3D Neurovascular Unit Tissue Model to Assess Responses to Traumatic Brain Injury.

The neurovascular unit (NVU) is a critical interface in the central nervous system that links vascular interactions with glial and neural tissue. Disruption of the NVU has been linked to the onset and progression of neurodegenerative diseases. Despite its significance the NVU remains challenging to study in a physiologically relevant manner. Here, a 3D cell triculture model of the NVU is developed that incorporates human primary brain microvascular endothelial cells, astrocytes, and pericytes into a tissue system that can be sustained in vitro for several weeks. This tissue model helps recapitulate the complexity of the NVU and can be used to interrogate the mechanisms of disease and cell-cell interactions. The NVU tissue model displays elevated cell death and inflammatory responses following mechanical damage, to emulate traumatic brain injury (TBI) under controlled laboratory conditions, including lactate dehydrogenase (LDH) release, elevated inflammatory markers TNF-α and monocyte chemoattractant cytokines MCP-2 and MCP-3 and reduced expression of the tight junction marker ZO-1. This 3D tissue model serves as a tool for deciphering mechanisms of TBIs and immune responses associated with the NVU.

Microbes affect gut epithelial cell composition through immune-dependent regulation of intestinal stem cell differentiation.

Gut microbes play important roles in host physiology; however, the mechanisms underlying their impact remain poorly characterized. Here, we demonstrate that microbes not only influence gut physiology but also alter its epithelial composition. The microbiota and pathogens both influence intestinal stem cell (ISC) differentiation. Intriguingly, while the microbiota promotes ISC differentiation into enterocytes (EC), pathogens stimulate enteroendocrine cell (EE) fate and long-term accumulation of EEs in the midgut epithelium. Importantly, the evolutionarily conserved Drosophila NFKB (Relish) pushes stem cell lineage specification toward ECs by directly regulating differentiation factors. Conversely, the JAK-STAT pathway promotes EE fate in response to infectious damage. We propose a model in which the balance of microbial pattern recognition pathways, such as Imd-Relish, and damage response pathways, such as JAK-STAT, influence ISC differentiation, epithelial composition, and gut physiology.

Multiscale analysis reveals that diet-dependent midgut plasticity emerges from alterations in both stem cell niche coupling and enterocyte size.

The gut is the primary interface between an animal and food, but how it adapts to qualitative dietary variation is poorly defined. We find that the Drosophila midgut plastically resizes following changes in dietary composition. A panel of nutrients collectively promote gut growth, which sugar opposes. Diet influences absolute and relative levels of enterocyte loss and stem cell proliferation, which together determine cell numbers. Diet also influences enterocyte size. A high sugar diet inhibits translation and uncouples intestinal stem cell proliferation from expression of niche-derived signals, but, surprisingly, rescuing these effects genetically was not sufficient to modify diet's impact on midgut size. However, when stem cell proliferation was deficient, diet's impact on enterocyte size was enhanced, and reducing enterocyte-autonomous TOR signaling was sufficient to attenuate diet-dependent midgut resizing. These data clarify the complex relationships between nutrition, epithelial dynamics, and cell size, and reveal a new mode of plastic, diet-dependent organ resizing.

Recruitment of Adult Precursor Cells Underlies Limited Repair of the Infected Larval Midgut in Drosophila.

Surviving infection requires immune and repair mechanisms. Developing organisms face the additional challenge of integrating these mechanisms with tightly controlled developmental processes. The larval Drosophila midgut lacks dedicated intestinal stem cells. We show that, upon infection, larvae perform limited repair using adult midgut precursors (AMPs). AMPs differentiate in response to damage to generate new enterocytes, transiently depleting their pool. Developmental delay allows for AMP reconstitution, ensuring the completion of metamorphosis. Notch signaling is required for the differentiation of AMPs into the encasing, niche-like peripheral cells (PCs), but not to differentiate PCs into enterocytes. Dpp (TGF-ß) signaling is sufficient, but not necessary, to induce PC differentiation into enterocytes. Infection-induced JAK-STAT pathway is both required and sufficient for differentiation of AMPs and PCs into new enterocytes. Altogether, this work highlights the constraints imposed by development on an organism's response to infection and demonstrates the transient use of adult precursors for tissue repair.

Hippo, TGF-ß, and Src-MAPK pathways regulate transcription of the upd3 cytokine in Drosophila enterocytes upon bacterial infection.

Cytokine signaling is responsible for coordinating conserved epithelial regeneration and immune responses in the digestive tract. In the Drosophila midgut, Upd3 is a major cytokine, which is induced in enterocytes (EC) and enteroblasts (EB) upon oral infection, and initiates intestinal stem cell (ISC) dependent tissue repair. To date, the genetic network directing upd3 transcription remains largely uncharacterized. Here, we have identified the key infection-responsive enhancers of the upd3 gene and show that distinct enhancers respond to various stresses. Furthermore, through functional genetic screening, bioinformatic analyses and yeast one-hybrid screening, we determined that the transcription factors Scalloped (Sd), Mothers against dpp (Mad), and D-Fos are principal regulators of upd3 expression. Our study demonstrates that upd3 transcription in the gut is regulated by the activation of multiple pathways, including the Hippo, TGF-ß/Dpp, and Src, as well as p38-dependent MAPK pathways. Thus, these essential pathways, which are known to control ISC proliferation cell-autonomously, are also activated in ECs to promote tissue turnover the regulation of upd3 transcription.

Regional Cell-Specific Transcriptome Mapping Reveals Regulatory Complexity in the Adult Drosophila Midgut.

Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, we generated RNA-sequencing transcriptomes of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, we identify transcriptional diversities among cell types and document regional differences within each cell type that define further specialization. We validate cell-specific and regional Gal4 drivers; demonstrate roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation, and study the transcriptional response of midgut cells upon infection. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions.

Methods to assess intestinal stem cell activity in response to microbes in Drosophila melanogaster.

Drosophila melanogaster presents itself as a powerful model for studying the somatic stem cells of the gut and how bacteria affect intestinal homeostasis. The Gal4/UAS/Gal80 (ts) system allows for temporally controlled expression of fluorescent proteins, RNAi knock-down, and other genetic constructs targeted to specific cell populations in the midgut. Similarly, FLP/FRT-mediated somatic recombinations in intestinal stem cells (ISCs) are utilized to visualize and analyze the clonal lineages of individual or populations of stem cells. Live imaging microscopy and immunofluorescence allow both qualitative and quantitative characterization of stem cell shape, proliferation, and differentiation. Here, we detail the use of these tools and techniques for studying gut performance during and following a bacterial infection in the adult fruit fly.