Epigenetic and transcriptional regulation of cortico-ponto-cerebellar circuit formation

The precerebellar system constitutes an array of nuclei located in the mammalian hindbrain and conveys movement and balance information from the cortex, spinal cord and periphery to the cerebellum (Sotelo, 2004). Within this system, the pontine nuclei (PN), including pontine gray and reticulotegmental nuclei, mostly relay cortical information (Schwarz and Thier, 1999). During the processing through the cortex, PN and cerebellum, continuous maps of sensorimotor information are transformed into a complex fractured map (Leergaard et al., 2006). To date, however, there is a paucity of knowledge on the molecular and cellular mechanisms organizing this complex circuitry. Previous work suggests an intrinsic topographic organization, according to rostro-caudal progenitor origin, that is maintained during migration and nucleation of the PN (Di Meglio et al., 2013). As a result, one of the hallmarks of the PN topography is a well-defined population of Hox paralogous group 5 (PG5) expressing neurons in the posterior part of the PN. However, the molecular mechanisms governing the spatial expression pattern of Hox PG5 genes in the PN and their functional impact on circuit formation remain largely unknown.
The first part of this thesis focuses on the molecular mechanisms of Hox PG5 induction in the precerebellar system. We find that the precise spatio-temporal expression pattern of Hox PG5 genes rely on the integration of environmental signaling and the resulting modifications of the epigenetic landscape. Unlike transcripts of more anterior Hox genes, expression of Hox PG5 genes is entirely excluded from progenitors in the rhombic lip (RL) and only induced in a subset of postmitotic neurons. Mapping and manipulation of signaling pathways show that the restriction of Hox PG5 induction to the ventrally located (i.e. posterior RL-derived) postmitotic pontine neuron subsets is due to an interplay between retinoic acid (RA) and Wnt environmental signaling. Assessment of histone profiles at Hox loci indicate that the induction of Hox PG5 genes through RA is tightly linked to a depletion of the histone mark H3K27me3. However, conditional inactivation of Ezh2, a member of the polycomb repressive complex 2 responsible for setting the H3K27me3 mark (Margueron and Reinberg, 2011), does not result in a de-repression of Hox PG5 genes in the progenitor domain. In contrast, removal of H3K27me3 in Ezh2 depleted PN neurons leads to an ectopic induction of Hox PG5 in rostral PN neuron subsets of the migratory stream showing an enhanced response to RA (Di Meglio et al., 2013). Moreover, high levels of RA-induced Hox PG5 expression in postmitotic PN neurons require Jmjd3, one of the enzymes known to catalyze the removal of methyl groups at H3K27 (Agger et al., 2007; De Santa et al., 2007). We show that Jmjd3 is physically present at RA responsive elements in proximity to the Hoxa5 promoter supporting the direct involvement of Jmjd3 in Hox PG5 induction. Thus, a central function of H3K27me3 regulation during late stages of precerebellar development is the establishment of a threshold for RA mediated activation of Hox PG5 genes to allow for diversification of PN neurons. Finally, we show how the integration of environmental signaling on the epigenetic level results in distinct changes of the three dimensional (3D) organization of chromatin at Hox PG5 loci in vivo. Together, the late specification of PN neurons employs a sophisticated sequence of interactions between signaling pathways such as RA and Wnt, and histone modifying enzymes like Ezh2 and Jmjd3.
The second part of the thesis addresses the functional significance of Hox PG5 genes in sub-circuit formation of PN neurons. Using multiple conditional overexpression strategies, we show that the expression of Hoxa5 is sufficient to shape the input-output relationship of PN neurons. Hoxa5 expressing neurons migrate into a posterior position in the PN and induce a distinct transcriptional program specific for topographic circuit formation. Together, this indicates a crucial role of Hoxa5 in the specification of the positional identity of PN subsets. We further describe a genetically identified Hox PG5 negative PN subset that primarily projects to the paraflocculus, a lobule in the cerebellum heavily concerned with visually related tasks. Conditional overexpression of Hoxa5 in this PN subset leads to the ectopic targeting of several other lobes in the cerebellum concerned with processing of somatosensory information. This matches with the input connectivity of the PN that has been shown to be antero-posteriorly patterned, such that visual/medioposterior projections target the anterior, Hox PG5 negative, and somatosensory projections target the posterior, Hox PG5 positive part of the PN (Di Meglio et al., 2013; Leergaard and Bjaalie, 2007). Consequently, Hoxa5 overexpressing PN neurons are largely devoid of input from the visual cortex and primarily engage in a somatosensory hindlimb specific circuitry. One single Hox gene is thus sufficient to position neurons in the posterior aspects of the PN, change their transcriptional program and rearrange both, output connectivity to the cerebellum and input connectivity from the cortex. These findings extend the function of Hox genes to orchestrating topographic circuit formation in the PN. Further, the presented results point towards an involvement of Hox genes in the longstanding problem of fracturing of the somatosensory map that is realized between the cortex and the cerebellum.