Deciphering HOXA5 functions in the establishment and maturation of the precerebellar circuits

Details

Supervisors

Gofflot, Françoise

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Hox genes are involved in many processes during embryonic development, but they also have been reported to have a role in adult life. Spatiotemporal characterization of Hoxa5 showed its expression, at postnatal and adult stages, in precerebellar nuclei located in the pons and medulla oblongata. The precerebellar system act as the set of relay neurons and provides the main input to the cerebellum. Moreover, postnatal loss of function of Hoxa5, during the period of establishment of the precerebellar system, revealed through transcriptomic analysis a downregulation of genes associated with synaptic functions. Those findings suggests that HOXA5 might have a role in neuritogenesis and synaptogenesis in precerebellar circuits. To investigate on this hypothesis, the objective is to evaluate the impact of Hoxa5 deletion on neuritogenesis and synaptogenesis at the neuronal level but also characterize its role in the precerebellar system. However, to achieve this, it’s necessary to have appropriate robust models. Therefore, the focus of my master thesis is on the development and optimization of the necessary tools to address these research questions e<ectively. To investigate the impact of Hoxa5 deletion at the neuronal level, we developed an in vitro neuronal model using primary cultures of hindbrain neurons derived from mice carrying the tamoxifen-inducible CreERT2 recombinase transgene and homozygous floxed Hoxa5 alleles. By employing the CreERT2/LoxP system and applying hydroxytamoxifen treatments we managed to successfully inactivate Hoxa5 in our primary cultures. In this master thesis, we show that administration, in primary cultures medium, of 10μL of [1μM] of hydroxytamoxifen at 1 day in vitro is enough to induce e<icient recombination. Moreover, immunofluorescence analysis suggests that hydroxytamoxifen or ethanol, its solvent, don’t have a detrimental e<ect on cell viability. To gain a deeper understanding of Hoxa5's role in the precerebellar circuit, it is essential to map the axonal projections of neurons expressing Hoxa5. Since the HOXA5 transcription factor is predominantly localized in the nucleus, a new transgenic reporter line has been generated. In this line, the coding sequence for the red fluorescent TdTomato protein is driven by Hoxa5 regulatory elements. This enables visualization of Hoxa5-expressing cells by detecting the fluorescence of the cytoplasmic TdTomato protein. The first step before mapping axonal projections, is to validate and characterize this line. In this master thesis, we show that out of nine Hoxa5::TdTomato reporter lines generated, four exhibit transgene transmission and show no detrimental phenotypes. Among these four lines, three accurately recapitulate Hoxa5 endogenous expression and dynamics at developmental stage E10.5, E12.5 and E13.5. By addressing the need for reliable and precise models, the findings presented in this master thesis establish a strong foundation for future investigations into the role of Hoxa5 in the establishment and maturation of the precerebellar circuits.