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Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding

ABSTRACT: Axon pathfinding is a key step during the formation of neural circuits but however the transcriptional mechanisms regulating its progression remain poorly understood. The binary decision of crossing or avoiding the midline that neurons make during developmente midline that mammalian retinal ganglion cell (RGC) axons take at the optic chiasm has classically represented a robust model to search for novel mechanisms controlling the selection of axonal trajectories. Here, in order to identify new axon pathfinding regulators, we compared the transcriptome and chromatin accessibility profiles of mammalian retinal ganglion cells RGCs projecting ipsilateral (iRGCs) or contralaterally (cRGCs). Overall, our analyses retrieved dozens of new molecules potentially involved in axon pathfinding and uncovered the regulatory logic behind axon trajectories selection.

ORGANISM(S): Mus musculus

PROVIDER: GSE184275 | GEO | 2022/08/22

REPOSITORIES: GEO

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Project description:Using targeted gene deletion, we have firmly established that the Class IV POU domain transcription factor Brn-3.2 controls a developmental program regulating axon pathfinding in the mouse visual system. We have isolated and identified downstream gene targets of Brn-3.2, using Representational Difference Analysis (RDA), on cDNA populations derived from wildtype and Brn-3.2-/- retina at the appropriate embryonic stage. One of these candidate genes, the Homeodomain Interacting Protein Kinase 2 (HIPK2), is postulated to be a transcriptional coregulator, based on its in vitro interactions with repressor homeodomain proteins of the NK class, as well as other components of repressor complexes. HIPK2 has also been shown to be involved in post-translational modification of two major transcriptional regulators, p53 and CtBP. Expression of a dominant-negative form of HIPK2 in sensory neurons affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect.,The aim of this project is to identify targets for the Homeodomain Interacting Protein Kinase 2 (HIPK2), that we isolated as a downstream gene target of the class IV POU domain transcription factor Brn-3.2, and to investigate their function in the Brn-3.2 dependent pathway regulating axon pathfinding. ,We hypothesize that the genes regulated by HIPK2 will play a critical role in axon pathfinding and that the results of this study will provide novel insights into the molecular mechanisms of axon pathfinding, and possibly neural plasticity and regeneration, and therefore, be of great interest to the field of neurobiology in general.,In order to uncover potential biological role(s) of HIPK2 in neural development, and particularly axon pathfinding, we generated transgenic mouse models for in vivo studies. Our preliminary observations in transgenic mice, indicate that a form of the molecule expected to act as a dominant-negative and designed to be expressed in sensory neurons, affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect. We plan to take advantage of this model to identify genes regulated by HIPK2, and which are likely to be involved in axon pathfinding. To this end, we will compare gene expression profiles in sensory neurons isolated wild-type and transgenic mice. In our experimental paradigm, the trigeminal ganglion represents the ideal sensory structure in which to perform such an analysis: 1) High levels of transgene can be expressed in the trigeminal ganglion, as assessed by the expression of LacZ from the bicistronic construct which contains IRES-LacZ downstream of a dominant negative form of HIPK2. 2) The trigeminal ganglion can be dissected at embryonic day 13.5, when the phenotype is apparent, with ease and free of contamination from surrounding tissues. 3) The amounts of RNA that can be isolated from a single ganglion are in the range of 200-300 ng, which should be sufficient for microarray analysis following linear amplification of RNA. One series will be carried out with transgenic embryos and a control series will be carried out with wildtype littermates. Animals will be prepared and sacrificed by a standard protocol. Tissue will be rapidly dissected from E13.5 trigeminal ganglion, frozen in liquid nitrogen, and stored at -80C until RNA is extracted. RNA will be prepared using RNeasy Micro Kit. We will be providing 4 tissue samples for each of the wildtype and transgenic animals to mitigate any expression differences resulting from mouse to mouse variation.

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Multiomic Analysis of Neurons with Divergent Projection Patterns Identifies Novel Regulators of Axon Pathfinding

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