Biology subjects

Forsthoefel, D. J.

Publications and source records attributed to Forsthoefel, D. J..

4 recordsLinked to original sources

bioRxiv2024

Extracellular vesicles promote proliferation in an animal model of regeneration

Extracellular vesicles (EVs) are secreted nanoparticles composed of a lipid bilayer that carry lipid, protein, and nucleic acid cargo between cells as a mode of intercellular communication. Although EVs can promote tissue repair in mammals, their roles in animals with greater regenerative capacity are not well understood. Planarian flatworms are capable of whole body regeneration due to pluripotent somatic stem cells called neoblasts that proliferate in response to injury. Here, using transmission electron microscopy, nanoparticle tracking analysis, and protein content examination, we showed that EVs enriched from the tissues of the planarian Schmidtea mediterranea had similar morphology and size as other eukaryotic EVs, and that these EVs carried orthologs of the conserved EV biogenesis regulators ALIX and TSG101. PKH67-labeled EVs were taken up more quickly by S/G2 neoblasts than G1 neoblasts/early progeny and differentiated cells. When injected into living planarians, EVs from regenerating tissue fragments enhanced upregulation of neoblast-associated transcripts. In addition, EV injection increased the number of F-ara-EdU-labelled cells by 49% as compared to buffer injection only. Our findings demonstrate that regenerating planarians produce EVs that promote stem cell proliferation, and suggest the planarian as an amenable in vivo model for the study of EV function during regeneration.

cell biology↗

bioRxiv2023

Egr-2 coordinates intestinal remodeling by promoting midline patterning and pharynx regeneration in planarians

Regeneration requires remodeling of pre-existing structures and their integration with new cells and tissue. Identification of injury-induced regulators of these processes is central to regenerative biology. In planarian flatworms, the intestine remodels after amputation to re-establish normal morphology and integrate with the regenerating pharynx, the centrally located feeding organ. How these events are initiated and coordinated is currently unclear. In a candidate screen for injury-induced regulators of intestinal regeneration, we found that the transcription factor Early growth response protein 2 (Egr-2) was required for collective migration and fusion of intestinal branches at the anterior midline. Even though inhibition of egr-2 by RNA interference increased dorsoventral (DV) muscle fibers at the midline between intestinal branches, bulk RNA sequencing of regenerating tissue revealed negligible dysregulation of DV-, body wall-, or intestinal-muscle-associated transcripts. Instead, the midline polarity cue slit-1 and subsets of pharynx-associated transcripts were dysregulated. Both egr-2 and slit-1 RNAi delayed early pharynx regeneration, consistent with reduced slit-1 expression in egr-2(RNAi) regenerates in the region of pharynx specification. Furthermore, inhibition of pharynx regeneration regulators foxF-1 and foxA delayed integration with the intestine and disrupted formation of the intestines midline anterior branch. Our findings suggest that the pharynx plays an inductive role in initiation of intestinal remodeling, and demonstrate how injury-induced reprogramming influences subsequent tissue assembly and integration during regeneration.

developmental biology↗

bioRxiv2021

Intestine-enriched apolipoprotein b orthologs regulate stem cell differentiation and regeneration in planarians

Lipid metabolism plays an instructive role in regulating stem cell state and differentiation. However, the roles of lipid mobilization and utilization in stem cell-driven regeneration are unclear. Planarian flatworms readily restore missing tissue due to injury-induced activation of pluripotent somatic stem cells called neoblasts. Here, we identify two intestine-enriched orthologs of apolipoprotein b, apob-1 and apob-2, which mediate transport of neutral lipid stores from the intestine to target tissues including neoblasts, and are required for tissue homeostasis and regeneration. Inhibition of apob function by RNAi causes head regression and lysis in uninjured animals, and delays body axis re-establishment and regeneration of multiple organs in amputated fragments. Furthermore, apob RNAi causes expansion of the population of differentiating neoblast progeny and dysregulates expression of genes enriched in differentiating and mature cells in eight major cell type lineages. We conclude that intestine-derived lipids serve as a source of metabolites required for neoblast differentiation.

developmental biology↗

bioRxiv2019

Cell-type diversity and regionalized gene expression in the planarian intestine revealed by laser-capture microdissection transcriptome profiling

Organ regeneration requires precise coordination of new cell differentiation and remodeling of uninjured tissue to faithfully re-establish organ morphology and function. An atlas of gene expression and cell types in the uninjured state is therefore an essential pre-requisite for understanding how damage is repaired. Here, we use laser-capture microdissection (LCM) and RNA-Seq to define the transcriptome of the intestine of Schmidtea mediterranea, a planarian flatworm with exceptional regenerative capacity. Bioinformatic analysis of 1,844 intestine-enriched transcripts suggests extensive conservation of digestive physiology with other animals, including humans. Comparison of the intestinal transcriptome to purified absorptive intestinal cell (phagocyte) and published single-cell expression profiles confirms the identities of known intestinal cell types, and also identifies hundreds of additional transcripts with previously undetected intestinal enrichment. Furthermore, by assessing the expression patterns of 143 transcripts in situ, we discover unappreciated mediolateral regionalization of gene expression and cell-type diversity, especially among goblet cells. Demonstrating the utility of the intestinal transcriptome, we identify 22 intestine-enriched transcription factors, and find that several have distinct functional roles in the regeneration and maintenance of goblet cells. Furthermore, depletion of goblet cells inhibits planarian feeding and reduces viability. Altogether, our results show that LCM is a viable approach for assessing tissue-specific gene expression in planarians, and provide a new resource for further investigation of digestive tract regeneration, the physiological roles of intestinal cell types, and axial polarity.

developmental biology↗