Embryonic origins of ZebrinII parasagittal stripes and establishment of topographic Purkinje cell projections

Abstract

The establishment of neural circuits involves both the precise positioning of cells within brain regions and projection of axons to specific target cells. In the cerebellum (Cb), the medial-lateral (M-L) and anterior-posterior (A-P) position of each Purkinje cell (PC) and the topography of its axon can be defined with respect to two coordinate systems within the Cb; one based on the pattern of lobules and the other on PC gene expression in parasagittal clusters in the embryo (e.g. Pcp2) and stripes in the adult (e.g. ZebrinII). The relationship between the embryonic clusters of molecularly defined PCs and particular adult PC stripes is not clear. Using a mouse genetic inducible fate mapping (GIFM) approach and a Pcp2-CreER-IRES-hAP transgene, we marked three bilateral clusters of PC clusters with myristolated Green fluorescent protein (mGfp) on approximately embryonic day (E) 15 and followed their fate into adulthood. We found that these three clusters contributed specifically to ZebrinII-expressing PCs, including nine of the adult stripes. This result suggests that embryonic PCs maintain a particular molecular identity, and that each embryonic cluster can contribute PCs to more than one adult M-L stripe. Each PC projects a primary axon to one of the deep cerebellar nuclei (DCN) or the vestibular nuclei in the brainstem in an organized fashion that relates to the position of the PCs along the M-L axis. We characterized when PC axons from the three M-L clusters acquire topographic projections. Using a combination of GIFM to mark the PC clusters with mGfp and staining for human placental alkaline phosphatase (hAP) in Pcp2-CreER-IRES-hAP transgenic embryos we found that axons from each embryonic PCs cluster intermingled with neurons within particular DCN or projected out of the Cb toward the vestibular nuclei by E14.5. These studies show that PC molecular patterning, efferent circuitry, and DCN nucleogenesis occur simultaneously, suggesting a link between these processes.

In vivo analysis of Purkinje cell firing properties during postnatal mouse development

“…Third, Swiss Webster mice are very resilient to surgery , which is a key component in electrophysiology studies because each mouse is often probed for single units over several hours. There are no major differences in the morphogenesis, developmental patterning, or adult architecture of the cerebellum in Swiss Webster compared with inbred C57BL/6 mice (although there are strain-specific features), which indicated to us that because of the advantages listed above they would be a good model to assess developing neural function (Sillitoe et al 2009Sillitoe and Joyner 2007).…”

“…There are several reasons for this choice. First, these mice have been the strain of choice for genetic inducible fate mapping and cell lineage tracing regimes (Sgaier et al 2005;Sillitoe et al 2009;Zervas et al 2005). This is important because a major motivation for our study was to provide a fundamental knowledge base for understanding how genetic and developmental cell lineage data relate to cell function.…”

“…Moreover, our observation of relatively regular firing at P30 may in fact coexist with the irregular burst-like activity reported by Lorenzetto et al (2009) in light of recent data from an awake preparation that reported the presence of tonic, bursting, and even quiescent modes of Purkinje cell firing (Cheron et al 2014). In the future, it would be interesting to use juxtacellular labeling (Pinault 1996) to anatomically identify recorded neurons that were developmentally "marked" based on their genetic lineage (Sillitoe et al 2009) and then examine whether each class of Purkinje cell, based on its firing pattern, has a distinct location in the cerebellum, specific afferent connectivity, and unique developmental fate within the zonal "stripe" expression map (Sillitoe and Joyner 2007;White and Sillitoe 2013a). Indeed, the formation of zones is critically dependent on proper Purkinje cell firing (White et al 2014).…”

Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development

“…Further work is required to identify the factors that regulate PC fate choice, cell-cycle progression and exit, differentiation (Zhao et al, 2007), migration (Goldowitz et al, 1997;Rice et al, 1998;Trommsdorff et al, 1999;Park and Curran, 2008), survival (e.g. Croci et al, 2006;Croci et al, 2011), axonogenesis (Sillitoe et al, 2009;Miyata et al, 2010) and dendritogenesis (Boukhtouche et al, 2006;Poulain et al, 2008).…”

ENGRAILED 2 (EN2) Genetic and Functional Analysis

“…Interestingly, these stripe-like expression domains are coincident with the innervation of cerebellar afferents (mossy and climbing fibers), suggesting that these patterning genes regulate the topographic mapping of axons. Consistent with this possibility, En2 mouse mutants display connectivity phenotypes disrupting the innervation of mossy fibers (Herrup and Kuemerle 1997;Baader, Sanlioglu et al 1998;Baader, Vogel et al 1999;Sillitoe, Stephen et al 2008;Sillitoe, Gopal et al 2009;Sillitoe, Vogel et al 2010). Thus En2 is important in establishing the cerebellar connectivity map during development.…”