Single-nucleus multiome (RNA+ATAC) sequencing of the axolotl pallium
Ontology highlight
ABSTRACT: In order to understand the genomic and transcriptomic variability of the axolotl pallium, as well as reconstruct their intrinsic gene regulatory networks, we performed single-nucleus multiome sequencing (RNA and open chromatin) of whole axolotl pallium.
Project description:In order to understand the relationship between cellular diversity and pallium regions, single-nucleus RNA-seq (snRNA-seq) was performed in 3 microdissected regions from the axolotl pallium: medial, dorsal, and lateral.
Project description:The goal of this experiment is to track cellular regeneration after a dorsal injury to the axolotl pallium. To this end, we employed Div-seq, that is, performed snRNA-seq on cells labelled with EdU, which have thus recently replicated. We performed this in a time course, in order to observed the cell populations that were generated as regeneration progressed.
Project description:The Mexican axolotl (Ambystoma mexicanum) is one member of a select group of vertebrate animals that has retained the amazing ability to regenerate multiple body parts. In addition to being an important model system for regeneration, the axolotl is also a leading model system for developmental biologists. Many genes used in development have been identified to be reused again during regeneration, however how this molecular circuitry is controlled during regeneration is unknown. In recent years microRNAs have been identified as key regulators of gene expression during development, in many diseases and also in regeneration. Here we have used deep sequencing combined with qRT-PCR to identify microRNAs that are involved in regulating regeneration in axolotl. This approach has enabled us to identify well known families of microRNAs and in addition to identify putative novel microRNAs that differentially regulated in the regenerating tissue. These findings suggest that microRNAs may play key roles in managing the spatial and temporal expression of genes important for ensuring that the correct tissues are regenerated. small RNA Sequencing (2 samples) in Ambystoma Mexicanum
Project description:We generated Multiome RNA+ATAC data from the same cell from human PBMC. This served as a gold benchmark for a novel integration method for multi-omics data that we developed.
Project description:Previous studies of appendage regeneration in the axolotl have shown that multiple genetic programs are modulated through regulatory factors. MicroRNAs are short highly conserved non-coding genes that suppress expression of target genes and thereby control multiple genetic programs. Given their important regulatory roles and evolutionary conservation, we hypothesize that microRNAs define a conserved genetic regulatory circuit important for appendage regeneration. We characterized microRNA expression during Axolotl forelimb regeneration using small RNA sequencing. The same samples were assayed for mRNA expression using mRNA sequencing. Small RNA and mRNA gene expression profiling during 0, 3, 6 and 14 days post amputation.
Project description:We generated Multiome RNA+ATAC data from the same cell from human PBMC. This served as a gold benchmark for a novel integration method for multi-omics data that we developed.
Project description:Salamanders, such as the Mexican axolotl, are some of the few vertebrates fortunate in their ability to regenerate diverse structures after injury. Unlike mammals they are able to regenerate a fully functional spinal cord after injury. However, the early signals required to initiate a pro-regenerative response after spinal cord injury is not well understood. To address this question we developed a spinal cord injury model in axolotls and used in vivo imaging of ion sensitive dyes and determined that spinal cord injury induces a rapid and dynamic change in the resting membrane potential of ependymoglial cells. Prolonged depolarization of ependymoglial cells after injury inhibits glial cell proliferation and subsequent axon regeneration. Using transcriptional profiling we identified c-Fos as a key voltage sensitive early response gene that is expressed specifically in the ependymoglial cells after injury. This data establishes that dynamic changes in the membrane potential after injury are essential for regulating the specific spatiotemporal expression of c-Fos that is critical for promoting faithful spinal cord regeneration in axolotl. All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2â3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed. Experimental Design: Ivermectin injection Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate.
Project description:In this project we studied the effect of Langat infection (LGTV strain TP21) on the brains of mice, both wild-type and Ifnar-/-. Nuclei of the brain were isolated and subjected to 10x 3' scRNAseq.
Project description:Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to specific Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (Axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2â3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed. There were 9 microarrays conducted, and these were ivermectin-treated (n=3). Experimental Design: Ivermectin injection Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate. Mature - Uninjured spinal cord tissue Sc Crush- spinal cord tissue 1 day after injury Ivermectin - Ivermectin injected spinal cord tissue 1 day after injury.
Project description:To investigate spatial heterogeneities in the axolotl forebrain, a coronal section of it was obtained for spatial transcriptomics using Visium V1.