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The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia in their L-R organizer, consistent with the loss of motile cilia being a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have oneNodalgene, veiled chameleon exhibits expression of two paralogs ofNodalin the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

Planarian flatworms are best known for their impressive regenerative capacity, yet this trait varies across species. In addition, planarians have other features that share morphology and function with the tissues of many other animals, including an outer mucociliary epithelium that drives planarian locomotion and is very similar to the epithelial linings of the human lung and oviduct. Planarians occupy a broad range of ecological habitats and are known to be sensitive to changes in their environment. Yet, despite their potential to provide valuable insight to many different fields, very few planarian species have been developed as laboratory models for mechanism-based research. Here we describe a previously undocumented planarian isolate, Girardia sp. (Guanajuato). After collecting this isolate from a freshwater habitat in central Mexico, we characterized it at the morphological, cellular, and molecular level. We show that Girardia sp. (Guanajuato) not only shares features with animals in the Girardia genus but also possesses traits that appear unique to this isolate. By thoroughly characterizing this new planarian isolate, our work facilitates future comparisons to other flatworms and further molecular dissection of the unique and physiologically-relevant traits observed in this Girardia sp. (Guanajuato) isolate.

The stingers of jellyfish, sea anemones and other cnidarians, known as nematocysts, are remarkable cellular weapons used for both predation and defense1. Nematocysts are specialized organelles which consist of a pressurized capsule containing a coiled harpoon-like thread2. These structures are in turn built within specialized cells known as nematocytes3. When triggered4, the capsule explosively discharges, ejecting the coiled thread which punctures the target and rapidly elongates by turning inside out in a process called eversion5,6. Due to the structural complexity of the thread and the extreme speed of discharge, the precise mechanics of nematocyst firing have remained elusive7. Here, using a combination of live and super-resolution imaging, 3D electron microscopy and genetic perturbations, we define the step-by-step sequence of nematocyst operation in the model sea anemone Nematostella vectensis. This analysis reveals the complex biomechanical transformations underpinning the operating mechanism of nematocysts, one of the nature’s most exquisite biological micro-machines. Further, this study will provide insight into the form and function of related cnidarian organelles and serve as a template for the design of bioinspired microdevices.

The stinging organelles of jellyfish, sea anemones, and other cnidarians, known as nematocysts, are remarkable cellular weapons used for both predation and defense. Nematocysts consist of a pressurized capsule containing a coiled harpoon-like thread. These structures are in turn built within specialized cells known as nematocytes. When triggered, the capsule explosively discharges, ejecting the coiled thread which punctures the target and rapidly elongates by turning inside out in a process called eversion. Due to the structural complexity of the thread and the extreme speed of discharge, the precise mechanics of nematocyst firing have remained elusive7. Here, using a combination of live and super-resolution imaging, 3D electron microscopy, and genetic perturbations, we define the step-by-step sequence of nematocyst operation in the model sea anemone Nematostella vectensis. This analysis reveals the complex biomechanical transformations underpinning the operating mechanism of nematocysts, one of nature’s most exquisite biological micro-machines. Further, this study will provide insight into the form and function of related cnidarian organelles and serve as a template for the design of bioinspired microdevices.

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

As the planarian research community expands, the need for an interoperable data organization framework for tool building has become increasingly apparent. Such software would streamline data annotation and enhance cross-platform and cross-species searchability. We created the Planarian Anatomy Ontology (PLANA), an extendable relational framework of defined Schmidtea mediterranea (Smed) anatomical terms used in the field. At publication, PLANA contains over 850 terms describing Smed anatomy from subcellular to system levels across all life cycle stages, in intact animals and regenerating body fragments. Terms from other anatomy ontologies were imported into PLANA to promote interoperability and comparative anatomy studies. To demonstrate the utility of PLANA as a tool for data curation, we created resources for planarian embryogenesis, including a staging series and molecular fate-mapping atlas, and the Planarian Anatomy Gene Expression database, which allows retrieval of a variety of published transcript/gene expression data associated with PLANA terms. As an open-source tool built using FAIR (findable, accessible, interoperable, reproducible) principles, our strategy for continued curation and versioning of PLANA also provides a platform for community-led growth and evolution of this resource., Summary: Description of the construction of an anatomy ontology tool for planaria with examples of its potential use to curate and mine data across multiple experimental platforms.

Mammalian inner ear and fish lateral line sensory hair cells depend on fluid motion to transduce environmental signals and elicit a response. In mammals, actively maintained ionic homeostasis of the cochlear and vestibular fluid (endolymph) is essential for hair cell function and numerous mammalian hearing and vestibular disorders arise from disrupted endolymph ion homeostasis. Lateral line hair cells, however, are openly exposed to the aqueous environment with fluctuating ionic composition. How sensory transduction in the lateral line is maintained during environmental changes of ionic composition is not fully understood. Using lineage labeling,in vivotime lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. Furthermore, the invasive behavior is adaptive as it is triggered by drastic fluctuations in environmental stimuli. Our findings challenge the notion of an entirely placodally-derived lateral line and identify Nm ionocytes as regulators of mechanosensory hair cell function by modulating the ionic microenvironment. The discovery of lateral line ionocytes provides an experimentally accessiblein vivosystem to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism to selectively kill meiotic products (spores) that do not inherit wtf4. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate trafficking, as mutations that disrupt either of these processes result in cell death in the presence of the Wtf4 proteins. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy spores., eLife digest Meiotic drivers are genes that break the normal rules of inheritance. Usually, a gene has a 50% chance of passing on to the next generation. Meiotic drivers force their way into the next generation by poisoning the gametes (the sex cells that combine to form a zygote) that do not carry them. Harnessing the power of genetic drivers could allow scientists to spread beneficial genes across populations. One group of meiotic drivers found in fission yeast is called the 'with transposon fission yeast', or 'wtf' gene family. The wtf drivers act during the production of spores, which are the fission yeast equivalent of sperm, and they encode both a poison that can destroy the spores and its antidote. The poison spreads through the sac holding the spores, and can affect all of them, while the antidote only protects the spores that make it. This means that the spores carrying the wtf genes survive, while the rest of the spores are killed. To understand whether it is possible to use the wtf meiotic drivers to spread other genes, perhaps outside of fission yeast, scientists must first establish exactly how the proteins coded for by genes behave. To do this, Nuckolls et al. examined a member of the wtf family called wtf4. Attaching a fluorescent label to the poison and antidote proteins produced by wtf4 made it possible to see what they do. This revealed that the poison clumps, forming toxic aggregates that damage yeast spores. The antidote works by mopping up these aggregates and moving them to the cell's main storage compartment, called the vacuole. Mutations that disrupted the ability of the antidote to interact with the poison or its ability to move the poison into storage stopped the antidote from working. Nuckolls et al. also showed that if genetic engineering was used to introduce wtf4 into a distantly related species of budding yeast the effects of this meiotic driver were the same. This suggests that the wtf genes may be good candidates for future genetic engineering experiments. Engineered systems known as 'gene drives' could spread beneficial genetic traits through populations. This could include disease-resistance genes in crops, or disease-preventing genes in mosquitoes. The wtf genes are small and work independently of other genes, making them promising candidates for this type of system. These experiments also suggest that the wtf genes could be useful for understanding why clumps of proteins are toxic to cells. Future work could explore why clumps of wtf poison kill spores, while clumps of poison plus antidote do not. This could aid research into human ailments caused by protein clumps, such as Huntington’s or Alzheimer’s disease.

The budding yeast centrosome, often called the spindle pole body (SPB), nucleates microtubules for chromosome segregation during cell division. An appendage, called the half bridge, attaches to one side of the SPB and regulates SPB duplication and separation. Like DNA, the SPB is duplicated only once per cell cycle. During meiosis, however, after one round of DNA replication, two rounds of SPB duplication and separation are coupled with homologue segregation in meiosis I and sister-chromatid segregation in meiosis II. How SPB duplication and separation are regulated during meiosis remains to be elucidated, and whether regulation in meiosis differs from that in mitosis is unclear. Here we show that overproduction of the half-bridge component Kar1 leads to premature SPB separation during meiosis. Furthermore, excessive Kar1 induces SPB overduplication to form supernumerary SPBs, leading to chromosome missegregation and erroneous ascospore formation. Kar1-­mediated SPB duplication bypasses the requirement of dephosphorylation of Sfi1, another half-bridge component previously identified as a licensing factor. Our results therefore reveal an unexpected role of Kar1 in licensing meiotic SPB duplication and suggest a unique mechanism of SPB regulation during budding yeast meiosis.

Sing et al. characterize an unanticipated role for the Saccharomyces cerevisiae RSC complex in ploidy maintenance. They show that RSC promotes the distribution of Nbp1 and Ndc1 to the spindle pole body (SPB) to facilitate SPB maturation and accurate chromosome segregation., Ploidy is tightly regulated in eukaryotic cells and is critical for cell function and survival. Cells coordinate multiple pathways to ensure replicated DNA is segregated accurately to prevent abnormal changes in chromosome number. In this study, we characterize an unanticipated role for the Saccharomyces cerevisiae “remodels the structure of chromatin” (RSC) complex in ploidy maintenance. We show that deletion of any of six nonessential RSC genes causes a rapid transition from haploid to diploid DNA content because of nondisjunction events. Diploidization is accompanied by diagnostic changes in cell morphology and is stably maintained without further ploidy increases. We find that RSC promotes chromosome segregation by facilitating spindle pole body (SPB) duplication. More specifically, RSC plays a role in distributing two SPB insertion factors, Nbp1 and Ndc1, to the new SPB. Thus, we provide insight into a role for a SWI/SNF family complex in SPB duplication and ploidy maintenance.

The cell nuclei of Ophisthokonts, the eukaryotic supergroup defined by fungi and metazoans, is remarkable in the constancy of both their double-membraned structure and protein composition. Such remarkable structural conservation underscores common and ancient evolutionary origins. Yet, the dynamics of disassembly and reassembly displayed by Ophisthokont nuclei vary extensively. Besides closed mitosis in fungi and open mitosis in some animals, little is known about the evolution of nuclear envelope break down (NEBD) during cell division. Here, we uncovered a novel form of NEBD in primary oocytes of the flatwormSchmidtea mediterranea. From zygotene to metaphase II, both nuclear envelope (NE) and peripheral endoplasmic reticulum (ER) expand notably in size, likely involvingde novomembrane synthesis. 3-D electron microscopy reconstructions demonstrated that the NE transforms itself into numerous double-membraned vesicles similar in membrane architecture to NE doublets in mammalian oocytes after germinal vesicle breakdown. The vesicles are devoid of nuclear pore complexes and DNA, yet are loaded with nuclear proteins, including a planarian homologue of PIWI, a protein essential for the maintenance of stem cells in this and other organisms. Our data contribute a new model to the canonical view of NE dynamics and support that NEBD is an evolutionarily adaptable trait in multicellular organisms.

Summary Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, in vivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible in vivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Few tools exist to examine the inner nuclear membrane (INM) in living cells, and the INM-specific proteome is poorly characterized. Smoyer et al. combine a split-GFP screen and fluorescence correlation spectroscopy analysis to identify membrane proteins that access the INM and study INM-specific interactions., Understanding the protein composition of the inner nuclear membrane (INM) is fundamental to elucidating its role in normal nuclear function and in disease; however, few tools exist to examine the INM in living cells, and the INM-specific proteome remains poorly characterized. Here, we adapted split green fluorescent protein (split-GFP) to systematically localize known and predicted integral membrane proteins in Saccharomyces cerevisiae to the INM as opposed to the outer nuclear membrane. Our data suggest that components of the endoplasmic reticulum (ER) as well as other organelles are able to access the INM, particularly if they contain a small extraluminal domain. By pairing split-GFP with fluorescence correlation spectroscopy, we compared the composition of complexes at the INM and ER, finding that at least one is unique: Sbh2, but not Sbh1, has access to the INM. Collectively, our work provides a comprehensive analysis of transmembrane protein localization to the INM and paves the way for further research into INM composition and function.

Organ morphogenesis depends on the precise orchestration of cell migration, cell shape changes and cell adhesion. We demonstrate that Notch signaling is an integral part of the Wnt and Fgf signaling feedback loop coordinating cell migration and the self-organization of rosette-shaped sensory organs in the zebrafish lateral line system. We show that Notch signaling acts downstream of Fgf signaling to not only inhibit hair cell differentiation but also to induce and maintain stable epithelial rosettes. Ectopic Notch expression causes a significant increase in organ size independently of proliferation and the Hippo pathway. Transplantation and RNASeq analyses revealed that Notch signaling induces apical junctional complex genes that regulate cell adhesion and apical constriction. Our analysis also demonstrates that in the absence of patterning cues normally provided by a Wnt/Fgf signaling system, rosettes still self-organize in the presence of Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.21049.001

SummaryAggregation of damaged or misfolded proteins is a protective mechanism against proteotoxic stress, abnormalities of which underlie many aging-related diseases. Here, we show that in asymmetrically dividing yeast cells, aggregation of cytosolic misfolded proteins does not occur spontaneously but requires new polypeptide synthesis and is restricted to the surface of ER, which harbors the majority of active translation sites. Protein aggregates formed on ER are frequently also associated with or are later captured by mitochondria, greatly constraining aggregate mobility. During mitosis, aggregates are tethered to well-anchored maternal mitochondria, whereas mitochondria acquired by the bud are largely free of aggregates. Disruption of aggregate-mitochondria association resulted in increased mobility and leakage of mother-accumulated aggregates into the bud. Cells with advanced replicative age exhibit gradual decline of aggregates-mitochondria association, likely contributing to their diminished ability to rejuvenate through asymmetric cell division.

Millions of people around the world are affected by cystic kidney diseases, which are amongst the most common inherited genetic disorders. Throughout their life, people with these diseases develop fluid-filled cysts in their kidneys, which stop these organs from working properly and can eventually lead to organ failure. Healthy kidneys perform many vital roles in the body, including removing waste products and keeping the concentration of salts in the blood in balance. These activities depend on the kidneys filtering the blood, and then reabsorbing useful chemicals from the filtered fluid as it passes through small tubes called tubules. Cysts disrupt both of these processes. Mutations in many different genes can cause cystic kidney diseases. Many of these genes encode proteins that are involved in the formation of cilia: hair-like structures that project from some cell membranes. Cilia on the cells that line tubules are thought to bend in response to the flow of fluid and then generate signals that dampen cell proliferation. This would explain how the loss of cilia could cause too many cells to develop, which would lead to the formation of cysts. But many of the molecular details are missing from this explanation. Previous studies in mammals and simple model organisms (such as fruit flies and roundworms) have sought to fill in the gaps, but each model has its own limitations. Now, Thi-Kim Vu et al. propose that a flat worm called a planarian could provide a new and experimentally accessible animal model to study cystic kidney diseases. These flat worms get rid of their waste products via an excretory system that consists of branched tubules that spread throughout the body. Thi-Kim Vu et al. found that, like the tubules in the kidney, these tubules filter and then reabsorb chemicals from body fluids. Moreover, these processes are performed in different parts of the tubules, exactly as they are in the tubules in kidneys. The genome of a flat worm called Schmidtea mediterranea contains many genes that cause cysts to form when their equivalents are mutated in humans. Reducing the expression of these genes (and others that are involved in cilia formation) also caused cysts to form in the flat worms. These findings indicate that it is likely that the excretory systems of different animals have a shared evolutionary history. If so, the findings support the idea that cilia in kidney tubules send signals in response to fluid flow that affect kidney-specific stem cells. They also suggest that problems with these signals could be at the core of some human cystic kidney diseases. One of the next challenges will be to identify these cilia-associated signals. Finally, given that studies involving thousands of flat worms can be carried out with minimal cost, the ultimate goal is to develop flat worms into a new model to discover and investigate genes linked to human kidney diseases.

Background & Aims Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. Methods We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. Results CD44 + CD24 lo CD166 + cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44 + CD24 lo CD166 + GRP78 lo/– putative stem cells from mouse small intestine included Lgr5-GFP hi and Lgr5-GFP med/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. Conclusions We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44 + CD24 lo CD166 + , GRP78 lo/– , and c-Kit − facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44 + CD24 −/lo CD166 + also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.