Permanent deiodinase type 2 deficiency alters local thyroid hormone levels, disturbs development and strongly reduces fertility

Introduction: Vertebrate development and reproduction are both thyroid hormone (TH) dependent processes and TH bioavailability within each tissue is tightly controlled by deiodinating enzymes. To decipher the function of deiodinase type 2 (Dio2), the main TH-activating enzyme in zebrafish, we generated two mutant lines. Methods: KO was accomplished by zinc finger nuclease technology and dio2 gene disruption was verified by enzyme activity tests. Phenotypical analysis of Dio2KO zebrafish was carried out in embryos and larvae, using morphometric analysis, scoring of swim bladder volume, locomotor assay and immunohistochemistry, as well as during adulthood, by means of quantitative PCR, histology and clutch size counting. Results: We have generated two homozygous Dio2KO zebrafish lines. The first line is characterized by an in-frame deletion of 9 base pairs, resulting in the elimination of 3 conserved amino acids. The other line possesses an insertion of 4 base pairs, leading to the introduction of a premature stop codon. Both mutations permanently and specifically abolish Dio2 activity and the resulting mutant phenotypes are equally severe. Levels of active TH are strongly reduced in all tissues tested and downregulation of the inactivating enzyme Dio3 and TH receptor transcript levels points towards a severe hypothyroid status. Measurements within the first 7 days post fertilization (dpf) show a disturbed overall early development of Dio2KO zebrafish: body length, absolute and relative eye and ear size are all significantly reduced. Swim bladder inflation is delayed in Dio2KO larvae, which correlates to an impaired locomotor behaviour. Also vision is affected in Dio2KO larvae as tested by light response and confirmed by a reduction in retinal photoreceptor number during early-larval stages. Linear growth of Dio2KO fish is obviously retarded starting from early-larval stages until adulthood (4 months). By the age of 7 months, male Dio2KO fish are still significantly smaller than wild-type fish. At this stage, the weight of female Dio2KO fish is however significantly increased. This weight gain may point to fertility problems, since Dio2KO zebrafish have a very limited time window of reproductive activity. They become sexually mature at a later stage than wild-type fish, i.e. around 5 months compared to 3 months, and also the duration of their reproductive period is shortened to 2 months instead of 1.5 years. Dio2KO females not only have problems with egg deposition, resulting in strongly reduced clutch sizes, but histological analysis of ovaries reveals a time shift in oocyte production. At 4 months, Dio2KO fish possess significantly more primary oocytes than wild-type fish and the number of mature oocytes is significantly reduced. The fact that fertilization percentages are low suggest that also Dio2KO males may have fertility defects. Conclusion: Our Dio2KO zebrafish represent the first non-mammalian model with permanent deiodinase deficiency. Our results demonstrate that Dio2, and hence active TH, is essential at early life stages, to assure normal development, motility and vision, but also during adulthood for crucial processes such as reproduction.