Decreased, Triiodothyronine (T3) leads to Reduced, Posterior swim bladder inflation

The mechanism through which altered TH levels result in impaired posterior chamber inflation still needs to be elucidated. It is currently unclear which aspect of swim bladder development and inflation is affected by TH disruption. Based on the developmental stages of the posterior chamber, several hypotheses could explain effects on posterior chamber inflation due to disrupted TH levels. A first hypothesis includes effects on the budding of the posterior chamber inflation. Secondly, the effect on posterior chamber inflation could also be caused by disturbing the formation and growth of the three tissue layers of this organ. It has been reported that the Hedgehog signalling pathway plays an essential role in swim bladder development and is required for growth and differentiation of cells of the swim bladder. The Wnt/β-catenin signalling pathway is required for the organization and growth of all three tissue layers (Yin et al., 2011, 2012, Winata 2009, Kress et al., 2009). Both signalling pathways have been related to THs in amphibian and rodent species (Kress et al., 2009; Plateroti et al., 2006; Stolow and Shi, 1995). Molla et al. (2019) showed that insulin-like growth factor (IGF‐1) plays a role in swim bladder inflation/maturation in zebrafish. Reinwald et al. (2021) showed that T3 and propylthiouracil treatment of zebrafish embryos altered expression of genes involved in muscle contraction and functioning in an opposing fashion. The authors suggested impaired muscle function as an additional key event between decreased T3 levels and reduced swim bladder inflation. Several other hypotheses include effects on the successful initial inflation of the posterior chamber, effects on lactic acid production that is required for the maintenance of the swim bladder volume, or effects on the production of surfactant that is crucial to maintain the surface tension necessary for swim bladder inflation.

Another uncertainty lies in the systemic versus local changes in T3 levels and the relative importance of the different T4 activating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that exposure of zebrafish embryos to seven strong DIO1 inhibitors (measured using in chemico enzyme inhibition assays), six out of seven compounds impaired posterior chamber inflation, but almost all of these compounds also inhibit DIO2. Tetrachlorobisphenol A (TCBPA), the only compound that inhibits DIO1 and not DIO2, had no effect on the posterior swim bladder. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. These results suggest that DIO2 enzymes may play a more important role in swim bladder inflation compared to DIO1 enzymes. In the ToxCast DIO2 inhibition single concentration assay, 304 out of 1820 chemicals were positive and 177 of these were also positive for DIO1 inhibition (viewed on 5/7/2022). This complicates the distinction between the relative contribution of DIO1 and DIO2 inhibition to reduced swim bladder inflation. It has been previously suggested that DIO2 is the major contributor to TH activation in developing zebrafish embryos (Darras et al., 2015; Walpita et al., 2010). It has been shown that a morpholino knockdown targeting DIO1 mRNA alone did not affect embryonic development in zebrafish, while knockdown of DIO2 delayed progression of otic vesicle length, head-trunk angle and pigmentation index (Houbrechts et al., 2016; Walpita et al., 2010, 2009). DIO1 inhibition may only become essential in hypothyroidal circumstances, for example when DIO2 is inhibited or in case of iodine deficiency, in zebrafish (Walpita et al., 2010) and mice (Galton et al., 2009; Schneider et al., 2006).

As reported by Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. Heijlen et al. (2014) additionally reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3. Wei et al. (2018) showed that exposure to bisphenol S in adult zebrafish decreased T4 levels and increased T3 levels, and these changes in thyroid hormone levels were transferred to the offspring, in which impaired swim bladder inflation was observed. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.

In the study of Cavallin et al. (2017) fathead minnow embryos were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed increased whole-body T3 concentrations in 4 and 6 day old embryos, together with impaired posterior chamber inflation. Transcript levels of DIO1, 2 and 3 remained unaltered and thus offered no proof of a compensatory mechanism that could explain these results.

The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, posterior swim bladder chamber inflation, which occurs early during development, appears to be less sensitive to inhibition of TH synthesis than to inhibition of the conversion of T4 to T3 (Stinckens et al., 2016, 2018; Nelson et al., 2016). There have however been a few reports of reduced posterior inflation upon inhibition of TH synthesis (Liu and Chan, 2002). It must however be noted that these observations could reflect delayed inflation due to a general delay in development rather than a direct effect on the swim bladder. Longer observations would have to clarify this.