The chicken cerebellum as a model to study the impact of monocarboxylate transporter 8 deficiency on early brain development

Thyroid hormones (THs) have been characterised as important regulatory factors of vertebrate brain development. As such, they contribute to a wide variety of neurodevelopmental processes that depend on adequate TH levels as well as a correct timing of TH access to distinct neural cells in order to obtain a fully functional brain. This strict regulation of local TH availability is the result of a complex interplay between TH transporters mediating TH passage across the plasma membrane and deiodinases which intracellularly activate or inactivate THs. While the consequences of TH deficiency on brain development have already been well described, the challenges for a better understanding of the underlying mechanisms of TH action in the developing brain are considerable. In this thesis, we focused on the functional involvement of the monocarboxylate transporter 8 (MCT8) in cerebellar circuit organisation by using a knockdown approach in the chicken embryo.

To build a general picture of the dependency of the developing cerebellum on TH signalling, we determined the cell-specific expression pattern of TH transporters and deiodinases. Our results showed that throughout embryonic development, the cerebellum expresses the genes needed for TH traffic between neural cells and TH (in)activation in a tissue- and cell-specific way. The differential expression of TH regulatory genes implies differing spatio-temporal sensitivities to TH signalling. Purkinje cells (PCs), which are the sole output neurons of the cerebellum and known to be TH-sensitive, showed pronounced expression of MCT8 supporting a model where the MCT8 transporter plays a decisive role in the uptake of receptor-active 3,5,3’-triiodothyronine (T3) in central neurons.

To study the role of this transporter in TH-dependent cellular events driving early cerebellar development, we developed a strategy to downregulate MCT8 expression and thereby alter local TH levels in the developing cerebellum. We created gene-specific RNA interference (RNAi) vectors based on the pRFPRNAiA and RCASARNAi vectors specifically designed for optimal gene silencing in chicken. The pRFPRNAiA vector is a standard plasmid vector, whereas the RCASARNAi vector is a precursor for a modified RCAS virus. In vitro testing in cell culture showed that both RNAi vectors efficiently silence MCT8 mRNA expression. To use these RNAi vectors in vivo, we developed a method for in vivo transfection of the cerebellum. By performing site-restricted in ovo electroporation at embryonic day 3, we were able to efficiently transfect distinct cerebellar cell types, as checked in embryos between 6 and 18 days old. Whereas the non-viral vector was predominantly expressed in PCs and cerebellar nuclei in one cerebellar half, the RCAS vector showed widespread expression in granule cells (GCs) throughout the cerebellum.

To study the effects of MCT8 deficiency on cerebellar circuit assembly, we used the non-viral MCT8 RNAi vector to specifically silence MCT8 expression in developing PCs. Our results showed that the MCT8 transporter seems to be an important factor throughout development in controlling neuronal T3 supply. This in turn regulates key cellular events of cerebellar development such as PC differentiation and GC proliferation via the cross-talk between TH signalling, Retinoic acid receptor-related orphan receptor alpha (RORα) and Sonic hedgehog (SHH). Our strategy thus not only provides novel insights into the molecular pathways possibly disturbed in MCT8-deficient cerebellar development in humans, it also opens up new opportunities to reveal the functional involvement of other TH regulatory genes, leading to a better understanding of the mechanisms underlying TH action in the entire developing brain.