverse brain sections were stained for BrdU. There were no differences in BrdU-ir between brains from control (panel 1) or T3 treated (panel 2) animals 24 h after treatment. However, after 48 h there was a significant increase in the number of BrdU-ir cells in brains of T3-treated animals (panel 4) compared to controls (panel 3). T3-treated animals exhibited more diffuse staining owing to the dilution of the BrdU by cell division. Many BrdU-ir cells seen in panel 4 exhibited elongate nuclei, suggesting that the newborn cells were migrating out of the proliferative zone. Scale bar = 50 μm.
Fig. 3. (A) Profiles of brain cell proliferation during spontaneous metamorphosis and following T3 treatment, measured by the number of BrdU-positive cells. Tadpoles were treated as described for Experiment 3 (see Fig. 1). During spontaneous metamorphosis (Control) cell proliferation was low at premetamorphic (Premet) stages, peaked during prometamorphosis (Promet; NF stage 56–57) and declined at metamorphic climax (Climax; NF stage 62–63). T3 treatment significantly increased cell proliferation at premetamorphic and early prometamorphic stages but failed to alter cell proliferation at later stages. (B) Developmental changes in BrdU labeling in the tadpole optic tectum. There were numerous BrdU-ir cells at prometamorphosis (NF stage 56; b); whereas, there were few BrdU-ir cells in premetamorphic (NF stage 52; a) or late prometamorphic (NF stage 62; c) brains. tect, optic tectum; V, ventricle. Scale bar = 50 μm.
Fig. 5. (A–L) Schematic coronal illustration of neurogenic zones (gray bands surrounding ventricles on left of sections; identified by the distribution of BrDU and pH3 immunoreactivity) and thyroid hormone receptor β (TRβ) immunoreactivity (-ir; large and small dots on right of sections) distribution in the brain of NF stage 52 X. laevis tadpoles following 48 h treatment with T3 (50 nM). TRα mRNA, which is not shown in the figure, is highly expressed in all neurogenic zones and many other areas throughout the tadpolebrain. The drawing at the top of the figure shows a dorsal view of the X. laevis brain. Letters correspond to the rostrocaudal location of sections as depicted in the whole-brain drawing. Large circles represent large cells that exhibited robust TRβ-ir, and small circles represent smaller TRβ-ir cells. The anatomical drawings are from Tuinhof et al. (1998), with modifications of basal ganglia subdivisions according to (Marín et al., 1998). Abbreviations are defined in Table 2.
Fig. 6. (A) Western blots testing the specificity of antisera raised against synthetic peptides corresponding to xTRα (top gel; 1:1000) and xTRβ (bottom gel; 1:1000). Polyacrylamide gels (10 %) were loaded with lysates of bacteria expressing xTRα (lanes 1,2,3: 5, 2.5, 1.25 μl induced lysate; lane 4, 5 μl uninduced lysate) or xTRβ (lanes 5,6,7: 5, 2.5, 1.25 μl induced lysate; lane 8, 5 μl uninduced lysate). Note that the anti-xTRα serum crossreacts with xTRβ (arrows showing two immunoreactive bands in both lysates) and was not used for further study; whereas, the anti-xTRβ serum was specific for xTRβ and was used for IHC. (B) Representative photomicrographs of transverse sections through the brain of a NF stage 52 tadpole that had been treated with T3 (50 nM) for 48 h showing the distribution of TRβ immunoreactive (TRβ-ir) cells. 1. Dorsal pallium (dp), medialpallium (mp) and lateralpallium (lp), and internal granule cell layer (igl; Fig. 5, panel B). 2. Dorsal, medial and lateralpallium, striatum (Str) and accumbens (Acc; Fig. 5, panel C). 3. Dorsal, medial and lateralpallium, lateralamygdala (LA), medialamygdala (MeA) and central amygdala (CeA; Fig. 5, panel D). 4. Anterior preoptic area (Fig. 5, panel D). 5. Ventral hypothalamic area (Fig. 5, panel G). 6. Optic tectum (Fig. 5, panel J). Scale bar = 200 μm.
Fig. 7. (A) Thyroid hormone receptor alpha (TRα) mRNA colocalizes with BrdU-ir in tadpolebrain. Premetamorphic (NF stage 52) tadpoles were treated as described for Experiment 3 (see Fig. 1). Dual labeling histochemistry was conducted for TRα mRNA (red; by ISHH) and BrdU (green; by IHC) on sagittal sections of T3-treated tadpolebrain (regions shown are the diencephalon and cerebellum). Sections were analyzed by confocal microscopy. Note the colocalization of TRα mRNA and BrdU-ir in cords of cells adjacent to the ventricles. Hypo, hypothalamus; Pit, pituitary. Scale bar = 50 μm. (B) Treatment with T3 increases TRβ immunoreactivity (ir) in tadpolebrain, and TRβ is excluded from the majority of BrdU-positive cells. Premetamorphic tadpoles (NF stage 52) were treated as described for Experiment 3 (see Fig. 1). Dual fluorescent immunohistochemistry was conducted for BrdU (green) and TRβ (red) without (1–3) or with (4–6) T3 treatment. Sections were analyzed by confocal microscopy. Shown is the region of the thalamus and optic tectum in the sagittal plane. In untreated tadpoles few BrdU-ir or TR-ir cells are detected (1 — TRβ, 2 — BrdU, 3 — merge), but T3 treatment significantly increased TRβ-ir (4 — TRβ) and cell proliferation (5 — BrdU). Only a small proportion of BrdU-ir cells express TRβ, as indicated by the yellow cells (6 — merge). Scale bar = 50 μm.
Fig. 8. Distinct populations of cells respond to T3 by proliferating (as evidenced by BrdU incorporation — green) or by upregulating the TrβA promoter (as evidenced by EGFP expression — red). Premetamorphic tadpoles (NF stage 52) were transfected with a X. laevis TrβA promoter-EGFP reporter plasmid by electroporation-mediated gene transfer, treated with T3 (50 nM) for 48 h, and then immersed in BrdU for 3 h before sacrifice and harvest of brains for dual labeling fluorescent immunohistochemistry for EGFP and BrdU. Transverse sections through the lateral ventricles were prepared and analyzed by confocal microscopy. 1. A majority of EGFP-positive cells (red — with white arrows) were BrdU negative. The yellow arrow shows one cell with colocalization of EGFP and BrdU. Scale bar = 100 μm. 2. High magnification image showing that most EGFP-positive cells are located at the periphery of the subventricular zone (SVZ), with few in the ventricular zone (VZ). Scale bar = 50 μm. 3. Some EGFP expressing cells exhibited long processes that extended out to the pial surface characteristic of radial glia. Scale bar = 200 μm. 4. Most radial glial cells expressing EGFP were BrdU positive (yellow arrows) with cell bodies located in the SVZ. Scale bar = 50 μm.
Fig. 9. The TRα selective agonist CO23, but not the TRβ selective agonists GC1 and GC24, increases cell proliferation in the premetamorphic tadpolebrain. (A) Quantification of the number of pH3-positive nuclei in the region of the lateral ventricles in tadpoles treated with vehicle (DMSO; control), T3 (10 nM), GC1 (100 nM), GC24 (100 nM) or CO23 (100 nM) for 48 h. Compounds were added to the aquarium water and were replenished after 24 h. The length of both lateral ventricles from each animal was analyzed. Shown are the data from one of two experiments that gave identical results. (P < 0.05; ANOVA; n = 4/treatment). (B) The GC compounds caused external metamorphic changes, thus showing that they had biological activity. Shown are representative tadpoles treated with vehicle (DMSO; control), GC1 or GC24 (100 nM each) for 6 days (all of the T3 treated tadpoles had died by 4 days and thus are not shown; CO23 animals were not analyzed — the effects on brain cell proliferation verified the biological activity of the CO23). The arrows point to the gills, which showed resorption following treatment with GC1 and GC24. Differentiation of the hindlimb digits was observed with both GC1 and GC24 treatment but not in controls (n = 6/treatment).
thrb (thyroid hormone receptor, beta) gene expression in Xenopus laevis embryos, NF stage 52, as assayed by immunohistochemistry, sagittal section, anteriorleft, dorsal up.
