Biology subjects

Bagnat, M.

Publications and source records attributed to Bagnat, M..

14 recordsLinked to original sources

bioRxiv2026

Clathrin-coated tubules mediate rapid and selective protein absorption by lysosome-rich enterocytes

In the digestive tract of neonatal mammals and in fish, lysosome-rich enterocytes (LREs) internalize dietary proteins and digest them intracellularly within a large lysosomal vacuole. Impaired LRE function leads to growth stunting and poor survival in zebrafish and mice. Previous work has shown that protein uptake relies on a scavenger receptor complex. However, how LREs can internalize proteins rapidly enough to support organismal growth is unclear. Here, using live and electron microscopy and genetic manipulations in zebrafish, we find that LREs contain apical micron-sized clathrin-coated endocytic tubules (ETs) that perform rapid internalization of proteins and other soluble cargoes, while excluding resident apical membrane proteins. Dab2, an endocytic adaptor linking clathrin to scavenger receptors, controls both the biogenesis and function of ETs by recruiting unconventional myosin Myo6 and by influencing dynamin activity. Our findings suggest that continuous pinching of vesicles from ETs allows for rapid, high-capacity internalization of dietary protein.

cell biology↗

bioRxiv2026

Hydrostatic pressure shapes and canalizes semicircular canal morphology to ensure vestibular function

How tissues acquire reproducible shapes to support their function is a fundamental question in developmental biology. Zebrafish semicircular canals form when epithelial pillars partition the lumen of the otic vesicle, the embryonic precursor of the inner ear, into the tubes that sense head rotation for balance. We show that hydrostatic pressure generated by the inflating otic vesicle shapes pillar geometry. Acute vesicle deflation shortens and broadens pillars, whereas pharmacologically induced inflation elongates and narrows them, effects captured by a physical model that treats the vesicle as a pressurized viscoelastic shell. Pillars initially form with variable curvature, but continued vesicle expansion drives them toward a common, straight geometry, a process accelerated by inflation and blocked by deflation. We identify Wnt signaling as crucial for sustaining this pressure by maintaining epithelial barrier integrity. Its disruption impairs pillar geometry and vestibular-dependent swimming behavior. Together, these findings identify hydrostatic pressure as a critical physical mechanism that canalizes tissue geometry to ensure robust organ formation and function.

developmental biology↗

bioRxiv2026

Genome wide transcriptional changes underlie gradual and recurrent adaptation to protein malnutrition in zebrafish

Lysosome-rich enterocytes (LREs) are a specialized population of intestinal cells that mediate the uptake and absorption of dietary proteins in fish and neonatal mammals. Loss of LRE function causes inhibited growth and poor survival due to protein malnutrition. Previously, we reported that in zebrafish loss of Plasmolipin (pllp), an endosomal membrane protein highly expressed in LREs, impairs LRE differentiation and dietary protein absorption, resulting in marked reduction in survival rates. Raising pllp homozygous mutants surviving to adulthood and in-crossing them for multiple generations resulted in their adaptation to malnutrition, with adapted pllp mutants showing no survival deficits. To uncover mechanisms underlying this phenomenon, we compared the older adapted pllp allele with genetically related wild type (WT) fish and a newly generated pllp mutant allele. Using transcriptome profiling and quantitative protein absorption assays, we found that adapted pllp mutants exhibit upregulation of LRE endocytic components, resulting in a capacity for protein absorption that exceeds that of WT. This hyperactivation of LRE endocytic and absorptive activity is aided by a fine-tuned transcriptional regulation of immune genes that may contribute to the enhanced survival of pllp mutants in the face of increased exposure to environmental antigens. Genetic analyses indicate that these adaptations emerge gradually and are recurrent as shown experimentally by the adaptation of a mutant allele upon inbreeding and natural selection. Overall, our study illustrates that genome wide transcriptional changes underly adaptation mechanisms that enhance intestinal function and organismal survival in response to protein malnutrition. Author SummaryIn this study, we investigated how zebrafish adapt to protein malnutrition when the function of specialized intestinal protein-absorbing enterocytes, also found in newborn mammals, is impaired. We observed that fish carrying a mutation that severely disrupts intestinal protein absorption gradually recovered their ability to survive over multiple generations of inbreeding, even though the underlying mutation remained intact. By comparing gene activity in these adapted fish with that of newly generated mutants, we found that adaptation involves a coordinated rewiring of two systems: the enterocytes themselves became hyperactivated, absorbing more protein than even wildtype fish, while the immune system was simultaneously recalibrated to dampen inflammation. We further showed that this adaptive process is recurrent by using a second, independently generated mutant line that underwent a strikingly similar recovery trajectory over successive generations. Together, our findings reveal that animals can overcome a severe, heritable nutritional deficit through a gradual, genome-wide transcriptional response that fundamentally reshapes intestinal function across generations.

developmental biology↗

bioRxiv2025

A self-limiting mechanotransduction feedback loop ensures robust organ formation

Organ morphogenesis uses mechanotransduction feedback loops to convert forces into gene expression changes that regulate cell mechanics. How these loops integrate with developmental programs to ensure robust outcomes remains unclear. We show Yap mechanotransduction establishes a self-limiting positive feedback loop for semicircular canal formation in zebrafish. Canal development proceeds through bud initiation, extension, and fusion within the otic epithelium. Local swelling of hyaluronan-rich extracellular matrix (ECM) in the bud activates Yap in a spatial pattern. Yap induces its target ccn1l1, promoting further ECM expansion to sustain bud extension. This feedback loop confers developmental robustness: graded knockdown of ccn1l1 reduces extension rate, yet canal formation persists and fails only with strong disruption. Critically, the loop contains its own termination mechanism. During bud fusion, PKA-CREB signaling, activated by an adhesion GPCR, gpr126, suppresses ccn1l1, ending the loop. These findings reveal how mechanotransduction loops with built-in termination provide developmental control by integrating mechanical forces, transcriptional responses, and morphogenetic outcomes.

developmental biology↗

bioRxiv2025

Tissue-wide, synchronous Erk oscillations time the segmentation of the zebrafish notochord

The generation of a segmented body plan is a hallmark of vertebrate development. Biological oscillators provide a mechanism for timing the formation of repeated structures, yet to date only a few examples of signaling oscillators have been identified in development. Here, we show that the periodic addition of mineralizing segments in the zebrafish notochord is timed by tissue-wide oscillations of Erk activity. We report delayed oscillations in the transcription of spry and dusp genes, suggesting that they act as negative feedback. Erk activity is regulated by EGFR signaling and increased egf expression marks the emergence of oscillations via an infinite-period bifurcation, revealing the mechanism controlling the onset of notochord segmentation and the tunability of the oscillator. Our results show a remarkable degree of synchronization across the notochord and reveal an instance of biological clocks timing a patterning process, crucial for the development of the vertebral column from the notochord.

developmental biology↗

bioRxiv2024

Unsaturated lipids as key control points for caveola formation and disassembly

Caveolae are specialized plasma membrane domains with a unique lipid composition. Recent work has implicated lipid peroxidation in triggering caveola disassembly, releasing caveolar proteins to regulate the cellular response to oxidative stress. Here we investigated the role of specific lipid species in caveola formation and response to oxidative stress. Systematic screening of lipid enzymes identified ACSL4, a critical enzyme in the synthesis of polyunsaturated fatty acid-containing lipids, and enzymes involved in the biosynthesis of pro-ferroptotic ether lipids, as key regulators of caveola formation. Omega-6 fatty acids promoted efficient caveola formation, while their oxidation or displacement by omega-3 or monounsaturated fatty acids disrupted this process. Moreover, we found that omega-6 fatty acid-containing membrane lipids were required for oxidative stress-induced caveola disassembly. These findings unveil a new model for caveola formation, highlighting specific unsaturated lipids as essential caveolar components and key control points for caveola disassembly in response to an oxidizing environment.

cell biology↗

bioRxiv2024

High-speed 4D fluorescence light field tomography of whole freely moving organisms

Volumetric fluorescence imaging techniques, such as confocal, multiphoton, light sheet, and light field microscopy, have become indispensable tools across a wide range of cellular, developmental, and neurobiological applications. However, it is difficult to scale such techniques to the large 3D fields of view (FOV), volume rates, and synchronicity requirements for high-resolution 4D imaging of freely behaving organisms. Here, we present reflective Fourier light field computed tomography (ReFLeCT), a new high-speed volumetric fluorescence computational imaging technique. ReFLeCT synchronously captures entire tomograms of multiple unrestrained, unanesthetized model organisms over multi-millimeter 3D FOVs at 120 volumes per second. In particular, we applied ReFLeCT to reconstruct 4D videos of fluorescently labeled zebrafish and Drosophila larvae, enabling us to study their heartbeat, fin and tail motion, gaze, jaw motion, and muscle contractions with nearly isotropic 3D resolution while they are freely moving. As a novel approach for snapshot tomographic capture, ReFLeCT is a major advance towards bridging the gap between current volumetric fluorescence microscopy techniques and macroscopic behavioral imaging.

animal behavior and cognition↗

bioRxiv2024

Protein absorption in the zebrafish gut is regulated by interactions between lysosome rich enterocytes and the microbiome

Dietary protein absorption in neonatal mammals and fishes relies on the function of a specialized and conserved population of highly absorptive lysosome rich enterocytes (LREs). The gut microbiome has been shown to enhance absorption of nutrients, such as lipids, by intestinal epithelial cells. However, whether protein absorption is also affected by the gut microbiome is poorly understood. Here, we investigate connections between protein absorption and microbes in the zebrafish gut. Using live microscopy-based quantitative assays, we find that microbes slow the pace of protein uptake and degradation in LREs. While microbes do not affect the number of absorbing LRE cells, microbes lower the expression of endocytic and protein digestion machinery in LREs. Using transgene assisted cell isolation and single cell RNA-sequencing, we characterize all intestinal cells that take up dietary protein. We find that microbes affect expression of bacteria-sensing and metabolic pathways in LREs, and that some secretory cell types also take up protein and share components of protein uptake and digestion machinery with LREs. Using custom-formulated diets, we investigated the influence of diet and LRE activity on the gut microbiome. Impaired protein uptake activity in LREs, along with a protein-deficient diet, alters the microbial community and leads to increased abundance of bacterial genera that have the capacity to reduce protein uptake in LREs. Together, these results reveal that diet-dependent reciprocal interactions between LREs and the gut microbiome regulate protein absorption.

cell biology↗

bioRxiv2024

Phased ERK-responsiveness and developmental robustness regulate teleost skin morphogenesis

Elongation of the vertebrate embryonic axis necessitates rapid expansion of the epidermis to accommodate the growth of underlying tissues. Here, we generated a toolkit to visualize and quantify signaling in entire cell populations of periderm, the outermost layer of the epidermis, in live developing zebrafish. We find that oriented cell divisions facilitate growth of the early periderm during axial elongation rather than cell addition from the basal layer. Activity levels of ERK, a downstream effector of MAPK pathway, gauged by a live biosensor, predicts cell cycle entry, and optogenetic ERK activation controls proliferation dynamics. As development proceeds, rates of peridermal cell proliferation decrease, ERK activity becomes more pulsatile and functionally transitions to promote hypertrophic cell growth. Targeted genetic blockade of cell division generates animals with oversized periderm cells, yet, unexpectedly, development to adulthood is not impaired. Our findings reveal stage-dependent differential responsiveness to ERK signaling and marked developmental robustness in growing teleost skin.

developmental biology↗

bioRxiv2024

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aortas (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces. HighlightsO_LIPlexin-D1 mechanosensing of blood flow tunes the caliber of the Dorsal Aorta (DA) C_LIO_LIThe DA widens without raising endothelial cell numbers, which can change separate from the caliber C_LIO_LIThe Kruppel-like transcription factor 2 (KLF2) is a key Plexin-D1 mechano-effector during development C_LIO_LIKLF2 increases endothelial cell size to expand the DA caliber C_LI

developmental biology↗

bioRxiv2023

Rediscovering the Rete Ovarii: a secreting auxiliary structure to the ovary

The rete ovarii (RO) is an appendage of the ovary that has been given little attention. Although the RO appears in drawings of the ovary in early versions of Grays Anatomy, it disappeared from recent textbooks, and is often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.

developmental biology↗

bioRxiv2023

Axial segmentation by iterative mechanical signaling

In bony fishes, formation of the vertebral column, or spine, is guided by a metameric blueprint established in the epithelial sheath of the notochord. Generation of the notochord template begins days after somitogenesis and even occurs in the absence of somite segmentation. However, patterning defects in the somites lead to imprecise notochord segmentation, suggesting these processes are linked. Here, we reveal that spatial coordination between the notochord and the axial musculature is necessary to ensure segmentation of the zebrafish spine both in time and space. We find that the connective tissues that anchor the axial skeletal musculature, known as the myosepta in zebrafish, transmit spatial patterning cues necessary to initiate notochord segment formation, a critical pre-patterning step in spine morphogenesis. When an irregular pattern of muscle segments and myosepta interact with the notochord sheath, segments form non-sequentially, initiate at atypical locations, and eventually display altered morphology later in development. We determine that locations of myoseptum-notochord connections are hubs for mechanical signal transmission, which are characterized by localized sites of deformation of the extracellular matrix (ECM) layer encasing the notochord. The notochord sheath responds to the external mechanical changes by locally augmenting focal adhesion machinery to define the initiation site for segmentation. Using a coarse-grained mathematical model that captures the spatial patterns of myoseptum-notochord interactions, we find that a fixed-length scale of external cues is critical for driving sequential segment patterning in the notochord. Together, this work identifies a robust segmentation mechanism that hinges upon mechanical coupling of adjacent tissues to control patterning dynamics.

developmental biology↗

bioRxiv2021

Knock-in tagging in zebrafish facilitated by insertion into non-coding regions

Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent markers by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, and the ECM receptor {beta}1 integrin. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies. Summary statementGeneration of endogenously tagged stable zebrafish knock-in lines is simplified by the integration of fluorescent protein cassettes with mRNA splicing elements into non-coding regions of genes.

developmental biology↗

bioRxiv2019

High fat diet induces microbiota-dependent silencing of enteroendocrine cells

Enteroendocrine cells (EECs) are specialized sensory cells in the intestinal epithelium that sense and transduce nutrient information. Consumption of dietary fat contributes to metabolic disorders, but EEC adaptations to high fat feeding were unknown. Here, we established a new experimental system to directly investigate EEC activity in vivo using a zebrafish reporter of EEC calcium signaling. Our results reveal that high fat feeding alters EEC morphology and converts them into a nutrient insensitive state that is coupled to endoplasmic reticulum (ER) stress. We called this novel adaptation \"EEC silencing\". Gnotobiotic studies revealed that germ-free zebrafish are resistant to high fat diet induced EEC silencing. High fat feeding altered gut microbiota composition including enrichment of Acinetobacter species, and we identified an Acinetobacter strain sufficient to induce EEC silencing. These results establish a new mechanism by which dietary fat and gut microbiota modulate EEC nutrient sensing and signaling.

developmental biology↗