Transcriptional and epigenetic regulation of neuronal migration and circuitry development in the murine hindbrain

Polycomb group proteins, including Ezh2, regulate many target genes, which control early cell fate decisions. We addressed whether Ezh2-dependent epigenetic regulation of transcriptional programs also orchestrates complex processes such as long-distance tangential neuronal migration in the developing nervous system.
We focused on the migratory behavior of the anterior extramural stream (AES) that contributes to the brainstem pontine nuclei (PN), the main relay between cortex and cerebellum.
We found that the PN neuron migratory behavior is largely pre-mapped at the progenitor stage. The AES stereotypic migratory pattern emerges through an inter-dependent interaction between: i) an intrinsic Ezh2-dependent transcriptional program established in PN progenitors, which is maintained in migrating neurons and enables appropriate response to environmental cues; and, ii) an Ezh2-dependent silencing program that regulates the spatial distribution of extrinsic signals in the migratory environment, such as Ntn1, also pre-mapped in ventricular progenitors.
Specifically, by restricting Netrin1 expression to ventral hindbrain, Ezh2 allows normal PN migration in a non-cell-autonomous manner. In conditional Ezh2 mutants, ectopic Netrin1 de-repression leads to abnormal migration and supernumerary nuclei that integrate the cortico-ponto-cerebellar circuitry. Importantly, we revealed for the first time an intrinsic topographic organization of the PN migratory stream, according to rostrocaudal progenitor origin. Neuronal position is maintained throughout migration and settling in the PN and correlates with patterned cortical input. By counteracting retinoid signaling, Ezh2 sets graded Hox expression in migrating neurons which in turn maintain graded activity of repulsive receptors Unc5b/Unc5c, generating subsets with distinct responsiveness to Netrin1.
These findings point to a migratory protomap established in progenitors, whereby Polycomb-mediated epigenetic silencing is fundamental to establish these intrinsic and extrinsic programs. On the one hand, Ezh2 sets the levels of environmental attractive/repulsive signals through dorsoventrally-restricted silencing of Ntn1. On the other hand, Ezh2-mediated repression contributes to set a heterogeneous Hox transcriptional program in the AES that, in turn, provides neuronal subsets with distinct Unc5b-mediated repulsion to environmental Ntn1.
Our results extend the involvement of Ezh2 beyond fate and subtype identity specification to a novel role in orchestrating epigenetic regulation of topographic neuronal guidance in the mammalian brain.
Lastly, the pontine gray nucleus is a fundamental relay station for the transformation of orderly motor and sensory maps in the cerebral cortex into ÔpatchyÕ representations of input in the granular layer of the cerebellar cortex. Little is known about the molecular and cellular mechanisms assembling these complex input-output wiring patterns in PN. Our results strongly suggest that the intrinsic pre-mapping of PN from progenitors of distinct rostrocaudal origin contributes to organize broad topographic input from distinct cortical areas.
To further investigate this, we established means to analyze cortico-ponto-cerebellar connectivity by using a) transgenic animals b) viral tracing and c) in utero electroporation. These methods will serve further in-depth analysis of the pontine nuclei circuitry and allow functional experiments.
This thesis encompasses the accepted publication ÒEzh2 orchestrates topographic tangential migration and connectivity of precerebellar neuronsÓ (Di Meglio et al., 2013), two manuscripts that resulted from collaborations about ÒPartial ipsilateral wiring of subcortical sensory inputs duplicates the facial mapÓ and ÒMouse Hoxa2 genetic analysis provides a model for human microtia and auricle duplicationÓ, a manuscript for a book chapter about ÒThe Cre/Lox system to assess the development of the mouse brainÓ as well as unpublished results that were part of previous versions of the Science publication (Di Meglio et al., 2013) and results from further investigations of cortico-ponto-cerebellar connectivity as well as in-depth analysis of generated and examined transgenic mouse lines.