Anne Grapin-Botton

The human pancreas secretes digestive fluid that flows through an extensive ductal network before emptying into the duodenum. However, little is known about how hydrodynamic forces influence pancreatic function and development. We hypothesized that pancreatic cells respond to physiological levels of fluid shear stress. In this study, we developed a microfluidic system featuring reversible sealing between the device layer and the cell culture substrate, enabling (1) conventional open-well cell culture prior to fluid shear stress application and (2) selective patterning of proteins and cells for migration studies. Channel dimensions were chosen to support 24-hour perfusion using standard syringe pumps. Using this platform, we investigated effects of physiological shear stress levels representing basal and activated pancreatic function (2, 8 and 33 mPa) on human iPSC-derived pancreatic progenitor cells. Pancreatic progenitor cells exhibit graded sensitivity to shear stress at the transcriptomic level. Specifically, shear stress upregulated expression of cell cycle-related gene sets; endocrine-associated genes including NEUROG3 ; and Notch receptor ligands ( DLL1 , DLL3 ) in a magnitude-dependent manner. The exposure to unidirectional fluid flow also resulted in a reduction in YAP1 protein content, decreased WWTR1 gene expression, and downregulation of YAP/TAZ target genes. Furthermore, barrier-based migration assays under shear stress revealed that cell patches expanded their boundaries both along and against the flow, with greater distance in the direction of flow. Our system and findings may help lay the foundation for understanding biophysical regulation of developmental processes in a physiologically important, yet hard-to-access, epithelial model system.

Although lumen formation is a key feature of organogenesis, luminal compartments – formed through the secretion of fluid, ions and macromolecules by the cells, usually on the apical side – are often viewed as passive by-products of epithelial polarity. Here, we discuss how luminal fluids actively contribute to morphogenesis in metazoans through mechanical, hydraulic and electrochemical interactions with the surrounding epithelia and the extracellular matrix (ECM). We first outline the diversity of lumen architectures and summarize conserved mechanisms of lumen nucleation, fusion, folding and growth across metazoans. We then present the physical principles governing lumen morphogenesis, highlighting how hydrostatic and osmotic pressures, cortical tension and matrix mechanics interact to shape tissues. Furthermore, theoretical modeling and numerical simulations provide a quantitative framework for predicting phenotypes related to lumen shape, exploring the parameter space of hydraulic and active tissue mechanics variables, and linking molecular perturbations to emergent tissue geometries. Finally, we examine feedback loops whereby lumen-associated forces regulate junctional integrity, proliferation, differentiation and tissue patterning. Together, these concepts position luminal fluids as active regulators of lumen morphogenesis.

In this issue of Genes & Development , Unger and colleagues (doi:10.1101/gad.353153.125) combined human pluripotent stem cell-derived in vitro models with targeted in vivo mouse models to reveal multiple developmental defects triggered by HNF1A mutations causing maturity-onset diabetes of the young. This work paints the picture of a disorder that starts well before diabetes manifests, highlighting its complexity arising from the diverse roles of HNF1A across distinct cell types, each potentially differentially impacted by different mutations.

Background: While pancreatic cysts have been described in syndromic ciliopathies, the pancreas is not commonly recognized as a target organ. However, several ciliary gene knockout mouse models develop a pancreatic phenotype combining acinar atrophy and adipocyte accumulation, hereby called adipopancreatosis, suggesting a link between ciliary dysfunction and pancreatic disease. Objective: We investigated whether mutations in ciliopathy-associated genes are linked to pancreatic dysfunction in humans. Design: We analyzed a cohort of 341 patients with pediatric-onset pancreatic anomalies and characterized the pancreatic phenotype of new mouse models with conditional Nphp3 inactivation or bearing Nphp3 mutations recapitulating human mutations. In patients, pancreatic fat content was quantified using Dixon-MRI. Results: Mutations in the cilium-related HNF1B and NPHP3 were identified in patients presenting with both renal and pancreatic dysfunction. Nphp3 mutant mice developed acinar atrophy, adipopancreatosis, and moderate inflammation. Adipocytes in the pancreas exhibited a white adipocyte-like profile and likely originated from mesothelial-derived fibroblasts. Reduced numbers and altered length of ductal cilia were monitored. Interestingly, secretory canaliculi, typically unnoticed structures found within and between acinar cells and connected to the acinar lumen, exhibited a microcystic morphology. Consistent with the mouse phenotype, Dixon-MRI revealed significantly increased pancreatic fat content in patients with HNF1B and NPHP3 mutations. Conclusion: We describe a previously unrecognized pancreatic manifestation of ciliopathies, which we name ciliogenic pancreatopathy. Patients with known ciliopathy-causing mutations should be evaluated for this pancreatic condition, particularly those with kidney disease, as concomitant exocrine pancreatic insufficiency may further compromise renal function or the outcome of kidney graft. What is already known on this topic: Ciliopathies, resulting from defects in primary cilia, are genetic disorders primarily affecting the kidney and liver. Pancreatic cysts have been sporadically reported in syndromic ciliopathies. The pancreas is not currently recognized as a major target organ of ciliary dysfunction. A clear link between ciliary gene mutations and pancreatic anomalies is still unknown. Animal studies have suggested a possible association between ciliary dysfunction and pancreatic anomalies. What this study adds: Identifies HNF1B and NPHP3 mutations as genetic causes of a pancreatic phenotype characterized by acinar atrophy and adipose replacement (adipopancreatosis). Demonstrates the presence of defective ductal cilia and moderate inflammation in the pancreas of Nphp3 mutant mice. Reveals that secretory canaliculi in the exocrine pancreas of Nphp3 mutant mice acquire a microcystic morphology. Shows that patients with HNF1B or NPHP3 mutations have significantly increased pancreatic fat content by Dixon-MRI. Defines a new disease entity, ciliogenic pancreatopathy, as a pancreatic manifestation of ciliopathies. How this study might affect research, practice or policy: Establishes the pancreas as a novel and clinically relevant target of ciliopathies. Expands the phenotypic spectrum of HNF1B- and NPHP3-related diseases to include exocrine pancreatic dysfunction. Suggests that patients with ciliopathy-causing mutations should be evaluated for exocrine pancreatic insufficiency. Highlights the need to consider pancreatic function monitoring in kidney disease and transplant settings. Opens new research avenues into the role of primary cilia in pancreatic homeostasis and disease.

Lumen formation in organ epithelia involves processes such as polarization, secretion, exocytosis and contractility, but what controls lumen shape remains unclear. Here we study how lumina develop spherical or complex structures using pancreatic organoids. Combining computational phase-field modelling and experiments, we found that lumen morphology depends on the balance between cell cycle duration and lumen pressure, low pressure and high proliferation produce complex shapes. Manipulating proliferation and lumen pressure can alter or reverse lumen development both in silico and in vitro. Increasing epithelial permeability reduces lumen pressure, converting from spherical to complex lumina. During pancreas development, the epithelium is initially permeable and becomes sealed, experimentally increasing permeability at late stages impairs ductal morphogenesis. Overall, our work underscores how proliferation, pressure and permeability orchestrate lumen shape, offering insights for tissue engineering and cystic disease treatment.

Many internal organs in the body harbor a fluid-filled lumen. Lumen nucleation and fusion have been reported as dependent on organ-type during organogenesis. In contrast, the physics of lumen suggests that force balance between luminal pressure and cell mechanics leads to generic rules. However, this hypothesis lacks experimental evidence. Here we compare lumen dynamics for three different systems (MDCK cysts, pancreatic spheres, and epiblast model) by using quantitative cell biology, microfabrication, and theory. We report that the initial cell number determines the maximum number of lumens but does not impact the steady state, which is a final single lumen. We show that lumen dynamics is determined by luminal hydrostatic pressure. We also use MDCK cysts to manipulate cell adhesion and lumen volume to successfully reproduce the fusion dynamics of pancreatic spheres and epiblasts. Our results reveal self-organisation rules of lumens with relevance for morphogenesis and tissue engineering.

Genetically encoded fluorescent biosensors are widely used to monitor small molecule and ion levels in living cells. Quantitative FRET and FLIM sensors and highly sensitive intensiometric sensors have been developed for many analytes. Notwithstanding notable advances over the last years, a universal high-performance sensor design for absolute quantification that does not require specialized equipment has yet to be developed. We here report the GEQO platform of quantitative biosensors featuring calcium, ATP, cAMP, and organelle-specific variants. We used GEQO sensors to follow calcium and cAMP transients in immortalised cells, human pancreatic progenitor cells, and zebrafish embryos. We show that GEQO-based absolute quantification allows to account for analyte buffering and retains information in time trace data lost during relative quantification. GEQO biosensors will enable quantitative analyte measurements across a wide range of imaging platforms, a key prerequisite for diagnostic applications and quantitative approaches in basic cell biology.