Immunofluorescence localization of thyroid hormone receptor protein β1 and variant α2 in selected tissues: Cerebellar Purkinje cells as a model for β1 receptor-mediated developmental effects of thyroid hormone in brain

Rat c-erbAβ1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T₃) levels and T₃-dependent brain development, these data suggest that the β1 receptor may mediate the T₃ effect. However, in cerebellum c-erbAβ1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T₃-sensitive process, we assessed the levels of the β1 receptor protein in cerebellum during development. Antisera to unique peptide regions of β1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T₃ binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-β1 antiserum stained liver nuclei but not testis nuclei, which contain no T₃ binding activity or β1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of β1 receptor protein despite the low β1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunopreeipitable β1 receptor binding capacity rose in the neonatal Period. Antiserum to the non-T₃ binding α2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The α2 fluorescence in testis was high, consistent with the high levels of α2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the β1 receptor plays a central role in T₃-induced brain development and strongly suggest that the Purkinje cell in a direct target for T₃.