Dual function model revised by thyroid hormone receptor alpha knockout frogs

Abstract

All vertebrates require thyroid hormone (TH) for normal growth and development. Plasma TH enters cells and alters gene expression via nuclear receptors TRα and TRβ. In-vitro studies showed that TRs function as repressors of TH-inducible genes in the absence of TH and as activators of those same genes in the presence of TH. A dual function model was proposed to harmonize these molecular TR actions with the dynamic expression of TRs and peak in production of TH experienced during development. Conclusive tests of the repression activity of TRs early in development as predicted by the model awaited gene knockout technology targeting TRα. At the molecular level, active repression of genes involved in metamorphosis by TRα in the absence of TH was confirmed in whole bodies and intestine from TRα knockout studies. As a consequence of this reduced repression in TRα knockout animals, initiation of limb morphogenesis occurs precociously. However, subsequent limb development is retarded during rising plasma TH levels due to reduced TR-dependent responsivity to TH. In contrast to the limbs, intestine remodeling is delayed by one to two developmental stages in TRα knockout animals, despite de-repressed levels of TH-induced genes during premetamorphosis. Surprisingly, in the absence of TRα, hind limbs do not require gene induction by TH signaling to complete morphological growth and development, which is contrary to prediction by the dual function model. Full evaluation of the dual function model for all organs awaits the production of TRα and TRβ double knockout frogs.

Keywords: Gene knockout; Metamorphosis; Tadpole; Xenopus tropicalis.

Figure 1

Diagrammatic representation of overall growth and development throughout the larval period and metamorphosis in wild-type and TRαKO tadpoles. A) TRαKO tadpoles (KO, dashed lines) initially grow faster than wild-type tadpoles (WT, solid lines) during premetamorphosis, but during the remainder of the larval period from the initiation of metamorphosis through to tail resorption, significant but small size differences are observed. B) TRαKO animals initially develop faster during premetamorphosis (< stage 54) (Nieuwkoop and Faber, 1994) than wild-type tadpoles based on hind limb criteria. Then, during prometamorphosis (stages 54 - 58) when TH levels begin to increase, TRαKO animals progress more slowly than wild-type tadpoles based on hind limb criteria. Then, during climax of metamorphosis (stages 58 - 66), when TH levels reach their peak, TRαKO and wild-type tadpoles develop at a similar rate. The vertical dotted lines from left to right in both graphs indicate initiation of feeding (stage 45), initiation of metamorphosis (stage 54), mid-point of plasma TH levels during metamorphosis (stage 58), and tail resorption (stage 66).

Figure 2

Mutations in TRα result in precocious initiation of hind limb morphogenesis. A) Injection of TALEN mRNAs targeting the DNA-binding domain of TRα was performed in one cell of a two-cell stage embryo of Xenopus tropicalis. mCherry mRNA was co-injected as a red fluorescent tracer to identify which side of the embryo (left in this case) was injected with TRα TALENs. B) Hind limbs of the resulting tadpole during premetamorphosis were examined for developmental progress on the injected and uninjected sides of the animal. As shown in B′, the hind limbs on the injected side are more advanced in size and stage, due to lack of TRα-mediated repression of genes important for metamorphic progression.

Figure 3

Hind limb and skin morphogenesis in the absence of TH signaling in TRαKO tadpoles. Wild-type (A) and TRαKO (B) tadpoles shown in dorsal and side views were reared in 1 μM methimazole (inhibits TH synthesis) for 10 weeks starting at the initiation of feeding when the thyroid gland is forming. Development in the methimazole-treated wild-type tadpole is blocked at the beginning of metamorphosis, at the point when plasma TH levels begin to rise in untreated tadpoles. Notably, hind limb morphogenesis has stalled just after the paddle stage, and the skin is still thin and transparent as it is before it remodels to adult skin. In the methimazole-treated TRαKO tadpole, hind limb morphogenesis is complete, and in locations where adult skin replaces larval skin (i.e., not skin covering gills and tail), the skin has become opaque obscuring view of internal structures.