Production and Application of Polyclonal Antibody to Human Thyroid Transcription Factor 2 Reveals Thyroid Transcription Factor 2 Protein Expression in Adult Thyroid and Hair Follicles and Prepubertal Testis

Abstract

Germline mutations in thyroid transcription factor 2 (TTF2) cause thyroid agenesis, spiky hair, and cleft palate, indicating thyroidal and extrathyroidal expression. We sought to investigate this by producing and applying an antibody to human TTF2. The coding region of human TTF2 was cloned into a bacterial expression vector, production of the soluble TTF2 protein optimized, and pure TTF2 obtained by nickel chromatography. Rabbits were immunized and the resulting TTF2 polyclonal titrated on formalin-fixed, paraffin-embedded sections of thyroid. The optimized protocol was applied to a range of tissues. Nine milligrams of TTF2 protein was obtained per liter of culture and a high-titer antibody produced. This displayed specific staining of thyroid follicular cell nuclei/cytoplasm and not of the interstitium, connective tissue, smooth muscle, or endothelium. No staining was obtained with the preimmune serum in the same conditions, or with the majority of other tissues tested with the TTF2 polyclonal. The exceptions were testis and skin, in which nuclear TTF2 immunoreactivity was present in the seminiferous tubules and cells in the follicular outer root sheath, respectively. In conclusion, we have produced a polyclonal antibody for human TTF2 and demonstrated immunoreactivity for this transcription factor in adult human thyroid and hair follicles and prepubertal testis.

Diagnostic utility of thyroid transcription factors Pax8 and TTF-2 (FoxE1) in thyroid epithelial neoplasms

“…23 The importance of these three transcription factors is evidenced by congenital thyroid agenesis, which results from mutations in any of the transcription factors. [26][27][28][29][30][31][32] We found consistent nuclear expressions of Pax8, TTF-1, and TTF-2 in well-differentiated neoplasms of follicular cell origin, that is, papillary carcinomas, follicular adenomas, follicular carcinomas, and poorly differentiated carcinomas. Seventy-nine percent of anaplastic carcinomas variably showed Pax8 reaction, whereas TTF-1 and TTF2 were expressed in only 18 and 7% of anaplastic carcinomas, respectively.…”

Application of Immunohistochemistry in Thyroid Pathology

“…15,70 Thyroid transcrip- tion factor 2 expression is restricted to the thyroid; anterior pituitary; epithelia of the oropharynx, trachea, and esophagus; exocrine cells of the seminiferous tubules of the testis; and epidermis and hair follicles. 12,[71][72][73][74][75][76][77][78] Thyroid transcription factor 2 is 1 of 3 TTFs that play critical roles in thyroid organogenesis and differentiation. However, TTF2 seems to play a role in the development of the negative controller of thyroid-specific gene expression.…”

Anatomy and Development of the Thyroid

“…151 In humans, FOXE1 is expressed in the thyroid, foregut embryonic thymus, 49 in the outer follicular hair sheath, and in the seminiferous tubules of prepubertal testis. 155 Studies in mutant mice have shown that Foxe1 is tightly regulated in the thyroid bud by Pax8 and in the pharyngeal cells by Shh. 50 Notably, in keratinocytes Fox1 is a direct target of Gli2, a key mediator of Shh signaling.…”