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Single-cell RNA-seq data for control and POU IV-misexpressed Ciona intestinalis embryos at late tailbud (LTB) stage

ABSTRACT: Purpose: Understand how the misexpression of key sensory cell determinant POU IV affects cell type specification of various sensory neurons in the peripheral nervous system in Ciona. Methods: CesA>POU IV (2.5ng/l) + CesA>GFP (5.0ng/l) injected eggs and control eggs were fertilized side by side, and allowed to develop to the late tailbud stage (13.5h after fertilization at 18°C). For each sample, 120 morphologically normal embryos were used for single cell RNA-seq assays. Dissociation of the embryos and single cell RNA-seq assays by 10x Genomics chromium system were done as described in 9. Horie R. et al., Shared evolutionary origin of vertebrate neural crest and cranial placodes. Nature 560, 228-232 (2018). Results: Misexpression of POU IV leads to the formation of Synthetic Cell Types that exhibit properties of other sensory neurons, especially those of bipolar tail neurons (BTNs) and a synthetic gene battery that is not found in any wildtype sensory neuron.

ORGANISM(S): Ciona intestinalis

PROVIDER: GSE192645 | GEO | 2021/12/29

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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Single-cell RNA-seq data for control and POU IV-misexpressed Ciona intestinalis embryos at late tailbud (LTB) stage

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