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Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria.

ABSTRACT: Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using successful feeding as a proxy for regeneration. We found that knockdown of 20 genes caused a wide range of regeneration phenotypes and that RNAi of the forkhead transcription factor FoxA, which is expressed in a subpopulation of stem cells, specifically inhibited regrowth of the pharynx. Selective amputation of the pharynx therefore permits the identification of genes required for organ-specific regeneration and suggests an ancient function for FoxA-dependent transcriptional programs in driving regeneration. DOI: http://dx.doi.org/10.7554/eLife.02238.001.

SUBMITTER: Adler CE

PROVIDER: S-EPMC3985184 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria.

Adler Carolyn E CE Seidel Chris W CW McKinney Sean A SA Sánchez Alvarado Alejandro A

eLife 20140415

Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using success ...[more]

PMID: 24737865

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Project description:Purpose: Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue. Methods: ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury. Results: MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology. Conclusions: Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.

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Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria.

Publications

Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria.

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