3,5-T2 and 3,3',5-T3 Regulate Cerebellar Thyroid Hormone Signalling and Myelin Molecular Dynamics in Tilapia

Abstract

In contrast to mammalian adults, myelination in teleosts occurs throughout their lifespan and most of the progenitor cells are originated in the cerebellum. To understand the role that thyroid hormones (THs) play in juvenile cerebellar myelination in teleosts, we identified and localised the expression of genes involved in TH signalling (mct8, oatp1c1, dio2, dio3, thraa and l-thrb1) and analysed the effects of the two bioactive THs, T2 and T3, upon their regulation, as well as upon some structural components of the myelination process. Ex vivo approaches using organotypic cerebellar cultures followed by FISH and qPCR showed gene-specific localisation and regulation of TH signalling genes in the cerebellar nuclei. In vivo approaches using methimazole (MMI)-treated juvenile tilapias replaced with low doses of T3 and T2 showed by immunofluorescence that myelin fibres in the cerebellum are more abundant in the granular layer and that their visible size is reduced after MMI treatment but partially restored with TH replacement, suggesting that low doses of TH promote the re-myelination process in an altered condition. Together, our data support the idea that T2 and T3 promote myelination via different pathways and prompt T2 as a target for further analysis as a promising therapy for hypomyelination.

Figure 1

Fluorescent in situ hybridisation analysis. Ex vivo experiments for the quantification of mRNA expression in confocal images of sagittal sections of cerebellum in control and treated groups show the localisation of the probes for each gene. mRNA expression of the different genes was detected with Cy3 in red and the signal of DAPI in blue. The scale bar represented 50 μm. (a) Transporters of THs, (b) deiodinases, (c) receptors of THs (d) table showing the abundance [low (+), medium (++) or high (+++)] or absence (−) of expression in each structure that conforms the cerebellum. e) Quantification of total fluorescence normalised with DAPI. For all graphs * is p < 0.05 and (f). Photomicrographs of the same sections with Nissl staining showing in pointed lines the definition of the different nuclei that comprise the tilapia cerebellum. The zone of expression of each gene in the control groups is marked in colour stars.

Figure 2

Cerebellar mRNA expression of mbp, p0, plp1b, olig2, sox10 tnks and GlialCAM. Tilapia were exposed to 4.5 mM MMI with or without simultaneous addition of 1 nM T2, T3 or T2 + T3 for 30 days. Values are means +/− S.E.M. Significance is indicated p < 0.05 with respect to control group.

Figure 3

Myelin distribution and composition in tilapia cerebella. (a,b) Coronal sections of cerebella (5 μm) in Kluver-Barrera staining. (c) Coronal sections of cerebella (50 μm) in black-gold staining. Bundles of myelin fibres pass through the granular layer. (d,e) Confocal microscopy of 20 μm coronal sections of the cerebellum of a control animal, immunostained to myelin basic protein (red) and counterstained with DAPI (blue). Note that stained myelin proteins and fibres are more abundant in the granular layer. (f) Frontal view of the reconstruction of 30 confocal sections showing the merge of red for myelin and blue for DAPI. (g) Frontal view of 30 confocal sections for a 3D reconstruction showing only the red channel for myelin. (h) Lateral view of a 3D reconstruction showing myelin fibres. (i) Zoom of myelin fibres in a frontal view. 3D reconstruction and measurement of myelin fibre diameters were performed with Amira software. A total of 9 images (3D reconstructions) within the granular layer from 3 different cerebella per group were analysed. (j) Myelin fibre apparent diameters of tilapia exposed to 4.5 mM MMI with or without simultaneous addition of 1 nM T2, T3 or T2 + T3 for 30 days. Values are means +/− S.E.M of ~700 single measurements per group. Significance is indicated as p = 0.001 compared to the control (a) or MMI-treated group (b) ared to the control (a) or MMI-treated group (b).