Blood myeloid cells differentiate to lung resident cells and respond to pathogen stimuli in a 3D human tissue-engineered lung model.

Introduction: Respiratory infections remain a leading global health concern. Models that recapitulate the cellular complexity of the lower airway of humans will provide important information about how the immune response reflects the interactions between diverse cell types during infection. We developed a 3D human tissue-engineered lung model (3D-HTLM) composed of primary human pulmonary epithelial and endothelial cells with added blood myeloid cells that allows assessment of the innate immune response to respiratory infection. Methods: The 3D-HTLM consists of small airway epithelial cells grown at air-liquid interface layered on fibroblasts within a collagen matrix atop a permeable membrane with pulmonary microvascular endothelial cells layered underneath. After the epithelial and endothelial layers had reached confluency, an enriched blood monocyte population, containing mostly CD14+ monocytes (Mo) with minor subsets of CD1c+ classical dendritic cells (cDC2s), monocyte-derived dendritic cells (Mo-DCs), and CD16+ non-classical monocytes, was added to the endothelial side of the model. Results: Immunofluorescence imaging showed the myeloid cells migrate through and reside within each layer of the model. The myeloid cell subsets adapted to the lung environment in the 3D-HTLM, with increased proportions of the recovered cells expressing lung tissue resident markers CD206, CD169, and CD163 compared with blood myeloid cells, including a population with features of alveolar macrophages. Myeloid subsets recovered from the 3D-HTLM displayed increased expression of HLA-DR and the co-stimulatory markers CD86, CD40, and PDL1. Upon stimulation of the 3D-HTLM with the toll-like receptor 4 (TLR4) agonist bacterial lipopolysaccharide (LPS), the CD31+ endothelial cells increased expression of ICAM-1 and the production of IL-10 and TNFα was dependent on the presence of myeloid cells. Challenge with respiratory syncytial virus (RSV) led to increased expression of macrophage activation and antiviral pathway genes by cells in the 3D-HTLM. Discussion: The 3D-HTLM provides a lower airway environment that promotes differentiation of blood myeloid cells into lung tissue resident cells and enables the study of respiratory infection in a physiological cellular context.

The cohesin modifier ESCO2 is stable during DNA replication.

Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.

Single-cell transcriptomics identifies Keap1-Nrf2 regulated collective invasion in a Drosophila tumor model.

Apicobasal cell polarity loss is a founding event in epithelial-mesenchymal transition and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, we discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, our findings describe the comprehensive transcriptome of cells within the follicle cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis.

In the body, most cells exhibit some form of spatial asymmetry: the compartments within the cell are not evenly distributed, thereby allowing the cells to know whether a surface is on the 'outside' or the 'inside' of a tissue or organ. In the cells of epithelial tissues, which line most of the cavities and the organs in the body, this asymmetry is known as apical-basal polarity. Maintaining apical-basal polarity in epithelial cells is one of the main barriers that stops cancer cells from invading other tissues, which is the first step of metastasis, the process through which cancer cells leave their tissue of our origin and spread to distant locations in the body. In the fruit fly Drosophila melanogaster, scientists have engineered cells in several tissues to stop producing the proteins that help establish apical-basal polarity, in an effort to study the earliest steps of tumor formation. Unfortunately, these experiments frequently lead to rampant metastasis, making it difficult to identify the earliest changes that make the tumor cells more likely to become invasive. Therefore, finding a tissue in which loss of apical-basal polarity does not cause aggressive cancer progression is necessary to address this gap in knowledge. The epithelial cell layer lining the ovaries of fruit flies may be such a tissue. When these cells lose their apical-basal polarity, rather than becoming metastatic and spreading to distant organs, they interleave with each other, forming a tumorous growth that only invades into the neighboring compartment. Chatterjee et al. used this system to study individual invasive cells. They wanted to know whether the genes that these cells switch on and off are known to be involved in human cancers, and if so, which of them control the invasive behavior of tumor cells. Chatterjee et al. determined that when cells in the fruit-fly ovary lost their polarity, they turned genes on and off in a pattern similar to that seen both in mammalian cancers and in tumors from other fly tissues. One of the notable changes they observed in the ovarian cells that lost apical-basal polarity was the activation of the Keap1/Nrf2 oxidative-stress signaling pathway, which normally protects cells from damage caused by excessive oxidation. In the ovarian cells, however, the activation of these genes also led to aggressive invasion of the collective tumor cells into the neighboring compartment. Interestingly, this increase in invasiveness was characterized by polarized changes within the cells, specifically in the scaffolding that allows cells to keep their shape and move: the edge of the cells leading the invasion had greater levels of a protein called actin, which enables the cells to protrude into the neighboring compartments. Chatterjee et al. have identified a new mechanism that impacts the migratory behavior of cells. Insights from their findings will pave the way for a better understanding of how and when this mechanism plays a role in metastasis.

Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells.

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs) are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in 12 â€‹h during midoogenesis. Previously, we reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. Here we report that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study we also used single-cell RNA sequencing data to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened.

Modeling Notch-Induced Tumor Cell Survival in the Drosophila Ovary Identifies Cellular and Transcriptional Response to Nuclear NICD Accumulation.

Notch is a conserved developmental signaling pathway that is dysregulated in many cancer types, most often through constitutive activation. Tumor cells with nuclear accumulation of the active Notch receptor, NICD, generally exhibit enhanced survival while patients experience poorer outcomes. To understand the impact of NICD accumulation during tumorigenesis, we developed a tumor model using the Drosophila ovarian follicular epithelium. Using this system we demonstrated that NICD accumulation contributed to larger tumor growth, reduced apoptosis, increased nuclear size, and fewer incidents of DNA damage without altering ploidy. Using bulk RNA sequencing we identified key genes involved in both a pre- and post- tumor response to NICD accumulation. Among these are genes involved in regulating double-strand break repair, chromosome organization, metabolism, like raptor, which we experimentally validated contributes to early Notch-induced tumor growth. Finally, using single-cell RNA sequencing we identified follicle cell-specific targets in NICD-overexpressing cells which contribute to DNA repair and negative regulation of apoptosis. This valuable tumor model for nuclear NICD accumulation in adult Drosophila follicle cells has allowed us to better understand the specific contribution of nuclear NICD accumulation to cell survival in tumorigenesis and tumor progression.

Analysis of the Temporal Patterning of Notch Downstream Targets during Drosophila melanogaster Egg Chamber Development.

Living organisms require complex signaling interactions and proper regulation of these interactions to influence biological processes. Of these complex networks, one of the most distinguished is the Notch pathway. Dysregulation of this pathway often results in defects during organismal development and can be a causative mechanism for initiation and progression of cancer. Despite previous research entailing the importance of this signaling pathway and the organismal processes that it is involved in, less is known concerning the major Notch downstream targets, especially the onset and sequence in which they are modulated during normal development. As timing of regulation may be linked to many biological processes, we investigated and established a model of temporal patterning of major Notch downstream targets including broad, cut, and hindsight during Drosophila melanogaster egg chamber development. We confirmed the sequential order of Broad upregulation, Hindsight upregulation, and Cut downregulation. In addition, we showed that Notch signaling could be activated at stage 4, one stage earlier than the stage 5, a previously long-held belief. However, our further mitotic marker analysis re-stated that mitotic cycle continues until stage 5. Through our study, we once again validated the effectiveness and reliability of our MATLAB toolbox designed to systematically identify egg chamber stages based on area size, ratio, and additional morphological characteristics.

A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis.

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.

The Ecdysone and Notch Pathways Synergistically Regulate Cut at the Dorsal-Ventral Boundary in Drosophila Wing Discs.

Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal-ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. Here, we show that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, while ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, we find that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, our findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, we show br promotes Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc.

Sponge hybridomas: applications and implications.

Many sponge-derived natural products with applications to human health have been discovered over the past three decades. In vitro production has been proposed as one biological alternative to ensure adequate supply of marine natural products for preclinical and clinical development of drugs. Although primary cell cultures have been established for many marine phyla, no cell lines with an extended life span have been established for marine invertebrates. Hybridoma technology has been used for production of monoclonal antibodies for application to human health. We hypothesized that a sponge cell line could be formed by fusing sponge cells of one species with those of another, or by fusing sponge cells with rapidly dividing, marine-derived, non-sponge cells. Using standard methods for formation of hybridomas, with appropriate modifications for temperature and salinity, cells from individuals of the same sponge species, as well as cells from individuals of two different sponge species were successfully fused. Research in progress is focused on optimizing fusion to produce a cell line and to stimulate expression of natural products with therapeutic relevance. Experimental hybridomas may also be used as models to test hypotheses related to naturally occurring sponge chimeras and hybridomas.