Biology subjects

Megason, S. G.

Publications and source records attributed to Megason, S. G..

16 recordsLinked to original sources

bioRxiv2025

Mini-Pcdh15b Gene Therapy Rescues Visual Deficits in a Zebrafish Model of Usher Syndrome Type 1F

Usher syndrome type 1F (USH1F) is a severe inherited disorder caused by mutations in PCDH15, resulting in congenital deafness, vestibular dysfunction, and progressive retinal degeneration leading to blindness. While cochlear implantation can restore hearing, no therapeutic interventions currently exist for vision loss. Gene augmentation therapy represents a promising approach; however, the PCDH15 coding sequence ([~]5.3 kb) exceeds the packaging capacity of adeno-associated virus (AAV) vectors. To overcome this limitation, we previously engineered shortened "mini-PCDH15" constructs that retain key structural and functional domains while fitting within a single AAV. Among these, the mini-PCDH15-V4 variant successfully restored hearing in Pcdh15-deficient mice. Here, we investigated the ability of a cone-targeted mini-pcdh15b-V4 transgene to rescue vision in a zebrafish model of USH1F. Untreated pcdh15b-deficient zebrafish exhibited severe structural and functional defects of the retina, including disorganized and shortened photoreceptor outer segments, disrupted calyceal processes, and markedly reduced electroretinogram (ERG) and optokinetic response (OKR) performance. Targeted expression of mini-pcdh15b-V4 recapitulated typical localization of Pcdh15b to calyceal processes and outer segment membranes, rescued photoreceptor architecture, and re-established both structural organization and functional output. Treated mutants exhibited improved visual tracking behavior and full recovery of ERG a-wave and b-wave amplitudes, indicating restoration of photoreceptor and synaptic function. Importantly, mini-pcdh15b-V4 expression produced no adverse effects in wild-type or heterozygous fish, supporting the safety of cone-specific expression. Together, these findings demonstrate that mini-pcdh15b-V4 can restore both photoreceptor structure and visual function in pcdh15b-deficient zebrafish. This work establishes the pcdh15b mutant zebrafish as a powerful preclinical model for studying USH1F retinopathy and supports the translational potential of rationally engineered mini-PCDH15 constructs as a feasible gene therapy approach for preventing or reversing vision loss in individuals with USH1F.

neuroscience↗

bioRxiv2025

Tissue Fluidity: A Double-Edged Sword for Multicellular Patterning

The organization of cells into spatial patterns is a fundamental aspect of multicellularity. One major mechanism underlying tissue patterning is adhesion-based cell sorting, in which a heterogeneous mixture of cell types spontaneously separates into distinct domains based on differences in adhesion protein expression. Here, we identify tissue fluidity--the extent to which cells can move freely within a tissue--as a critical regulator of adhesion-based sorting. First, we describe a physically well-understood minimal tissue model that can integrate both tissue fluidity and adhesion-based sorting, and demonstrate that this model can quantitatively reproduce experimentally measured sorting dynamics in a fibroblast cell culture assay. We go on to show that altering tissue fluidity by any mechanism in the model leads to substantial changes in the rate or accuracy of sorting (or both). We further demonstrate that the balance between cell motility, which acts to fluidize the tissue, and homotypic cell-cell adhesion, which acts to solidify the tissue, sensitively tunes a fundamental trade-off between the rate and accuracy of sorting--such that sorting can only occur when motility and adhesion are tightly coupled. Intriguingly, best fits of the simulations to the experiments across a range of adhesion protein expression conditions suggest that cells may naturally scale their motility strength with their adhesion strength - thereby maintaining a permissive fluidity for sorting. Overall, our results indicate that tissue fluidity must be tightly regulated for sorting to occur, and that cells may have evolved a mechanism to naturally co-regulate their mechanical properties in order to sustain a patterning-competent fluidity. Statement of SignificanceTissue fluidity, or the ability of cells to freely rearrange within a tissue, is a universal property of multicellular organisms that plays central roles in development, cancer, and wound healing. Here, we identify tissue fluidity as a critical regulator of a major mechanism of multicellular patterning - adhesion-based cell sorting. The results of our combined experimental-computational investigation suggest that tissues can readily tune their fluidity in order to freeze, catalyze, or erase multicellular patterns - carrying significant implications for our understanding of how patterns are formed in development, lost in diseases affecting tissue organization (e.g., cancer), regained through the processes of wound healing and regeneration, and can be engineered in the creation of synthetic organoid and embryoid systems. One sentence summaryBiophysical modeling demonstrates how tissue fluidity is a key regulator of the rate and accuracy of adhesion-based sorting.

cell biology↗

bioRxiv2025

Neural plate pre-patterning enables specification of intermediate neural progenitors in the spinal cord

Spinal cord organization begins with the specification of a series of neural progenitor domains along the dorsal-ventral axis of the neural tube. Patterning at the dorsal and ventral ends is established by canonical morphogen gradients. Intermediate progenitor domains are located distally to morphogen sources yet display similar organization. How this is achieved is unclear and the relevant positional cues have not been identified. Here we show that intermediate progenitor domains are established through a distinct mechanism that operates in the preceding neural plate stage and leverages its multiple signaling inputs and morphogenesis. An integrated spatial atlas of transcription factor expression, signaling activity, and cell movement reveals that the neural plate comprises an array of cell states, defined by combinatorial signaling and morphogenesis-driven transitions between local signaling environments. Specifically, we find that p0 and p1 intermediate progenitors, located immediately outside the morphogen-patterned region, arise from specific precursor states that are established in the posterior neural plate by tailbud-derived Wnt signaling. Convergent-extension movements subsequently drive progenitor specification by displacing precursors out of Wnt/FGF signaling fields while shaping them into stripe-like domains. Thus, by controlling signaling exposure, tissue dynamics convert positional information encoded by signaling history into downstream fate specification. This mode of patterning may operate broadly across developmental contexts, with implications for tissue engineering and patterning-related disorders.

developmental biology↗

bioRxiv2024

Zebrafish glial-vascular interactions progressively expand over the course of brain development

Glial-vascular interactions are critical for the formation and maintenance of brain blood vessels and the blood-brain barrier (BBB) in mammals, but their role in zebrafish is not well understood. Our previous work has detailed the timeline of BBB functional maturation in zebrafish, revealing a conserved mechanism of BBB induction through the suppression of endothelial transcytosis. Yet, as opposed to extensive research on glial-vascular interactions in rodents, such interactions remain largely overlooked in the zebrafish model system. Here, we focus on glial-vascular development in the zebrafish brain, leveraging three glial gene promoters: gfap (glial fibrillary acidic protein), glast (an astrocyte-specific glutamate transporter), and glastini (a new, shortened, equally effective version of the Glast promoter). Using these glial promoters, sparse labeling revealed fewer glial-vascular interactions during early larval stages, with both glial coverage and contact area increasing as the zebrafish brain matured. We then generated stable transgenic lines for both the Glast and Glastini promoters and observed similar increases in glial coverage during larval development, starting at [~]30% coverage at 3 days post-fertilization (dpf) and peaking at [~]60% at 10 dpf. Ultrastructural assessment of glial-vascular interactions using electron microscopy (EM) confirmed a progressive increase in glial coverage over larval development, with maximal coverage reaching [~]70% in adult zebrafish, significantly lower than the nearly 100% coverage observed in mammals. Finally, immunogold-EM labeling confirmed that cells identified as glia in aforementioned morphological analyses were indeed Glast-positive. Taken together, our results identify the temporal profile of glial-vascular maturation in the zebrafish brain.

developmental biology↗

bioRxiv2023

Global decoupling of cell differentiation from cell division in early embryo development

As tissues develop, cells divide and differentiate concurrently. Conflicting evidence shows that cell division is either dispensable or required for formation of cell types. To determine the role of cell division in differentiation, we arrested the cell cycle in zebrafish embryos using two independent approaches and profiled them at single-cell resolution. We show that cell division is dispensable for differentiation of all embryonic tissues during initial cell type differentiation from early gastrulation to the end of segmentation. In the absence of cell division, differentiation slows down in some cell types, and cells exhibit global stress responses. While differentiation is robust to blocking cell division, the proportions of cells across cell states are not. This work simplifies our understanding of the role of cell division in development and showcases the utility of combining embryo-wide perturbations with single-cell RNA sequencing to uncover the role of common biological processes across multiple tissues.

developmental biology↗

bioRxiv2022

A bioelectrical phase transition patterns the first beats of a vertebrate heart

The heart is among the first organs to function in vertebrate development, but its transition from silent to beating has not been directly characterized. Using all-optical electrophysiology, we captured the very first zebrafish heartbeat and analyzed the development of cardiac excitability around this singular event. The first beats appeared suddenly and propagated coherently across the primordial heart. Targeted optogenetic perturbations mapped the development of excitability and conduction before and after the first heartbeats. Measured bioelectrical dynamics support a noisy saddle-node on invariant circle (SNIC) bifurcation as the critical phase transition that starts the heart. Simple models of this bifurcation quantitatively capture cardiac dynamics in space and time through early development, including coherent beating before transcriptional specification of pacemakers. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

developmental biology↗

bioRxiv2021

The secreted neuronal signal Spock1 regulates the blood-brain barrier

The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restricts its permeability to proteins and small molecules. Classic chick-quail chimera studies showed that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally-expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 induces BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression. One-Sentence SummarySpock1 is a signal secreted by neurons that induces barrier properties in the brain vasculature

neuroscience↗

bioRxiv2021

Bmper is required for morphogenesis of the anterior and posterior semicircular canal ducts in the developing zebrafish inner ear

BMP signalling is known to have a conserved function in development of the semicircular canal system of the vertebrate inner ear, but its regulation, target genes and effects on cell behaviour during otic morphogenesis are not fully understood. We have characterised the effects of mutations in the zebrafish gene bmper, which codes for a regulator of BMP signalling with both pro- and anti-BMP roles in different developmental contexts. The inner ears of bmper mutant embryos develop with truncations of the anterior and posterior semicircular canal ducts. To image the developing ear in live embryos, we have exploited a new transgenic line, Tg(smad6b:EGFP), which exhibits strong GFP expression in the otic epithelium. Morphometric analysis indicates defects in the bmper mutant ear from early stages of semicircular canal formation, correlating with a specific reduction in BMP signalling activity and specific loss of dlx5a expression in dorsal otic epithelium. Subsequent changes to cell shape occur at the truncation site and the dorsolateral septum. The bmper mutations that we describe are adult viable; truncation of the anterior and posterior semicircular canal ducts persists into adulthood. Our results argue against a major role for Bmper in specification of the pre-placodal region, induction of the otic placode, or development of the neural crest, processes in which Bmper function has previously been implicated. Instead, we conclude that a key requirement for Bmper function in the zebrafish is to promote BMP signalling during patterning and morphogenesis of the semicircular canal system.

developmental biology↗

bioRxiv2019

An adhesion code ensures robust pattern formation during tissue morphogenesis

An outstanding question in embryo development is how spatial patterns are formed robustly. In the zebrafish spinal cord, neural progenitors form stereotypic stripe-like patterns despite noisy morphogen signaling and large-scale cellular rearrangement required for tissue growth and morphogenesis. We set out to understand the mechanisms underlying this patterning robustness. Our adhesion assays revealed a preference for three neural progenitor types to stabilize contacts with cells of the same type. Genetic analysis uncovered a three-molecule adhesion code, composed of N-cadherin, Cadherin 11, and Protocadherin 19, with unique gene expression profiles for each cell type. Perturbation of the adhesion code results in loss of homotypic preference ex vivo and patterning errors in vivo. Both the cell fate and adhesion code are co-regulated by the common upstream morphogen signal Shh. We propose that robust patterning in tissues undergoing morphogenesis results from a previously unappreciated interplay between morphogen gradient-based patterning and adhesion-based self-organization.

developmental biology↗

bioRxiv2019

Suppression of transcytosis regulates zebrafish blood-brain barrier development

As an optically transparent model organism with an endothelial blood-brain barrier (BBB), zebrafish offer a powerful tool to study the vertebrate BBB. However, the precise developmental profile of functional zebrafish BBB acquisition and the subcellular and molecular mechanisms governing the zebrafish BBB remain poorly characterized. Here we find a spatiotemporal gradient of barrier acquisition. Moreover, we capture the dynamics of developmental BBB leakage using live imaging, revealing a combination of steady accumulation in the parenchyma and sporadic bursts of tracer leakage. Electron microscopy studies further reveal that this steady accumulation results from high levels of transcytosis that are eventually suppressed, sealing the BBB. Finally, we demonstrate a key mammalian BBB regulator Mfsd2a, which inhibits transcytosis, plays a conserved role in zebrafish. Mfsd2aa mutants display increased larval and adult BBB permeability due to increased transcytosis. Our findings indicate a conserved developmental program of barrier acquisition between zebrafish and mice.

developmental biology↗

bioRxiv2018

Cortical Column and Whole Brain Imaging of Neural Circuits with Molecular Contrast and Nanoscale Resolution

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.\n\nOne Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.

neuroscience↗

bioRxiv2018

Size control of the inner ear via hydraulic feedback

Animals make organs of precise size, shape, and symmetry despite noise in underlying molecular and cellular processes. How developing organs manage this noise is largely unknown. Here, we combine quantitative imaging, physical theory, and physiological measurement of hydrostatic pressure and fluid transport in zebrafish to study size control of the developing inner ear. We find that fluid accumulation creates hydrostatic pressure in the lumen leading to stress in the epithelium and expansion of the otic vesicle. Pressure, in turn, inhibits fluid transport into the lumen. This negative feedback loop between pressure and transport allows the otic vesicle to change growth rate to control natural or experimentally-induced size variation. Spatiotemporal patterning of contractility modulates pressure-driven strain for regional tissue thinning. Our work connects moleculardriven mechanisms, such as osmotic pressure driven strain and actomyosin tension, to the regulation of tissue morphogenesis via hydraulic feedback to ensure robust control of organ size.

developmental biology↗

bioRxiv2018

Feedback control of neurogenesis by tissue packing

Balancing the rate of differentiation and proliferation in developing tissues is essential to produce organs of robust size and composition. Whilst many molecular regulators have been established, how these connect to physical and geometrical aspects of tissue architecture is poorly understood. Here, using high-resolution timelapse imaging, we find that dense tissue packing and complex cell geometries play a significant role in regulating differentiation rate in the zebrafish neural tube. Specifically, in regions of high cell density, progenitors are physically pushed away from the apical surface, which, in a Notch-dependent manner, leads to their differentiation. Using simulations we show that this naturally performs negative feedback control on cell number. Our results suggest a model whereby differentiation rate is carefully tuned to correct fluctuations in cell number, originating from variable cell cycle progression and inherently probabilistic differentiation programs.

developmental biology↗

bioRxiv2018

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.\n\nOne Sentence SummaryCombining lattice light sheet microscopy with adaptive optics enables high speed, high resolution in vivo 3D imaging of dynamic processes inside cells under physiological conditions within their parent organisms.

cell biology↗

bioRxiv2017

Size-reduced embryos reveal a gradient scaling based mechanism for zebrafish somite formation

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed, and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a \"clock and scaled gradient\" model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we experimentally confirm--the formation of periodic \"echoes\" in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role both in progression and size control of somitogenesis.\n\nSummary statementBy comparing patterning in zebrafish embryos of different size we show that a dynamically scaling gradient in the presomitic mesoderm regulates somite size control.

developmental biology↗

bioRxiv2017

Lamellar Junctions In The Endolymphatic Sac Act As A Relief Valve To Regulate Inner Ear Pressure

The inner ear is a fluid-filled closed-epithelial structure whose normal function requires maintenance of an internal hydrostatic pressure and fluid composition by unknown mechanisms. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation every 1-3 hours. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of thin overlapping basal lamellae that dynamically extend over neighboring cells before rupturing under pressure leading to ES collapse. The unexpected discovery of a physical relief valve in the ear emphasizes the need for further study into how organs control fluid pressure, volume, flow, and ion homeostasis in development and disease.

developmental biology↗