Identification of regulators of germ stem cell enwrapment by its niche in C. elegans.

Many stem cell niches contain support cells that increase contact with stem cells by enwrapping them in cellular processes. One example is the germ stem cell niche in C. elegans, which is composed of a single niche cell termed the distal tip cell (DTC) that extends cellular processes, constructing an elaborate plexus that enwraps germ stem cells. To identify genes required for plexus formation and to explore the function of this specialized enwrapping behavior, a series of targeted and tissue-specific RNAi screens were performed. Here we identify genes that promote stem cell enwrapment by the DTC plexus, including a set that specifically functions within the DTC, such as the chromatin modifier lin-40/MTA1, and others that act within the germline, such as the 14-3-3 signaling protein par-5. Analysis of genes that function within the germline to mediate plexus development reveal that they are required for expansion of the germ progenitor zone, supporting the emerging idea that germ stem cells signal to the niche to stimulate enwrapping behavior. Examination of wild-type animals with asymmetric plexus formation and animals with reduced DTC plexus elaboration via loss of two candidates including lin-40 indicate that cellular enwrapment promotes GLP-1/Notch signaling and germ stem cell fate. Together, our work identifies novel regulators of cellular enwrapment and suggests that reciprocal signaling between the DTC niche and the germ stem cells promotes enwrapment behavior and stem cell fate.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the C. elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor 2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

Definitive hematopoietic stem cells minimally contribute to embryonic hematopoiesis.

Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

Comprehensive Endogenous Tagging of Basement Membrane Components Reveals Dynamic Movement within the Matrix Scaffolding.

Basement membranes (BMs) are supramolecular matrices built on laminin and type IV collagen networks that provide structural and signaling support to tissues. BM complexity, however, has hindered an understanding of its formation, dynamics, and regulation. Using genome editing, we tagged 29 BM matrix components and receptors in C. elegans with mNeonGreen. Here, we report a common template that initiates BM formation, which rapidly diversifies during tissue differentiation. Through photobleaching studies, we show that BMs are not static-surprisingly, many matrix proteins move within the laminin and collagen scaffoldings. Finally, quantitative imaging, conditional knockdown, and optical highlighting indicate that papilin, a poorly studied glycoprotein, is the most abundant component in the gonadal BM, where it facilitates type IV collagen removal during BM expansion and tissue growth. Together, this work introduces methods for holistic investigation of BM regulation and reveals that BMs are highly dynamic and capable of rapid change to support tissues.

Ectopic Germ Cells Can Induce Niche-like Enwrapment by Neighboring Body Wall Muscle.

Niche cell enwrapment of stem cells and their differentiating progeny is common and provides a specialized signaling and protective environment. Elucidating the mechanisms underlying enwrapment behavior has important basic and clinical significance in not only understanding how niches are formed and maintained but also how they can be engineered and how they are misregulated in human pathologies, such as cancer. Previous work in C. elegans found that, when germ cells, which are enwrapped by somatic gonadal niche cells, are freed into the body cavity, they embed into other tissues. We investigated this phenomenon using live-cell imaging and discovered that ectopic germ cells preferentially induce body-wall muscle to extend cellular processes that enwrap the germ cells, the extent of which was strikingly similar to the distal tip cell (DTC)-germ stem cell niche. Enwrapment was specific for escaped germ cells, and genetic analysis revealed it did not depend on pathways that control cell death and engulfment or muscle arm extension. Instead, using a large-scale RNAi screen and GFP knockin strains, we discovered that the enwrapping behavior of muscle relied upon the same suite of cell-cell adhesion molecules that functioned in the endogenous niche: the C. elegans E-cadherin HMR-1, its intracellular associates α-catenin (HMP-1) and ß-catenin (HMP-2), and the L1CAM protein SAX-7. This ectopic niche-like behavior resembles the seed-and-soil model of cancer metastasis and offers a new model to understand factors regulating ectopic niche formation.

Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome.

Pericentromeric satellite repeats are enriched in 5-methylcytosine (5mC). Loss of 5mC at these sequences is common in cancer and is a hallmark of Immunodeficiency, Centromere and Facial abnormalities (ICF) syndrome. While the general importance of 5mC is well-established, the specific functions of 5mC at pericentromeres are less clear. To address this deficiency, we generated a viable animal model of pericentromeric hypomethylation through mutation of the ICF-gene ZBTB24. Deletion of zebrafish zbtb24 caused a progressive loss of 5mC at pericentromeres and ICF-like phenotypes. Hypomethylation of these repeats triggered derepression of pericentromeric transcripts and activation of an interferon-based innate immune response. Injection of pericentromeric RNA is sufficient to elicit this response in wild-type embryos, and mutation of the MDA5-MAVS dsRNA-sensing machinery blocks the response in mutants. These findings identify activation of the innate immune system as an early consequence of pericentromeric hypomethylation, implicating derepression of pericentromeric transcripts as a trigger of autoimmunity. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Spliceosomal component Sf3b1 is essential for hematopoietic differentiation in zebrafish.

SF3B1 (Splicing factor 3b, subunit 1) is one of the most commonly mutated factors in myelodysplastic syndrome (MDS). Although the genetic correlation between SF3B1 mutations and MDS etiology are quite strong, no in vivo model currently exists to explore how SF3B1 loss alters blood cell development. Using zebrafish mutants, we show here that proper function of Sf3b1 is required for all hematopoietic lineages. As in MDS patients, zebrafish sf3b1 mutants develop a macrocytic-anemia-like phenotype due to a block in maturation at a late progenitor stage. The mutant embryos also develop neutropenia, because their primitive myeloid cells fail to mature and turn on differentiation markers such as l-plastin and myeloperoxidase. In contrast, production of definitive hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelial cells within the dorsal aorta is greatly diminished, whereas arterial endothelial cells are correctly fated. Notch signaling, imperative for the endothelial-to-hematopoietic transition, is also normal, indicating that HSPC induction is blocked in sf3b1 mutants downstream or independent of Notch signaling. The data demonstrate that Sf3b1 function is necessary during key differentiation fate decisions in multiple blood cell types. Zebrafish sf3b1 mutants offer a novel animal model with which to explore the role of splicing in hematopoietic development and provide an excellent in vivo system with which to delve into the question of why and how Sf3b1 dysfunction is detrimental to hematopoietic differentiation, which could improve MDS diagnosis and treatment.

Digging the New York City Skyline: soil fungal communities in green roofs and city parks.

In urban environments, green roofs provide a number of benefits, including decreased urban heat island effects and reduced energy costs for buildings. However, little research has been done on the non-plant biota associated with green roofs, which likely affect their functionality. For the current study, we evaluated whether or not green roofs planted with two native plant communities in New York City functioned as habitats for soil fungal communities, and compared fungal communities in green roof growing media to soil microbial composition in five city parks, including Central Park and the High Line. Ten replicate roofs were sampled one year after planting; three of these roofs were more intensively sampled and compared to nearby city parks. Using Illumina sequencing of the fungal ITS region we found that green roofs supported a diverse fungal community, with numerous taxa belonging to fungal groups capable of surviving in disturbed and polluted habitats. Across roofs, there was significant biogeographical clustering of fungal communities, indicating that community assembly of roof microbes across the greater New York City area is locally variable. Green roof fungal communities were compositionally distinct from city parks and only 54% of the green roof taxa were also found in the park soils. Phospholipid fatty acid analysis revealed that park soils had greater microbial biomass and higher bacterial to fungal ratios than green roof substrates. City park soils were also more enriched with heavy metals, had lower pH, and lower quantities of total bases (Ca, K, and Mg) compared to green roof substrates. While fungal communities were compositionally distinct across green roofs, they did not differentiate by plant community. Together, these results suggest that fungi living in the growing medium of green roofs may be an underestimated component of these biotic systems functioning to support some of the valued ecological services of green roofs.