Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts.National Institutes of Health (U.S.) (R01GM080639)National Science Foundation (U.S.). Graduate Research Fellowship Program (Grant 1122374

Neoblast specialization during regeneration of the planarian S. mediterranea

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.Cataloged from PDF version of thesis. "Due to the condition of the original material, there are unavoidable flaws in this reproduction. Table 3.1 is missing from page 167"-- Disclaimer Notice page.Includes bibliographical references (pages 175-186).Planarians are well known for their ability to regenerate an entire animal from small tissue fragments. Planarian regeneration requires a population of dividing cells called neoblasts that are distributed throughout the body. Historically, neoblasts have been considered a homogeneous population of stem cells capable of differentiating into all cell types. Most studies, however, analyze neoblasts at the population rather than the single cell level, making it difficult to determine how heterogeneous the neoblast population is. A bulk RNA sequencing approach with expression screening identified 33 new transcription factors transcribed in specific differentiated cells that were also expressed in small fractions of neoblasts during regeneration. Transcription factors of distinct differentiated tissues were expressed in different subsets of neoblasts, whereas transcription factors expressed in the same differentiated tissues were expressed in the same neoblasts. These results suggest roles for neoblast-expressed transcription factors in the specification of distinct tissues. Furthermore, the transcription factors klf, Pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine- beta-hydroxylase-expressing neurons, and the pharynx, respectively. The planarian nervous system is comprised of numerous different cell types, providing an opportunity to study how neoblasts acquire the diverse cell fates that comprise a particular tissue. We used single-cell sequencing to identify the transcriptomes of hundreds of planarian neurons and neoblasts. Using computational analysis of these data we identified the transcriptomes of several specific types of planarian neuronal cells, including cholinergic, dopaminergic, and serotonergic neurons, as well as glial cell types. In neoblasts, we identified a population of cells that expressed both markers of differentiated neurons and transcription factors expressed in various neural cell types, which we hypothesize to be neural specialized neoblasts. We found a number of unique populations of neural neoblasts that correspond with specific neural sub-types. Interestingly, however, these neural specialized neoblasts do not express a detectable unified gene regulatory network. These results are consistent with direct specification of neural sub-types in neoblasts and suggest that neoblasts do not differentiate down a highly hierarchical lineage path as has been described for many developmental lineages.by Kellie M. Kravarik.Ph. D

Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea

Planarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts

foxF-1 Controls Specification of Non-body Wall Muscle and Phagocytic Cells in Planarians

Planarians are flatworms capable of regenerating any missing body part in a process requiring stem cells and positional information. Muscle is a major source of planarian positional information and consists of several types of fibers with distinct regulatory roles in regeneration. The transcriptional regulatory programs used to specify different muscle fibers are poorly characterized. Using single-cell RNA sequencing, we define the transcriptomes of planarian dorsal-ventral muscle (DVM), intestinal muscle (IM), and pharynx muscle. This analysis identifies foxF-1, which encodes a broadly conserved Fox-family transcription factor, as a master transcriptional regulator of all non-body wall muscle. The transcription factors encoded by nk4 and gata4/5/6-2 specify two different subsets of DVM, lateral and medial, respectively, whereas gata4/5/6-3 specifies IM. These muscle types all express planarian patterning genes. Both lateral and medial DVM are required for medial-lateral patterning in regeneration, whereas medial DVM and IM have a role in maintaining and regenerating intestine morphology. In addition to the role in muscle, foxF-1 is required for the specification of multiple cell types with transcriptome similarities, including high expression levels of cathepsin genes. These cells include pigment cells, glia, and several other cells with unknown function. cathepsin+ cells phagocytose E. coli, suggesting these are phagocytic cells. In conclusion, we describe a regulatory program for planarian muscle cell subsets and phagocytic cells, both driven by foxF-1. FoxF proteins specify different mesoderm-derived tissues in other organisms, suggesting that FoxF regulates formation of an ancient and broadly conserved subset of mesoderm derivatives in the Bilateria.National Institutes of Health (U.S.) (Grant R01GM080639

Single-cell isolation from full-thickness human intestinal tissue resections for single-cell RNA sequencing

Summary: Single-cell isolation techniques allow the investigation of physical and functional relationships between individual cells within a complex cell population. Here, we present a protocol for single-cell isolation from full-thickness intestinal tissue resections. We describe steps for pre-processing specimens, isolation of lamina propria and muscular layers, and red blood cell lysis. We then detail fixation of isolated cells and assessment of cell quality. The resulting cell suspension can be subjected to RNA sequencing on the 10× Chromium platform.For complete details on the use and execution of this protocol, please refer to Mukherjee et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics