Biology subjects

Carney, T. J.

Publications and source records attributed to Carney, T. J..

13 recordsLinked to original sources

bioRxiv2025

TGF-beta coordinates changes in Keratin gene expression during complex tissue regeneration.

Zebrafish larval tail regeneration can be broadly broken down into three phases: wound repair, initiation of regeneration and redevelopment of the tail. We and others have shown that activation of the Hedgehog and TGF-beta signalling pathways is required to initiate tail regeneration. In this study we initially focus on these two pathways to better understand their role regeneration. We have found that Hedgehog signalling acts upstream of the TGF-beta pathway by activating transcription of the TGF-beta ligand tgfb1a. We have identified differentially expressed genes that are regulated after tail excision as well as those that are dependent upon TGF-beta signalling. From this analysis we found that Keratin genes fall into categories of co-expression: one set that is upregulated by TGF-beta signalling after injury (krtup) and a second set that is downregulated (krtdown). To see whether these two sets show co-expression more broadly, we analysed single cell RNA sequencing data and found that krts fall into the same categories during zebrafish development. We have mapped the expression of a representative member of krtup (krt97) and krtdown (krt5) sets during development and regeneration and show that krt97 is found in epithelia that is moving and krt5 is expressed in epithelia that is stationary. Based upon our results we propose that the krtup set allows for collective epithelial migration while the krtdown set inhibits mobility. These results reveal one mechanism by which TGF-beta may initiate regeneration and could help to explain how changes in cytoskeletal composition influence cell movement during development and repair.

developmental biology↗

bioRxiv2025

Measuring morphogen transport over multiple spatial scales in live zebrafish embryos

Morphogenesis is controlled by signalling morphogens that form gradients across the embryo. The gradient formation requires that the low molecular weight morphogens slow down their diffusion by at least one order of magnitude. However, the precise slow-down of diffusion across the relevant micrometre scales has not been directly observed. Here, we develop and employ Single-Plane Illumination Microscope based spatial Fluorescence Cross-Correlation Spectroscopy (SPIM-sFCCS) to directly measure the diffusion coefficient of the morphogen Squint in early zebrafish embryos as a function of topography and length scale. We show that Squints diffusion coefficient changes on length scales that are commensurate with the diameter of the intercellular spaces in the embryo and that the slow-down is regulated by receptor binding. The slowdown can be reduced by either the knockdown of Activin receptor 2b, a receptor for Squint, or the overexpression of Lefty2, an inhibitor of Squint. Based on our results and supporting simulations, we propose an interstitial space-dependent transient receptor binding and diffusing mechanism to explain this slowdown, which is crucial for gradient formation and embryonic patterning.

developmental biology↗

bioRxiv2024

BMP signalling attenuates intercellular adhesion to drive mesenchyme migration during fin fold morphogenesis

The development of the vertebrate limb bud is a multifactorial process involving the coordinated activity of diverse signalling pathways and cell types. BMP signalling has been consistently shown to play an integral role during the patterning of mouse and chick limbs. However, the molecular mechanisms and cellular processes underlying its contribution to limb development are poorly understood. Using zebrafish embryos, we demonstrate a novel role of BMPs in driving median fin fold morphogenesis, an evolutionary related structure. Our findings indicated that a graded BMP signal is established in the emerging fin fold along the proximo-distal axis, with the fin mesenchymal cells being identified as the primary target of the signalling cue. Pharmacological and genetic ablation of BMP signalling in the fin fold inhibited the normal migration of the mesenchymal cells into the fin fold. The observed inhibition of migration was accompanied by the elongation in mesenchymal cell shape, which hinted at the possibility of altered physical forces acting on the cell population. We observed that loss of BMP signalling led to the cellular re-distribution and altered membrane dynamics of N-cadherin in the fin mesenchymal cells, which manifested as an increase in homotypic cell adhesion. This change in cell-adhesion serves as the mechanistic basis for the observed defect in migration. Furthermore, inability to detach due to loss of BMP signalling occurs independently of sphingosine-1-phosphate signalling that generates a gradient of mesenchyme adhesion to the underlying matrix. Overall, this study highlights the cellular and molecular mechanism underlying the role of BMPs in coordinating early fin bud morphogenesis.

developmental biology↗

bioRxiv2024

Protease-activated receptor 2 links protease activity with calcium waves during egg activation and blastomere cleavage

Successful initiation of animal development requires activation of the egg immediately prior to fusion of gamete pronucleii. In all taxa, this is initiated by waves of calcium transients which transverse across the egg. Calcium waves also occur at cleavage furrows during later blastula cytokinesis. Calcium is released from the endoplasmic reticulum through activation of 1,4,5-trisphosphate (IP3) receptors. Only a subset of the mechanisms employed to generate IP3 during vertebrate egg activation are defined, with strong evidence that other critical mechanisms exist. Serine proteases have been long implicated in egg activation and fertilisation. Here we report that treatment of zebrafish eggs with serine protease inhibitors leads to defective calcium wave propagation and failed egg activation. We further show that mutation of zebrafish Protease-activated receptor 2a (Par2a) also results in severe disruption of egg activation, leading to failed chorion elevation and ooplasmic segregation. Milder par2a mutants progress further, but then show abnormal blastomere cleavage. We observed that par2a mutants show decreased amplitude and duration of calcium transients. Restoring Ca++ or direct injection of IP3 ligand rescues egg activation aborted by either serine protease inhibitor treatment or by mutation of Par2a. We thus show that serine protease activity is a critical regulator of IP3 and subsequent calcium wave amplification during zebrafish egg activation, and link this to intracellular calcium release via the protease receptor, Par2a. This constitutes a novel signalling pathway critical for successful fertilisation. Significance StatementBoth sperm and egg must undergo a series of important steps to become competent for successful fertilisation. Defining these steps is central to our understanding of reproductive biology and our ability to improve fertility treatments. As the process of gamete maturation and fertilisation has highly conserved principles across the animal kingdom, there are also important implications for aqua- and agriculture. One of the first signalling events of your life leads to the release of bursts of calcium in the egg. We know the importance of this for fertilisation but have only a partial picture of how this occurs. Our work here, using fish genetics, identifies a new signalling pathway regulating these first important flashes of calcium in the egg.

developmental biology↗

bioRxiv2024

Genetic regulation of injury induced heterotopic ossification in adult zebrafish

Heterotopic ossification is the inappropriate formation of bone in soft tissues of the body. It can manifest spontaneously in rare genetic conditions or as a response to injury, known as acquired heterotopic ossification. There are several experimental models for studying heterotopic ossification from different sources of damage. However, their tenuous mechanistic relevance to the human condition, invasive and laborious nature and/or lack of amenability to chemical and genetic screens, limit their utility. To address these limitations, we developed a simple zebrafish injury model that manifests heterotopic ossification in response to micro-fractures in combination with muscle injury. These findings indicate that clinically-emulated injuries in zebrafish can lead to osteo-induction and proliferation as observed in heterotopic ossification in myositis ossificans traumatica. Exploiting this model, we analysed the penetrance and expressivity of heterotopic ossification and defined the transcriptional response to trauma, identifying differentially regulated genes. Taking advantage of defined mutants in several of these candidates, we explored their impact on heterotopic bone formation. Our findings revealed that an increase in potassium channel Kcnk5b activity potentiates injury response. In contrast, we demonstrate that inflammatory responses are essential for the ectopic bone growth, as mutations in Interleukin 11 receptor paralogue (Il11ra) exhibit a drastically reduced ossification response. Based on these findings, we postulate that enhanced ionic signaling, specifically through Kcnk5b, regulates the intensity of the skeletogenic injury response, which, in part, requires immune response regulated by Il11ra.

genetics↗

bioRxiv2022

Echolocation-like model of directed cell migrationwithin growing tissues

During development and regeneration, cells migrate to specific locations within growing tissues. These cells can respond to both biochemical signals and mechanical cues, resulting in directed migration. Such migration is often highly stereotypic. Yet, how cells respond to migratory signals in a robust manner within a growing domain remains an open problem. Here, we propose a model of directed migration in growing tissues motivated by echolocation. The migrating cells generate a signaling gradient that induces a response signal from the moving system boundary. This response signal mediates cellular adhesion to the surrounding matrix and hence modulates the cell migration. We find that such a mechanism can align a series of cells at stable positions within growing systems and can effectively scale to system size. Finally, we discuss the relevance of such a model to fibroblast migration and location within the developing zebrafish caudal fin, which may be regulated by opposing signaling gradients of Slit-Robo pathway components. Significance StatementHow do cells reliably migrate within growing environments? Here, we show that cells can take advantage of an echolocation-like process, whereby they induce a response from the tissue boundary. As they approach the boundary, the response signal strengthens and brings the cell to a fixed position from the boundary. This simple system may be applicable to fibroblast migration in the fin.

biophysics↗

bioRxiv2021

Colorimetric and fluorescent TRAP assays for visualising and quantifying fish osteoclast activity

Histochemical detection of tartrate-resistant acid phosphatase (TRAP) activity is a fundamental technique for visualizing osteoclastic bone resorption and assessing osteoclast activity status in tissues. This approach has mostly employed colorimetric detection, which has limited quantification of activity in situ and co-labelling with other skeletal markers. Here we report simple colorimetric and fluorescent TRAP assays in zebrafish and medaka, two important model organisms for investigating the pathogenesis of bone disorders. We show fluorescent TRAP staining, utilising the ELF97 substrate, is a rapid, robust and stable system to visualise and quantify osteoclast activity in zebrafish, and is compatible with other fluorescence stains, transgenic lines and antibody approaches. Using this approach, we show that TRAP activity is predominantly found around the base of the zebrafish pharyngeal teeth, where osteoclast activity state appears to be heterogeneous.

cell biology↗

bioRxiv2021

Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

cell biology↗

bioRxiv2021

Zebrafish pigment cells develop directly from persistent highly multipotent progenitors.

Neural crest cells (NCCs) are highly multipotent stem cells. A long-standing controversy exists over the mechanism of NCC fate specification, specifically regarding the presence and potency of intermediate progenitors. The direct fate restriction (DFR) model, based on early in vivo clonal studies, hypothesised that intermediates are absent and that migrating cells maintain full multipotency1-6. However, most authors favour progressive fate restriction (PFR) models, with fully multipotent early NCCs (ENCCs) transitioning to partially-restricted intermediates before committing to individual fates7-12. Here, single cell transcriptional profiling of zebrafish pigment cell development leads to us proposing a Cyclical Fate Restriction mechanism of NCC development that reconciles the DFR and PFR models. Our clustering of single NCC Nanostring transcriptional profiles identifies only broadly multipotent intermediate states between ENCCs and differentiated melanocytes and iridophores. Leukocyte tyrosine kinase (Ltk) marks the multipotent progenitor and iridophores, consistent with biphasic ltk expression13-15. Ltk inhibitor and constitutive activation studies support expression at an early multipotent stage, whilst lineage-tracing of ltk-expressing cells reveals their multipotency extends beyond pigment cell-types to neural fates. We conclude that pigment cell development does not involve a conventional PFR mechanism, but instead occurs directly and more dynamically from a broadly multipotent intermediate state.

developmental biology↗

bioRxiv2021

CD9 tetraspanins convey robustness to CXCR4b signalling during collective cell migration

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

developmental biology↗

bioRxiv2021

Aerobic glycolysis is important for zebrafish larval wound closure and tail regeneration

The underlying mechanisms of appendage regeneration remain largely unknown and uncovering these mechanisms in capable organisms has far-reaching implications for potential treatments in humans. Recent studies implicate a requirement for metabolic reprogramming reminiscent of the Warburg effect during successful appendage and organ regeneration. As changes are thus predicted to be highly dynamic, methods permitting direct, real-time visualization of metabolites at the tissue and organismal level, would offer a significant advance in defining the influence of metabolism on regeneration and healing. We sought to examine whether glycolytic activity was altered during larval fin regeneration, utilising the genetically encoded biosensor, Laconic, enabling the spatiotemporal assessment of lactate levels in living zebrafish. We present evidence for a rapid increase in lactate levels within minutes following injury, with a role of aerobic glycolysis in actomyosin contraction and wound closure. We also find a second wave of lactate production, associated with overall larval tail regeneration. Chemical inhibition of glycolysis attenuates both contraction of the wound and regrowth of tissue following tail amputation, suggesting aerobic glycolysis is necessary at two distinct stages of regeneration.

developmental biology↗

bioRxiv2021

Matriptase generates a tissue damage response via promoting Gq signalling, leading to RSK and DUOX activation

Tissues respond to damage by increasing inflammation and epithelial cell motility. How damage detection and responses are orchestrated is unclear. Overexpression of the membrane bound protease, Matriptase, or mutation of its inhibitor, Hai1, results in inflamed epithelia, in which cells have increased motility and are prone to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H2O2, Nf{kappa}B signalling, and IP3R-mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit rescues both the hai1a inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated pERK, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as pERK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

cell biology↗

bioRxiv2018

Functional analysis of a hypomorphic allele shows that MMP14 catalytic activity is the prime determinant of the Winchester syndrome phenotype

Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14s transport to the cell membrane. Instead, it partially impairs MMP14s proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14s catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density due to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.

genetics↗