Biology subjects

Poss, K. D.

Publications and source records attributed to Poss, K. D..

16 recordsLinked to original sources

bioRxiv2026

Asymmetric regeneration dynamics in zebrafish pectoral fins

Appendage amputation in regeneration-capable systems results in the full re-formation of lost tissue and cell populations. Despite an appreciation of this phenomenon in a select number of organisms, there is still much to quantitatively understand about the tissue growth dynamics during limb regeneration. To address these gaps in knowledge, we developed novel methods that enable longitudinal, quantitative imaging of zebrafish pectoral fin regeneration and uncovered a persistent spatial asymmetry in growth dynamics between anterior and posterior regions of the regenerating fin. Spatiotemporally resolved transcriptomics suggests differential anteroposterior regulation of the Mitogen-activated protein kinase (MAPK) and Wnt signaling pathways in pectoral fins. This asymmetry was also detected using quantitative imaging of biosensors reporting canonical Wnt and Erk pathway activity. In addition to known static spatially asymmetric genes, bulk transcriptomic analysis uncovered a large subset of genes with temporally asymmetric peaks in expression levels between anterior and posterior regions of the fin across regeneration timepoints, suggesting a global differential regulation of the regenerative program. Taken together, our results establish pectoral fins as a platform for quantitative analyses of appendage regeneration, and they identify persistent spatial asymmetries in growth, gene expression, and signaling activity during regeneration.

cell biology↗

bioRxiv2026

Microenvironmental and transcriptional determinants of the robustness of spinal cord regeneration in zebrafish

Unlike mammals, adult zebrafish can regenerate spinal cord tissue after complete transection. We report here that a substantial proportion of zebrafish receiving this injury fail to regain locomotor function, and we analyzed this critically. After stratifying large cohorts of injured animals according to functional recovery, we directly compared recovered and non-recovered individuals at matched post-injury stages. We find that permanently paralyzed animals form a tissue bridge across the lesion yet exhibit impaired axonal invasion and regrowth to downstream targets. Integrative single-nucleus profiling, spatial transcriptomics, and regulatory network analyses revealed that regenerative success is characterized by the coordinated establishment of a permissive multicellular regenerative state rather than major differences in cellular composition. Furthermore, we find that regenerative failure involves persistent non-permissive regulatory programs, including extracellular matrix remodeling, impaired adaptive T-cell activation, and reduced axon growth-associated signaling. Gene regulatory network and cellular trajectory analyses identified candidate transcriptional regulators that could not be resolved through differential expression analysis alone. Functional CRISPR F0 screening of several of these candidates identified hoxb5a as a critical determinant of regenerative outcome. Stable hoxb5a mutants exhibited impaired swimming recovery, disrupted bridge formation, and limited axon bridging after spinal cord injury. Together, our findings reveal key differences between success and failure in a model of elevated regenerative capacity, with hoxb5a acting as a regulator linking multicellular tissue remodeling to axonal regeneration and functional recovery.

developmental biology↗

bioRxiv2025

Enhancer-directed gene delivery for digit regeneration based on conserved epidermal factors

Limb loss remains a significant clinical challenge, but regenerative medicine approaches such as gene therapy offer a promising strategy to trigger endogenous regeneration programs. Optimal vector configurations and molecular targets for appendicular skeletal repair are not well defined. Here, we leveraged insights from species with a high endogenous capacity for appendage regeneration to design an enhancer-directed gene delivery platform that functions during mouse digit regeneration, a well characterized model for partial limb regeneration in mammals. Single-cell RNA sequencing of zebrafish caudal fin regeneration, combined with expression data in regenerating salamander limbs and mouse digit tips, implicated the SP family of transcription factors as conserved, epidermally-expressed mediators of appendage regrowth. Null mutants of Sp8 demonstrated impaired limb regeneration in salamanders, while conditional knockout of Sp6 and/or Sp8 in the mouse basal epidermis resulted in defective bony digit tip regeneration, involving an IL-17 mediated osteoclastogenic program. Spatiotemporally focused expression of FGF8, a known target of SP factors, using a zebrafish-derived tissue regeneration enhancer element via adeno-associated viral vectors, could partially rescue digit tip regeneration in SP knockout mice and accelerate digit regeneration in wildtype mice. Our results demonstrate a contextual gene therapy approach to address limb loss based on genes like SP transcription factors conserved across multiple contexts of appendage regeneration. Significance StatementInstructing regeneration of complex structures in mammals remains an unsolved problem. Gene therapy offers a compelling approach to foster endogenous regeneration by delivering therapeutic gene products to specific cells post injury. We identified a conserved regeneration-linked epidermal transcriptional program in mouse digit regeneration centered on the SP6 and SP8 transcription factors, involving inflammatory responses from osteoclasts. We engineered AAVs harboring a zebrafish tissue regeneration enhancer to direct FGF8 expression in the epidermis after amputation. This enhancer directed delivery partially rescued impaired digit regeneration in Sp6 and Sp8 conditional knockout mice and accelerated regrowth in wildtype digits. Our work links developmental signaling to adult regeneration and establishes a modular, injury site specific gene therapy framework that enables new interventions for limb healing.

genetics↗

bioRxiv2025

Live visualization of extracellular matrix dynamics during development and regeneration in zebrafish

Extracellular matrix (ECM) plays fundamental roles in animal development, regeneration, and disease. The difficulty of tagging endogenous matrix proteins in vertebrates has limited the understanding of ECM composition and dynamics in complex tissues. To visualize vertebrate ECM components, we tagged zebrafish Laminin, gamma 1 (Lamc1), Collagen, type I, alpha 2 (Col1a2), and Transforming growth factor, beta-induced (Tgfbi) using C-terminus in-fusion genome editing. Analysis of these knock-in lines revealed distinct expression of each protein in various tissues during development and regeneration. Fluorescent recover after photobleaching (FRAP) analysis further indicated that Lamc1 is stable in fin fold matrix but more dynamic in myoseptal matrix of developing zebrafish, while Col1a2 and Tgfbi are stable matrix components in myosepta. Strikingly, we found that Col1a2-mScarlet protein accumulates at the amputation plane during tailfin regeneration, where it remains concentrated for several days and distant from the regeneration blastema. This "foundation" region also displayed a distinct transcriptome suggesting active and dedicated events at the base of the regenerating appendage. Our resource enables live capture of ECM dynamics that can identify new events in developing and regenerating zebrafish. Summary statementExtracellular matrix resources for zebrafish development and tissue regeneration.

developmental biology↗

bioRxiv2025

Buffering of genetic defects in animal development by regeneration programs

Regeneration programs enable animals to restore damaged or lost tissues, and the range of stimuli for these programs is incompletely understood. Here, we used zebrafish, a vertebrate species with exceptional regenerative capacity, to identify chemically induced mutations that alter regeneration-associated gene activation. Transgenic zebrafish with a permissive promoter and EGFP cassette inserted in the vicinity of the pro-regenerative factor gene fgf20a were mutagenized, and larvae homozygous for ENU-induced mutations were assessed for disruptions in fgf20a-directed reporter gene expression following fin fold amputation. One line was identified with heritable, elevated fgf20a:EGFP presence in the absence of experimental injury, localized to regions of fin fold tissue undergoing degeneration. Whole-genome sequencing (WGS) identified a mutation within exon 72 of the fraser syndrome 1 (fras1) gene, mutated in patients with inherited skin disease. fras1 mutant larvae spontaneously displayed elevated expression of other known injury/regeneration-responsive reporter lines in fin fold, and zebrafish crispants for homologs of other genes mutated in human developmental diseases also displayed regeneration-associated gene expression in regions of dysmorphology. Tempering Fgf signaling by transgenic expression of a dominant-negative Fgf receptor in fras1 mutants exacerbated the disease phenotype. Our findings provide evidence that regeneration programs are harnessed in response to developmental defects caused by genetic mutations, potentially buffering deleterious phenotypes.

developmental biology↗

bioRxiv2025

Epicardial Tcf21 facilitates cardiomyocyte dedifferentiation and heart regeneration in zebrafish

Unlike mammals, zebrafish (Danio rerio) are able to regenerate their hearts after injury, making them an excellent model organism for studying the molecular mechanisms underlying heart regeneration. Epicardium, the outermost layer of the heart, is an essential player in this process. Injury-induced epicardium activation, characterized by the expression of embryonic epicardial marker genes including tcf21 supports cardiac regeneration by providing various cell types and releasing paracrine signals that promote the restoration of damaged tissue. However, the molecular mechanisms involved in this process are insufficiently understood. In this study, we describe a conditional tcf21flox allele and use it to investigate the role of Tcf21 in heart regeneration. By employing 4-hydroxytamoxifen inducible CreERT2 recombinase, we eliminated tcf21 expression in adult fish. Our findings indicate that loss of this transcription factor reduces the presence of dedifferentiated cardiomyocytes in the injury area and impairs heart regeneration. This work provides new insights into the molecular basis of the epicardial response to heart injury and its role in guiding heart regeneration.

developmental biology↗

bioRxiv2025

Spatial and longitudinal tracking of enhancer-AAV vectors that target transgene expression to injured mouse myocardium

Tissue regeneration enhancer elements (TREEs) direct expression of target genes in injured and regenerating tissues. Additionally, TREEs of zebrafish origin were shown to direct the expression of transgenes in border zone regions after cardiac injury when packaged into recombinant AAV vectors and introduced into mice. Future implementation of TREEs into AAV-based vectors as research tools and potential gene therapy modalities will require a more detailed understanding of expression dynamics and potential off-target effects. Here, we applied in vivo bioluminescent imaging to mice systemically injected with AAV vectors containing different combinations of capsids, enhancers, and timing of delivery. Longitudinal tracking of TREE-based expression over time revealed distinct amplitudes and durations of reporter gene expression in the injured heart directed by different TREEs. The liver-de-targeted AAV capsid, AAV.cc84, was able to deliver TREEs either pre- or post-cardiac injury to negate off-target expression in the liver while maintaining transduction in the heart. By screening AAV9-based capsid libraries dosed systemically in mice post-cardiac injury, we discovered a new capsid variant, AAV.IR41, with enhanced transduction at cardiac border zone regions, and with elevated transduction of TREE driven transgenes versus conventional AAV9 vectors. In vivo bioluminescence imaging offers insights into how enhancers and engineered capsids can be implemented to modulate spatiotemporal transgene expression for future targeted therapies.

developmental biology↗

bioRxiv2025

Decaying and expanding Erk gradients process memory of skeletal size during zebrafish fin regeneration

Regeneration of an amputated salamander limb or fish fin restores pre-injury size and structure, illustrating the phenomenon of positional memory. Although appreciated for centuries, the identity of position-dependent cues and how they control tissue growth are not resolved. Here, we quantify Erk signaling events in whole populations of osteoblasts during zebrafish fin regeneration. We find that osteoblast Erk activity is dependent on Fgf receptor signaling and organized into millimeter-long gradients that extend from the distal tip to the amputation site. Erk activity scales with the amount of tissue amputated, predicts the likelihood of osteoblast cycling, and predicts the size of regenerated skeletal structures. Mathematical modeling suggests gradients are established by the transient deposition of long-lived ligands that are transported by tissue growth. This concept is supported by the observed scaling of expression of the essential epidermal ligand fgf20a with extents of amputation. Our work provides evidence that localized, scaled expression of pro-regenerative ligands instructs long-range signaling and cycling to control skeletal size in regenerating appendages.

developmental biology↗

bioRxiv2023

A Cardiac Transcriptional Enhancer is Repurposed During Regeneration to Activate an Anti-proliferative Program

Zebrafish have a high capacity to regenerate their hearts. Several studies have surveyed transcriptional enhancers to understand how gene expression is controlled during heart regeneration. We have identified REN or the runx1 enhancer that during regeneration regulates the expression of the nearby runx1 gene. We show that runx1 mRNA is reduced with deletion of REN ({Delta}REN) and cardiomyocyte proliferation is enhanced in both {Delta}REN and {Delta}runx1 mutants only during regeneration. Interestingly, in uninjured hearts, {Delta}REN mutants have reduced expression of adamts1, a nearby gene that encodes a Collagen protease. This results in excess Collagen within cardiac valves of uninjured hearts. The {Delta}REN Collagen phenotype is rescued even by {Delta}runx1 mutations, suggesting that in uninjured hearts REN regulates adamts1 independently of runx1. Taken together, this suggests that REN is rewired from adamts1 in uninjured hearts to stimulate runx1 transcription during regeneration. Our data point to a previously unappreciated mechanism for gene regulation during zebrafish heart regeneration. We report that an enhancer is rewired from expression in a distal cardiac domain to activate a different gene in regenerating tissue.

developmental biology↗

bioRxiv2022

The Translation Initiation Factor Homolog, eif4e1c, Regulates Cardiomyocyte Metabolism and Proliferation During Heart Regeneration

The eIF4E family of translation initiation factors bind 5 methylated caps and act as the limiting-step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration.

developmental biology↗

bioRxiv2021

DeepProjection: Rapid and structure-specific projections of tissue sheets embedded in 3D microscopy stacks using deep learning

The efficient extraction of local high-resolution content from massive amounts of imaging data remains a serious and unsolved problem in studies of complex biological tissues. Here we present DeepProjection, a trainable projection algorithm based on deep learning. This algorithm rapidly and robustly extracts image content contained in curved manifolds from time-lapse recorded 3D image stacks by binary masking of background content, stack by stack. The masks calculated for a given movie, when predicted, e.g., on fluorescent cell boundaries on one channel, can subsequently be applied to project other fluorescent channels from the same manifold. We apply DeepProjection to follow the dynamic movements of 2D-tissue sheets in embryonic development. We show that we can selectively project the amnioserosa cell sheet during dorsal closure in Drosophila melanogaster embryos and the periderm layer in the elongating zebrafish embryo while masking highly fluorescent out-of-plane artifacts.

developmental biology↗

bioRxiv2021

Identification of enhancer regulatory elements that direct epicardial gene expression during zebrafish heart regeneration

The epicardium is a mesothelial tissue layer that envelops the heart. Cardiac injury activates dynamic gene expression programs in epicardial tissue, which in the case of zebrafish enables subsequent regeneration through paracrine and vascularizing effects. To identify tissue regeneration enhancer elements (TREEs) that control injury-induced epicardial gene expression during heart regeneration, we profiled transcriptomes and chromatin accessibility in epicardial cells purified from regenerating zebrafish hearts. We identified hundreds of candidate TREEs, defined by increased chromatin accessibility of non-coding elements near genes with increased expression during regeneration. Several of these candidate TREEs were incorporated into stable transgenic lines, with 5 of 6 elements directing injury-induced epicardial expression but not ontogenetic epicardial expression in hearts of larval animals. Whereas two independent TREEs linked to the gene gnai3 showed similar functional features of gene regulation in transgenic lines, two independent ncam1a-linked TREEs directed distinct spatiotemporal domains of epicardial gene expression. Thus, multiple TREEs linked to a regeneration gene can possess either matching or complementary regulatory controls. Our study provides a new resource and principles for understanding the regulation of epicardial genetic programs during heart regeneration.

developmental biology↗

bioRxiv2021

Generation of specialized blood vessels through transdifferentiation of lymphatic endothelial cells

The lineage and developmental trajectory of a cell are key determinants of cellular identity. Yet, the functional relevance of deriving a specific cell type from ontologically distinct progenitors, remains an open question. In the case of the vascular system, blood and lymphatic vessels are composed of endothelial cells (ECs) that differentiate and diversify to cater the different physiological demands of each organ. While lymphatic vessels have been shown to originate from multiple cell sources, lymphatic ECs (LECs) themselves seem to have a unipotent cell fate. In this work we uncover a novel mechanism of blood vessel formation through transdifferentiation of LECs. Using advanced long-term reiterative imaging and lineage-tracing of ECs in zebrafish, from embryonic development through adulthood, we reveal a hitherto unknown process of LEC-to-BEC transdifferentiation, underlying vascularization of the anal fin (AF). Moreover, we demonstrate distinct functional implications for deriving AF vessels from either LECs or BECs, uncovering for the first time a clear link between cell ontogeny and functionality. Molecularly, we identify Sox17 as a negative regulator of lymphatic fate specification, whose specific expression in AF LECs suppresses its lymphatic cell fate. Finally, we show that akin to the developmental process, during adult AF regeneration the vasculature is re-derived from lymphatics, demonstrating that LECs in the mature fish retain both potency and plasticity for generating specialized blood vessels. Overall, our work highlights a novel mechanism of blood vessel formation through LEC trans-differentiation, and provides the first in vivo evidence for a link between cell ontogeny and functionality in ECs.

developmental biology↗

bioRxiv2021

Mathematical modeling of Erk activity waves in regenerating zebrafish scales

Erk signaling regulates cellular decisions in many biological contexts. Recently, we have reported a series of Erk activity traveling waves that coordinate regeneration of osteoblast tissue in zebrafish scales. These waves originate from a central source region, propagate as expanding rings, and impart cell growth, thus controlling tissue morphogenesis. Here, we present a minimal reaction-diffusion model for Erk activity waves. The model considers three components: Erk, a diffusible Erk-activator, and an Erk-inhibitor. Erk stimulates both its activator and inhibitor, forming a positive and negative feedback loop, respectively. Our model shows that this system can be excitable and propagate Erk activity waves. Waves originate from a pulsatile source which is modeled by adding a localized basal production of the activator that switches the source region from an excitable to an oscillatory state. As Erk activity periodically rises in the source, it can trigger an excitable wave which travels across the entire tissue. Analysis of the model finds that positive feedback controls the properties of the traveling wavefront and that negative feedback controls the duration of Erk activity peak and the period of Erk activity waves. The geometrical properties of the waves facilitate constraints on the effective diffusivity of the activator, indicating that waves are an efficient mechanism to transfer growth factor signaling rapidly across a large tissue. Significance statementSignaling waves represent a possible mechanism of spatiotemporal organization of multicellular tissues. We have recently shown that waves of activity of the kinase Erk control osteoblast regeneration in adult zebrafish scales. Here, we present a detailed characterization of a mathematical model of these signaling waves. We show that a source region poised in an oscillatory state can broadcast traveling waves of Erk activity in the surrounding excitable tissue. The dynamics of the source control the number and frequency of waves. Geometrical arguments support the notion that excitable Erk waves are an effective mechanism to transport growth factor signaling across a large regenerating tissue.

developmental biology↗

bioRxiv2020

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EECs). EECs can communicate nutritional information to the nervous system, but similar mechanisms for microbial information are unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda specifically activates EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increases intestinal motility in an EEC-dependent manner. Microbial, pharmacological, or optogenetic activation of Trpa1+EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons through 5-HT. We identified a subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes that potently activates zebrafish EEC Trpa1 signaling and also directly stimulates human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to specific microbial signals.

physiology↗

bioRxiv2019

An Array of 60,000 Antibodies for Proteome-Scale Antibody Generation and Target Discovery

Antibodies are essential for elucidating the roles of genes decoded by genome sequencing. However, affordable technology for proteome-scale antibody generation does not exist. To address this, we developed the Proteome Epitope Tag Antibody Library (PETAL) and its array. PETAL consists of 62,208 mAbs against 15,199 peptides from diverse proteomes. PETAL harbors binders for a great multitude of proteins in nature due to antibody multispecificity, an intrinsic feature of an antibody. Distinctive combinations of 10,000-20,000 mAbs were found to target specific proteomes by array screening. Phenotype-specific mAb-target pairs were discovered for maize and zebrafish samples. Immunofluorescence and flow cytometry mAbs for human membrane proteins and ChIP-seq mAbs for transcription factors were identified from respective proteome-binding PETAL mAbs. Differential screening of cell surface proteomes of tumor and normal tissues discovered internalizing tumor antigens for antibody-drug conjugates. By discovering high affinity mAbs at a fraction of current time and cost, PETAL enables proteome-scale antibody generation and target discovery.

systems biology↗