Biology subjects

Sagasti, A.

Publications and source records attributed to Sagasti, A..

10 recordsLinked to original sources

bioRxiv2026

Mucus cell and ionocyte precursors migrate between epithelial cells to disperse through the zebrafish epidermis

Mucus-secreting cells and ionocytes play critical roles in many organs. Both cell types are usually distributed as scattered, solitary cells within an epithelium, a pattern presumably optimal for their function. To investigate how they attain their dispersed distributions, we imaged mucus cell and ionocyte precursors in the epidermis of developing zebrafish. Previous reports found that precursors of both cell types are first detected in the ventral epidermis (covering the yolk) before spreading dorsally, but the mechanism driving this progression was unknown. Photoconverting basal epidermal cells in the ventral embryo revealed that some cells actively migrate away from this area to populate the rest of the epidermis. These cells lose their basal cell identity when they start migrating and begin expressing markers of mature mucus cells or ionocytes during migration. These cells travel entirely between the two epithelial layers of the epidermis, occasionally pause migration to divide, and repel one another through contact inhibition of locomotion, behaviors that likely aid in their dispersal. After migrating for about a day, mucus cell and ionocyte precursors intercalate into the superficial epithelial layer of the epidermis, where they complete differentiation. These observations reveal how mucus cells and ionocytes achieve their scattered distributions in the zebrafish epidermis, suggesting that similar processes promote their distribution in other mucosal organs.

developmental biology↗

bioRxiv2024

Caliber of sensory axons in vivo varies spatially and temporally and is influenced by the cellular microenvironment

Cell shape is crucial to cell function, particularly in neurons. The cross-sectional diameter, also known as caliber, of axons and dendrites is an important parameter of neuron shape, best appreciated for its influence on the speed of action potential propagation. Many studies of axon caliber focus on cell-wide regulation and assume that caliber is static. Here, we have characterized local variation and dynamics of axon caliber in vivo using the peripheral axons of zebrafish touch-sensing neurons at embryonic stages, prior to sex determination. To obtain absolute measurements of caliber in vivo, we paired sparse membrane labeling with super- resolution microscopy of neurons in live fish. We observed that axon segments had varicose or "pearled" morphologies, and thus vary in caliber along their length, consistent with reports from mammalian systems. Sister axon segments originating from the most proximal branch point in the axon arbor had average calibers that were uncorrelated with each other. Axon caliber also tapered across the branch point. Varicosities and caliber, overall, were dynamic on the timescale of minutes, and dynamicity changed over the course of development. By measuring the caliber of axons adjacent to dividing epithelial cells, we found that skin cell division is one aspect of the cellular microenvironment that may drive local differences and dynamics in axon caliber. Our findings support the possibility that spatial and temporal variation in axon caliber could significantly influence neuronal physiology. Significance StatementAxon caliber directly influences how quickly neurons send messages to other cells and likely plays a role in neurons overall health. In the peripheral nervous system, where neurons cover particularly long distances, cell shape can determine whether an animal successfully executes behaviors such as escape responses. We found that axon caliber can vary between locations within the same cell and is highly dynamic. Taking these variations into account may allow neuroscientists to better estimate transmission speeds for cells in neural circuits. We observed that axon caliber is distorted when nearby skin cells change shape. Thus, cells not classically considered part of the nervous system can also contribute to caliber dynamics, broadening our view of axon caliber determinants.

neuroscience↗

bioRxiv2022

Skin-resident immune cells engulf axonal debris in adult epidermis

Somatosensory neurons extend enormous peripheral axons to the skin, where they detect diverse environmental stimuli. Somatosensory peripheral axons are easily damaged due to their small caliber and superficial location. Axonal damage results in Wallerian degeneration, creating vast quantities of cellular debris that phagocytes must remove to maintain organ homeostasis. The cellular mechanisms that ensure efficient clearance of axon debris from stratified adult skin are unknown. Here, we establish zebrafish scales as a tractable model to study axon degeneration in the adult epidermis. Using this system, we demonstrate that skin-resident immune cells known as Langerhans cells engulf the majority of axon debris. In contrast to immature skin, adult keratinocytes do not significantly contribute to debris removal, even in animals lacking Langerhans cells. Our study establishes a powerful new model for studying Wallerian degeneration and identifies a new function for Langerhans cells in maintenance of adult skin homeostasis following injury. These findings have important implications for pathologies that trigger somatosensory axon degeneration.

cell biology↗

bioRxiv2021

Sensory axons induce epithelial lipid microdomain remodeling and determine the distribution of junctions in the epidermis

Epithelial cell properties are determined by the polarized distribution of membrane lipids, the cytoskeleton, and adhesive junctions. Epithelia are often profusely innervated, but little work has addressed how contact with neurites affects the polarized organization of epithelial components. In previous work, we found that basal keratinocytes in the larval zebrafish epidermis wrap around axons to enclose them in ensheathment channels sealed by autotypic cell junctions. In this study, we used live imaging to characterize how sensory axons remodel cell membranes, the actin cytoskeleton, and adhesive junctions in basal keratinocytes. At the apical surface of basal keratinocytes, axons promoted the formation of lipid microdomains quantitatively enriched in reporters for PI(4,5)P2 and liquid-ordered (Lo) membranes. Lipid microdomains supported the formation of cadherin-enriched F-actin protrusions, which wrapped around axons, likely initiating the formation of ensheathment channels. Lo reporters, but not reporters of liquid-disordered (Ld) membranes, became progressively enriched at axon-associated membrane domains as autotypic junctions matured at ensheathment channels. In the absence of axons, cadherin-enriched lipid microdomains still formed on basal cell membranes, but were not organized into the contiguous domains normally associated with axons. Instead, these isolated domains formed ectopic heterotypic junctions with overlying periderm cells, a distinct epithelial cell type in the epidermis. Thus, axons inhibit the formation of epithelial heterotypic junctions by recruiting cadherin-rich lipid microdomains to form autotypic junctions at ensheathment channels. These findings demonstrate that sensory nerve endings dramatically remodel polarized epithelial components and regulate the adhesive properties of the epidermis.

cell biology↗

bioRxiv2021

The MAP3Ks DLK and LZK direct diverse responses to axon damage in zebrafish peripheral neurons

The MAP3Ks Dual Leucine Kinase (DLK) and Leucine Zipper Kinase (LZK) are essential mediators of axon damage responses, but their responses are varied, complex, and incompletely understood. To characterize their functions in axon injury, we generated zebrafish mutants of each gene, labeled motor neurons (MN) and touch-sensing neurons in live zebrafish, precisely cut their axons with a laser, and assessed the ability of mutant axons to regenerate. DLK and LZK were required redundantly and cell autonomously for axon regeneration in MNs, but not in larval Rohon-Beard (RB) or adult dorsal root ganglion (DRG) sensory neurons. Surprisingly, in dlk lzk double mutants, the spared branches of wounded RB axons grew excessively, suggesting that these kinases inhibit regenerative sprouting in damaged axons. Uninjured trigeminal sensory axons also grew excessively in mutants when neighboring neurons were ablated, indicating that these MAP3Ks are general inhibitors of sensory axon growth. These results demonstrate that zebrafish DLK and LZK promote diverse injury responses, depending on the neuronal cell identity and type of axonal injury. Significance statementThe MAP3Ks DLK and LZK are damage sensors that promote diverse outcomes to neuronal injury, including axon regeneration. Understanding their context-specific functions is a prerequisite to considering these kinases as therapeutic targets. To investigate DLK and LZK cell-type specific functions, we created zebrafish mutants in each gene. Using mosaic cell labeling and precise laser injury we found that both proteins were required for axon regeneration in motor neurons, but, unexpectedly, were not required for axon regeneration in Rohon-Beard or dorsal root ganglion (DRG) sensory neurons, and negatively regulated sprouting in the spared axons of touch-sensing neurons. These findings emphasize that animals have evolved distinct mechanisms to regulate injury site regeneration and collateral sprouting, and identify differential roles for DLK and LZK in these processes.

neuroscience↗

bioRxiv2020

Cortical myosin minifilaments orchestrate the arrangement of microridge protrusions on epithelial cell surfaces

Actin-based protrusions vary in morphology, stability, and arrangement on cell surfaces. Microridges are laterally-elongated protrusions arranged in maze-like patterns on mucosal epithelial cells that rearrange dynamically by fission and fusion. To characterize how microridges mature and investigate the mechanisms driving fission and fusion, we imaged microridges in the maturing skin of zebrafish larvae. After their initial development, microridges continued to lengthen and microridge alignment became increasingly well ordered. Imaging F-actin and Non-Muscle Myosin II (NMII) revealed that microridge fission and fusion were associated with local NMII activity in the apical cortex. Inhibiting NMII blocked rearrangements, reduced microridge density, and altered microridge spacing. High-resolution imaging revealed that individual cortical NMII minifilaments are tethered to protrusions, often connecting adjacent microridges. NMII minifilaments connecting the ends of microridges fused them together, whereas minifilaments oriented perpendicular to microridges severed them or pulled them closer together. Our findings demonstrate that as cells mature, microridges continue to remodel and form an increasingly orderly arrangement through a process orchestrated by cortical NMII contraction.

cell biology↗

bioRxiv2020

Keratins and Plakin family cytolinker proteins control the length of epithelial microridge protrusions

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable class of cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that Keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions from mucosal epithelial cells arranged in elaborate fingerprint-like patterns. Developing microridges on zebrafish skin cells contained both Actin and Keratin filaments. Keratin filaments maintained microridges upon F-actin disruption, and overexpressing Keratins lengthened microridges. Envoplakin and Periplakin, Plakin family cytolinkers that bind to F-actin and Keratins, localized to microridges and were required for their morphogenesis. Strikingly, Plakin protein levels directly determined microridge length. An actin-binding domain of Periplakin was required to initiate microridge morphogenesis, whereas Periplakin-Keratin binding was required to stabilize and elongate microridges. Our results thus separate microridge morphogenesis into two steps with differential requirements for cytoskeletal elements, expand our understanding of intermediate filament functions, and identify microridges as cellular protrusions that integrate actin and intermediate filaments.

cell biology↗

bioRxiv2019

Cortical contraction drives the 3D patterning of epithelial cell surfaces

Cellular protrusions create complex cell surface topographies, but biomechanical mechanisms regulating their formation and arrangement are largely unknown. To study how protrusions form, we focused on the morphogenesis of microridges, elongated actin-based structures projecting from the apical surfaces of zebrafish skin cells that are arranged in labyrinthine patterns. Microridges form by accreting simple finger-like precursors. Live imaging demonstrated that microridge morphogenesis is linked to apical constriction. A non-muscle myosin II (NMII) reporter revealed pulsatile contractions of the actomyosin cortex; inhibiting NMII demonstrated that contractions are required for apical constriction and microridge formation. A biomechanical model suggested that contraction reduces surface tension to permit the fusion of precursors into microridges. Indeed, reducing surface tension with hyperosmolar media promoted microridge formation. In anisotropically stretched cells, microridges formed by precursor fusion along the stretch axis, which computational modeling explained as a consequence of stretch-induced cortical flow. Collectively, our results demonstrate how contraction within the 2D plane of the cortex patterns 3D cell surfaces.\n\nSUMMARYMicroridges, elongated 3D protrusions arranged in maze-like patterns on zebrafish skin cells, form by the accretion of simple precursor projections. Modeling and in vivo experiments showed that cortical contractions promote the coalescence of precursors into microridges by reducing membrane tension.

cell biology↗

bioRxiv2018

A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites

Interactions between epithelial cells and neurons influence a range of sensory modalities including taste, touch, and smell. Vertebrate and invertebrate keratinocytes/keratinocyte-like epidermal cells ensheath peripheral arbors of somatosensory neurons, including nociceptors, yet the developmental origins and functional roles of this ensheathment are largely unknown. Here, we describe an evolutionarily conserved morphogenetic mechanism for epidermal ensheathment of somatosensory neurites. We found that somatosensory neurons in Drosophila and zebrafish induce formation of epidermal sheaths, which wrap neurites of different types of neurons to different extents. Neurites induce formation of plasma membrane phosphatidylinositol 4,5-bisphosphate microdomains at nascent sheaths, followed by a filamentous actin network, and recruitment of junctional proteins that likely form autotypic junctions to seal sheaths. Finally, blocking epidermal sheath formation destabilized dendrite branches and reduced nociceptive sensitivity in Drosophila. Epidermal somatosensory neurite ensheathment is thus a deeply conserved cellular process that contributes to the morphogenesis and function of nociceptive sensory neurons.

neuroscience↗

bioRxiv2017

A Macrophage Response To Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage In Leprosy

Mycobacterium leprae causes leprosy, and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interactions of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia, and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.

microbiology↗