Sean McKinney

The FATP1–DGAT2 complex facilitates lipid droplet expansion at the ER–lipid droplet interface

A complex between the ER resident protein FATP1 and the lipid droplet–localized DGAT2 protein facilitates lipid droplet expansion in C. elegans and mammalian cells.

Epithelial junctions maintain tissue architecture by directing planar spindle orientation

During epithelial cell proliferation, planar alignment of the mitotic spindle coordinates the local process of symmetric cell cleavage with the global maintenance of polarized tissue architecture. Although the disruption of planar spindle alignment is proposed to cause epithelial to mesenchymal transition and cancer, the in vivo mechanisms regulating mitotic spindle orientation remain elusive. Here we demonstrate that the actomyosin cortex and the junction-localized neoplastic tumour suppressors Scribbled and Discs large 1 have essential roles in planar spindle alignment and thus the control of epithelial integrity in the Drosophila imaginal disc. We show that defective alignment of the mitotic spindle correlates with cell delamination and apoptotic death, and that blocking the death of misaligned cells is sufficient to drive the formation of basally localized tumour-like masses. These findings indicate a key role for junction-mediated spindle alignment in the maintenance of epithelial integrity, and also reveal a previously unknown cell-death-mediated tumour-suppressor function inherent in the polarized architecture of epithelia.

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

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

TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis

Non-bilaterian phyla represent key lineages for exploring the evolutionary history of early animals. However, despite an increasing number of sequenced genomes from early-branching metazoans, efficient and reproducible methodologies for analysis of gene function remain a major challenge. Here we report the utilization of the TALEN and CRISPR/Cas9 systems to induce targeted mutations and homologous recombination-mediated transgenesis in the sea anemone Nematostella vectensis. We also present a new method to isolate genetically modified animals using engineered selection cassettes introduced by homologous recombination. Taken together, these methods will permit sophisticated gain- and loss-of-function analyses in Nematostella and perhaps other early metazoan species that allow for zygotic injection.

Egr-5 is a post-mitotic regulator of planarian epidermal differentiation

Neoblasts are an abundant, heterogeneous population of adult stem cells (ASCs) that facilitate the maintenance of planarian tissues and organs, providing a powerful system to study ASC self-renewal and differentiation dynamics. It is unknown how the collective output of neoblasts transit through differentiation pathways to produce specific cell types. The planarian epidermis is a simple tissue that undergoes rapid turnover. We found that as epidermal progeny differentiate, they progress through multiple spatiotemporal transition states with distinct gene expression profiles. We also identified a conserved early growth response family transcription factor, egr-5, that is essential for epidermal differentiation. Disruption of epidermal integrity by egr-5 RNAi triggers a global stress response that induces the proliferation of neoblasts and the concomitant expansion of not only epidermal, but also multiple progenitor cell populations. Our results further establish the planarian epidermis as a novel paradigm to uncover the molecular mechanisms regulating ASC specification in vivo. DOI: http://dx.doi.org/10.7554/eLife.10501.001

Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ

Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models (Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies. DOI: http://dx.doi.org/10.7554/eLife.07405.001

Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001

Pathogenic shifts in endogenous microbiota impede tissue regeneration via distinct activation of TAK1/MKK/p38.

The interrelationship between endogenous microbiota, the immune system, and tissue regeneration is an area of intense research due to its potential therapeutic applications. We investigated this relationship in Schmidtea mediterranea, a model organism capable of regenerating any and all of its adult tissues. Microbiome characterization revealed a high Bacteroidetes to Proteobacteria ratio in healthy animals. Perturbations eliciting an expansion of Proteobacteria coincided with ectopic lesions and tissue degeneration. The culture of these bacteria yielded a strain of Pseudomonas capable of inducing progressive tissue degeneration. RNAi screening uncovered a TAK1 innate immune signaling module underlying compromised tissue homeostasis and regeneration during infection. TAK1/MKK/p38 signaling mediated opposing regulation of apoptosis during infection versus normal tissue regeneration. Given the complex role of inflammation in either hindering or supporting reparative wound healing and regeneration, this invertebrate model provides a basis for dissecting the duality of evolutionarily conserved inflammatory signaling in complex, multi-organ adult tissue regeneration. DOI: http://dx.doi.org/10.7554/eLife.16793.001

An adaptable chromosome preparation methodology for use in invertebrate research organisms

BackgroundThe ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed.ResultsWe optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization.ConclusionsOur simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.

A nuclear hormone receptor and lipid metabolism axis are required for the maintenance and regeneration of reproductive organs

Understanding how stem cells and their progeny maintain and regenerate reproductive organs is of fundamental importance. The freshwater planarian Schmidtea mediterranea provides an attractive system to study these processes because its hermaphroditic reproductive system (RS) arises post-embryonically and when lost can be fully and functionally regenerated from the proliferation and regulation of experimentally accessible stem and progenitor cells. By controlling the function of a nuclear hormone receptor gene (nhr-1), we established conditions in which to study the formation, maintenance and regeneration of both germline and somatic tissues of the planarian RS. We found that nhr-1(RNAi) not only resulted in the gradual degeneration and complete loss of the adult hermaphroditic RS, but also in the significant downregulation of a large cohort of genes associated with lipid metabolism. One of these, Smed-acs-1, a homologue of Acyl-CoA synthetase, was indispensable for the development, maintenance and regeneration of the RS, but not for the homeostasis or regeneration of other somatic tissues. Remarkably, supplementing nhr-1(RNAi) animals with either bacterial Acyl-CoA synthetase or the lipid metabolite Acetyl-CoA rescued the phenotype restoring the maintenance and function of the hermaphroditic RS. Our findings uncovered a likely evolutionarily conserved role for nuclear hormone receptors and lipid metabolism in the regulation of stem and progenitor cells required for the long-term maintenance and regeneration of animal reproductive organs, tissues and cells.