Periodic Reporting for period 1 - MECHANOGUT (Probing the role of mechanosensory pathways in regulating intestinal stem cell activity and gut homeostasis)
Periodo di rendicontazione: 2024-02-01 al 2026-01-31
Sintesi del contesto e degli obiettivi generali del progetto
The intestinal epithelium maintains remarkable resilience against diverse environmental and physiological challenges through the adaptive proliferation and turnover of intestinal stem cells (ISCs). This dynamic renewal is tightly regulated to preserve gut barrier integrity, as dysregulated ISC activity can lead to pathological hyperplasia. Although extensive work has identified numerous biochemical factors, autocrine, paracrine, and juxtacrine, that govern ISC homeostasis, a key unresolved question is how tissue-scale mechanical forces are sensed and transduced into biochemical signals to maintain ISC-niche equilibrium.
The gut is continuously exposed to stretch, strain, and other biomechanical stresses, and accumulating evidence suggests that mechanical cues are central regulators of adult stem cell behavior. To dissect these mechanisms, this project leverages the adult Drosophila melanogaster midgut, a powerful and tractable model with a pseudostratified epithelial organization and stem cell architecture strikingly similar to the mammalian intestine. As a foundation for this work, an unbiased RNAi-based genetic screen targeting niche-expressed receptors was conducted under conditions of gut challenge using Erwinia carotovora carotovora infection. Survival assays revealed a significant enrichment of receptors involved in mechanotransductive signaling pathways, components essential for maintaining epithelial integrity during stress.
Building on these preliminary findings, this project aims to uncover how mechanosensory inputs regulate ISC behavior and preserve midgut homeostasis. By integrating genetic, biomechanical, and imaging approaches, the work will elucidate fundamental principles by which mechanical forces interface with stem cell regulatory networks. These insights will advance our understanding of epithelial resilience and may reveal conserved mechanisms relevant to mammalian gut health and disease.
The gut is continuously exposed to stretch, strain, and other biomechanical stresses, and accumulating evidence suggests that mechanical cues are central regulators of adult stem cell behavior. To dissect these mechanisms, this project leverages the adult Drosophila melanogaster midgut, a powerful and tractable model with a pseudostratified epithelial organization and stem cell architecture strikingly similar to the mammalian intestine. As a foundation for this work, an unbiased RNAi-based genetic screen targeting niche-expressed receptors was conducted under conditions of gut challenge using Erwinia carotovora carotovora infection. Survival assays revealed a significant enrichment of receptors involved in mechanotransductive signaling pathways, components essential for maintaining epithelial integrity during stress.
Building on these preliminary findings, this project aims to uncover how mechanosensory inputs regulate ISC behavior and preserve midgut homeostasis. By integrating genetic, biomechanical, and imaging approaches, the work will elucidate fundamental principles by which mechanical forces interface with stem cell regulatory networks. These insights will advance our understanding of epithelial resilience and may reveal conserved mechanisms relevant to mammalian gut health and disease.
Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti
The project was organized into three main work programs:
WP1 – How Mechanical Forces Influence Stem Cells in the Intestine
In this part of the project, I studied how physical forces in the gut affect the way intestinal stem cells divide and turn into mature cell types. To do this, I first developed a method to keep the fruit fly gut alive outside the body so I could film how the cells behave in real time.
To create gentle stretching forces in the gut, I fed the flies tiny plastic beads (3 micrometers in size). These beads cannot be digested, so they stretch the gut lining as they pass through. I then used a marker called pH3 (phosphorylated Histone3) to measure how much stem cells divide under these conditions.
To understand how mechanical stress affects the process of replacing old cells with new ones, I used a special genetic system called esg‑REDDM. This system uses two fluorescent colors that fade at different speeds, allowing me to see which cells are active stem/progenitor cells and which ones have recently matured. I also used an antibody called Prospero to specifically identify one of the major mature cell types in the gut. Combining these tools allowed me to measure how mechanical stretching affects the formation of both major gut cell lineages.
I also triggered stronger mechanical disturbances by creating tiny wounds in the gut. I did this either by using a laser or by briefly feeding flies very high doses of bacteria, which creates small holes in the gut lining. With live imaging, I compared how stem cells respond to these injuries versus how they behave in a healthy gut.
WP2 – How Specific Genes (Pak3 and Cirl) Help Cells Sense Mechanical Stress
In this work package, I examined two molecules, Pak3 and Cirl, that are thought to help gut cells sense and respond to mechanical forces. I used RNA interference (RNAi) to reduce the levels of these molecules in different cell types of the gut.
At first, I targeted enterocytes (the main absorptive cells), but the results were inconsistent. I discovered that one of the genetic tools I used was unintentionally active in stem cells as well. After switching to cleaner tools, I knocked down cirl specifically in stem and progenitor cells and showed that Cirl helps control how stem cells are arranged and maintained in the gut.
Using detailed imaging, I studied how epithelial cells behave when Pak3 or Cirl is removed. In the context of injury, I identified a signaling pathway (Pvf1–PVR) that guides stem cells to move toward wounds so they can divide and repair the damaged area. This part of the work was conducted together with a PhD student in the lab, and the results were published in a high‑impact open‑access journal. I supervised and trained the PhD student during this phase.
After injury, stem cells usually migrate, divide, and then spread back out into a normal pattern. I tested whether Cirl in stem cells and its partner Toll‑8 in enterocytes help restore this pattern after damage. By reducing these molecules in the relevant cell types, I showed how they influence the re‑establishment of normal stem cell spacing during recovery.
WP3 – The Role of CCN Proteins in Mechanically Controlled Stem Cell Behavior
In the final part of the project, I identified a new candidate called CCN (Cellular Communication Network) as a possible regulator of how stem cells respond to mechanical forces. To study this, I reduced CCN levels in stem and progenitor cells while again applying mechanical stress through plastic‑bead feeding.
Using quantitative imaging, I measured how losing CCN affects stem‑cell division and differentiation when the gut is under mechanical load. This work is still ongoing, and I am currently supervising a PhD student who is finishing the experimental and analytical components.
WP1 – How Mechanical Forces Influence Stem Cells in the Intestine
In this part of the project, I studied how physical forces in the gut affect the way intestinal stem cells divide and turn into mature cell types. To do this, I first developed a method to keep the fruit fly gut alive outside the body so I could film how the cells behave in real time.
To create gentle stretching forces in the gut, I fed the flies tiny plastic beads (3 micrometers in size). These beads cannot be digested, so they stretch the gut lining as they pass through. I then used a marker called pH3 (phosphorylated Histone3) to measure how much stem cells divide under these conditions.
To understand how mechanical stress affects the process of replacing old cells with new ones, I used a special genetic system called esg‑REDDM. This system uses two fluorescent colors that fade at different speeds, allowing me to see which cells are active stem/progenitor cells and which ones have recently matured. I also used an antibody called Prospero to specifically identify one of the major mature cell types in the gut. Combining these tools allowed me to measure how mechanical stretching affects the formation of both major gut cell lineages.
I also triggered stronger mechanical disturbances by creating tiny wounds in the gut. I did this either by using a laser or by briefly feeding flies very high doses of bacteria, which creates small holes in the gut lining. With live imaging, I compared how stem cells respond to these injuries versus how they behave in a healthy gut.
WP2 – How Specific Genes (Pak3 and Cirl) Help Cells Sense Mechanical Stress
In this work package, I examined two molecules, Pak3 and Cirl, that are thought to help gut cells sense and respond to mechanical forces. I used RNA interference (RNAi) to reduce the levels of these molecules in different cell types of the gut.
At first, I targeted enterocytes (the main absorptive cells), but the results were inconsistent. I discovered that one of the genetic tools I used was unintentionally active in stem cells as well. After switching to cleaner tools, I knocked down cirl specifically in stem and progenitor cells and showed that Cirl helps control how stem cells are arranged and maintained in the gut.
Using detailed imaging, I studied how epithelial cells behave when Pak3 or Cirl is removed. In the context of injury, I identified a signaling pathway (Pvf1–PVR) that guides stem cells to move toward wounds so they can divide and repair the damaged area. This part of the work was conducted together with a PhD student in the lab, and the results were published in a high‑impact open‑access journal. I supervised and trained the PhD student during this phase.
After injury, stem cells usually migrate, divide, and then spread back out into a normal pattern. I tested whether Cirl in stem cells and its partner Toll‑8 in enterocytes help restore this pattern after damage. By reducing these molecules in the relevant cell types, I showed how they influence the re‑establishment of normal stem cell spacing during recovery.
WP3 – The Role of CCN Proteins in Mechanically Controlled Stem Cell Behavior
In the final part of the project, I identified a new candidate called CCN (Cellular Communication Network) as a possible regulator of how stem cells respond to mechanical forces. To study this, I reduced CCN levels in stem and progenitor cells while again applying mechanical stress through plastic‑bead feeding.
Using quantitative imaging, I measured how losing CCN affects stem‑cell division and differentiation when the gut is under mechanical load. This work is still ongoing, and I am currently supervising a PhD student who is finishing the experimental and analytical components.
Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)
Using a tissue-stretcher, I showed that different epithelial cells of the gut lining have different biomechanical properties. The absorptive enterocytes are elastic and can be stretched in response to mechanical stretching. Stem and progenitor cells are solid-like and are more resistant to stretching forces. When the fruit flies were subjected to mechanical stress by feeding them with micron-sized beads, I observed an increase in ISC division and a specific increase in newly produced enteroendocrine cells. Such biomechanical signaling-dependent differentiation of ISCs to secretory enteroendocrine cells required CCN function in the stem and progenitor cells. Currently, we are testing the role of CCN downstream of mechanosensory channel proteins like Piezo and TrpA1, to regulate force-dependent upregulation of secretory cell production.
Although ISCs normally remain relatively stationary within their niche, I showed that localized epithelial damage triggers rapid, directed ISC migration toward the wound within just 2–6 hours. Mechanical and chemical insults, such as short‑term exposure to DSS or brief oral infection with Pseudomonas entomophila, generate small epithelial breaches identified by the loss of E‑Cadherin and leakage of fluorescent dextran through the gut barrier. Using live imaging of whole-mount gut explants, the authors demonstrate that ISCs actively move toward these disrupted regions, forming clusters around damaged sites. Importantly, this migration occurs before any detectable ISC divisions, indicating that motility is the earliest regenerative response. The PDGF/VEGF‑related receptor Pvr was identified as an essential regulator of ISC motility. When Pvr is specifically depleted in ISCs, both ISC migration and the formation of actin-based cellular protrusions are completely abolished. These protrusions are normally induced within hours after injury and reflect dynamic cytoskeletal remodeling required for movement. As a consequence of Pvr loss, infection‑ or damage‑induced ISC proliferation and epithelial turnover are also strongly suppressed, demonstrating a tight link between ISC motility and regenerative divisions. Conversely, artificial activation of Pvr within ISCs is sufficient to trigger broad, flattened cell morphologies, robust Rac1 activation, lamellipodia formation, and accelerated tissue turnover even in the absence of damage. Together, these findings show that Pvr signaling is both necessary and sufficient to drive ISC activation during regeneration.
The gut lining is made of a single layer of cells that must constantly replace themselves to maintain proper function. This replacement is driven by special cells called stem cells. For the tissue to work correctly, these stem cells need to be evenly spaced so they can produce new cells wherever they are needed. When the gut is injured, for example, by infection or toxins, this organized pattern is disrupted. Stem cells leave their usual positions and move toward the damaged area to help repair it. Once the wound is healed, the tissue must carefully rebuild its original pattern of evenly spaced stem cells. My work sheds light on two proteins on the surfaces of gut cells, called Cirl and Toll‑8, that play an essential role in rebuilding this organization. These two proteins are found in different cell types, Cirl on stem cells and the Toll-8 on mature enterocytes. Because of this, they act like a “matching pair” that helps neighboring cells recognize each other. When stem cells and mature cells interact through these proteins, they create tiny mechanical forces at their shared boundaries. These forces help maintain proper spacing and ensure that stem cells remain in the right places.
Overall, my findings show that Cirl and Toll‑8 together act as a system that helps the gut maintain its architecture and restore it after injury. By converting differences in cell identity into physical signals, they help the tissue organize itself during both normal upkeep and regeneration. Because the molecules we studied are evolutionarily conserved, these results may shed light on how other organs in humans maintain their structure and recover from damage.
Although ISCs normally remain relatively stationary within their niche, I showed that localized epithelial damage triggers rapid, directed ISC migration toward the wound within just 2–6 hours. Mechanical and chemical insults, such as short‑term exposure to DSS or brief oral infection with Pseudomonas entomophila, generate small epithelial breaches identified by the loss of E‑Cadherin and leakage of fluorescent dextran through the gut barrier. Using live imaging of whole-mount gut explants, the authors demonstrate that ISCs actively move toward these disrupted regions, forming clusters around damaged sites. Importantly, this migration occurs before any detectable ISC divisions, indicating that motility is the earliest regenerative response. The PDGF/VEGF‑related receptor Pvr was identified as an essential regulator of ISC motility. When Pvr is specifically depleted in ISCs, both ISC migration and the formation of actin-based cellular protrusions are completely abolished. These protrusions are normally induced within hours after injury and reflect dynamic cytoskeletal remodeling required for movement. As a consequence of Pvr loss, infection‑ or damage‑induced ISC proliferation and epithelial turnover are also strongly suppressed, demonstrating a tight link between ISC motility and regenerative divisions. Conversely, artificial activation of Pvr within ISCs is sufficient to trigger broad, flattened cell morphologies, robust Rac1 activation, lamellipodia formation, and accelerated tissue turnover even in the absence of damage. Together, these findings show that Pvr signaling is both necessary and sufficient to drive ISC activation during regeneration.
The gut lining is made of a single layer of cells that must constantly replace themselves to maintain proper function. This replacement is driven by special cells called stem cells. For the tissue to work correctly, these stem cells need to be evenly spaced so they can produce new cells wherever they are needed. When the gut is injured, for example, by infection or toxins, this organized pattern is disrupted. Stem cells leave their usual positions and move toward the damaged area to help repair it. Once the wound is healed, the tissue must carefully rebuild its original pattern of evenly spaced stem cells. My work sheds light on two proteins on the surfaces of gut cells, called Cirl and Toll‑8, that play an essential role in rebuilding this organization. These two proteins are found in different cell types, Cirl on stem cells and the Toll-8 on mature enterocytes. Because of this, they act like a “matching pair” that helps neighboring cells recognize each other. When stem cells and mature cells interact through these proteins, they create tiny mechanical forces at their shared boundaries. These forces help maintain proper spacing and ensure that stem cells remain in the right places.
Overall, my findings show that Cirl and Toll‑8 together act as a system that helps the gut maintain its architecture and restore it after injury. By converting differences in cell identity into physical signals, they help the tissue organize itself during both normal upkeep and regeneration. Because the molecules we studied are evolutionarily conserved, these results may shed light on how other organs in humans maintain their structure and recover from damage.