The UNC-83/UNC-84 LINC members are required for body wall muscle nuclei positioning in C. elegans

From a mutagenesis screen in the nematode C. elegans we isolated the mutant bar18, showing an accumulation of muscle cell nuclei around the posterior pharyngeal bulb of the worm. Quantification of the overall amount of body wall muscle nuclei, based on the muscle-specific reporter myo-3p::gfp::NLS, revealed that the number of nuclei in bar18 mutants is unchanged compared to WT worms. The accumulation of muscle nuclei around the posterior pharyngeal bulb is due to a positioning defect, which can be precisely quantified by subdividing the worm into head, neck, and posterior body segments. Whole-genome sequencing revealed that bar18 animals carry a mutation in the KASH-domain gene unc-83 causing a premature STOP. An additional unc-83 mutant allele recapitulates the phenotype, as does a mutant allele of UNC-84, a SUN-domain containing protein that interacts with UNC-83. UNC-83 and UNC-84 belong to a Linker of Nucleoskeleton and Cytoskeletonnuclear (LINC) complex that bridges the nuclear lamina with the cytoskeleton. SUN and KASH domain proteins are conserved in mammals and mutations in the corresponding genes have been linked to cancer, autism, muscular dystrophy and other diseases. Additionally, LINC complexes that function in nuclear migration have also been identified in mammals. We were able to rescue the unc-83 mutant phenotype by expressing the WT gene under a muscle-specific (myo-3p) promoter, demonstrating that the effect is cell autonomous. Mutations in either unc-83 or unc-84 have previously been linked to nuclear migration defects in P cells, intestinal cells and hyp7 hypodermal precursors but not in muscles. Whether the mis-positioning of muscle nuclei is due to migration or anchoring defects still needs to be determined

Strategies for in vivo reprogramming

Reprogramming has the potential to provide specific cell types for regenerative medicine applications aiming at replacing tissues that have been lost or damaged due to degenerative diseases and injury. In this review we discuss the latest strategies and advances of in vivo reprogramming to convert cell identities in living organisms, including reprogramming induced by transcription factors (TFs) and CRISPR/dCas9 synthetic TFs, as well as by cell fusion and small molecules. We also provide a brief recap of reprogramming barriers, the effect of senescence on reprogramming efficiency, and strategies to deliver reprogramming factors in vivo. Because of the limited space, we omit dwelling on naturally occurring reprogramming phenomena such as developmentally programmed transdifferentiation found in the nematode Caenorhabditis elegans

A novel, low-volume method for organ culture of embryonic kidneys that allows development of cortico-medullary anatomical organization

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1-3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium-around 85 microliters-using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle. © 2010 Sebinger et al

The C. elegans pseudogene sspt-16 (F55A3.7) is required to safeguard germ cells against reprogramming

We recently identified FAcilitates Chromatin Transcription (FACT) as a reprogramming barrier of transcription factor (TF) mediated conversion of germ cells into neurons in C. elegans. FACT is a conserved heterodimer consisting of SPT16 and SSRP1 in mammals. Duplication events during evolution in C. elegans generated two SSRP1 homologs named HMG-3 and HMG-4, while SPT-16 is the only homolog of SPT16. Yet, the pseudogene F55A3.7 has nearly complete nucleotide sequence homology to the spt-16 gene. However, F55A3.7 lacks some spt-16 exons and DNA pieces so we named it sspt-16 (short spt-16). Surprisingly, the deletion mutant ok1829, which affects only the sspt-16 pseudogene, shows similar germ cell reprogramming effects as described previously for FACT-depleted animals. We examined whether lack of sspt-16 affects other genes or chromatin accessibility, which may explain the permissiveness for germ cell reprogramming

The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique 'CONJUDOR'. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput

FACT sets a barrier for cell fate reprogramming in Caenorhabditis elegans and human cells

The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection

Proteomic and transcriptomic changes in hibernating grizzly bears reveal metabolic and signaling pathways that protect against muscle atrophy

Muscle atrophy is a physiological response to disuse and malnutrition, but hibernating bears are largely resistant to this phenomenon. Unlike other mammals, they efficiently reabsorb amino acids from urine, periodically activate muscle contraction, and their adipocytes differentially responds to insulin. The contribution of myocytes to the reduced atrophy remains largely unknown. Here we show how metabolism and atrophy signaling are regulated in skeletal muscle of hibernating grizzly bear. Metabolic modeling of proteomic changes suggests an autonomous increase of non-essential amino acids (NEAA) in muscle and treatment of differentiated myoblasts with NEAA is sufficient to induce hypertrophy. Our comparison of gene expression in hibernation versus muscle atrophy identified several genes differentially regulated during hibernation, including Pdk4 and Serpinf1. Their trophic effects extend to myoblasts from non-hibernating species (including C. elegans), as documented by a knockdown approach. Together, these changes reflect evolutionary favored adaptations that, once translated to the clinics, could help improve atrophy treatment

A Novel, Low-Volume Method for Organ Culture of Embryonic Kidneys That Allows Development of Cortico-Medullary Anatomical Organization

Here, we present a novel method for culturing kidneys in low volumes of medium that offers more organotypic development compared to conventional methods. Organ culture is a powerful technique for studying renal development. It recapitulates many aspects of early development very well, but the established techniques have some disadvantages: in particular, they require relatively large volumes (1–3 mls) of culture medium, which can make high-throughput screens expensive, they require porous (filter) substrates which are difficult to modify chemically, and the organs produced do not achieve good cortico-medullary zonation. Here, we present a technique of growing kidney rudiments in very low volumes of medium–around 85 microliters–using silicone chambers. In this system, kidneys grow directly on glass, grow larger than in conventional culture and develop a clear anatomical cortico-medullary zonation with extended loops of Henle

ECM modulated early kidney development in embryonic organ culture

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases

The CONDOR pipeline for simultaneous knockdown of multiple genes identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in C. elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique ‘CONDOR’. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput