Immunofluorescence localization of thyroid hormone receptor protein beta 1 and variant alpha 2 in selected tissues: cerebellar Purkinje cells as a model for beta 1 receptor-mediated developmental effects of thyroid hormone in brain.
Rat c-erbA beta 1 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 beta 1 receptor may mediate the T3 effect. However, in cerebellum c-erbA beta 1 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 beta 1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta 1 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-beta 1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta 1 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 beta 1 receptor protein despite the low beta 1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta 1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha 2 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 alpha 2 fluorescence in testis was high, consistent with the high levels of alpha 2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta 1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.
Strait
K A
KA
Department of Medicine, University of Minnesota, Minneapolis 55455.
Schwartz
H L
HL
Seybold
V S
VS
Ling
N C
NC
Oppenheimer
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000378:metabolism*; D011689:Purkinje Cells / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D010455:Peptides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine