The scale of zebrafish pectoral fin buds is determined by intercellular K+ levels and consequent Ca2+-mediated signaling via retinoic acid regulation of Rcan2 and Kcnk5b.

K+ channels regulate morphogens to scale adult fins, but little is known about what regulates the channels and how they control morphogen expression. Using the zebrafish pectoral fin bud as a model for early vertebrate fin/limb development, we found that K+ channels also scale this anatomical structure, and we determined how one K+-leak channel, Kcnk5b, integrates into its developmental program. From FLIM measurements of a Förster Resonance Energy Transfer (FRET)-based K+ sensor, we observed coordinated decreases in intracellular K+ levels during bud growth, and overexpression of K+-leak channels in vivo coordinately increased bud proportions. Retinoic acid, which can enhance fin/limb bud growth, decreased K+ in bud tissues and up-regulated regulator of calcineurin (rcan2). rcan2 overexpression increased bud growth and decreased K+, while CRISPR-Cas9 targeting of rcan2 decreased growth and increased K+. We observed similar results in the adult caudal fins. Moreover, CRISPR targeting of Kcnk5b revealed that Rcan2-mediated growth was dependent on the Kcnk5b. We also found that Kcnk5b enhanced depolarization in fin bud cells via Na+ channels and that this enhanced depolarization was required for Kcnk5b-enhanced growth. Lastly, Kcnk5b-induced shha transcription and bud growth required IP3R-mediated Ca2+ release and CaMKK activity. Thus, we provide a mechanism for how retinoic acid via rcan2 can regulate K+-channel activity to scale a vertebrate appendage via intercellular Ca2+ signaling.

Apela promotes blood vessel regeneration and remodeling in zebrafish.

In contrast to adult mammals, zebrafish display a high capacity to heal injuries and repair damage to various organs. One of the earliest responses to injury in adult zebrafish is revascularization, followed by tissue morphogenesis. Tissue vascularization entails the formation of a blood vessel plexus that remodels into arteries and veins. The mechanisms that coordinate these processes during vessel regeneration are poorly understood. Hence, investigating and identifying the factors that promote revascularization and vessel remodeling have great therapeutic potential. Here, we revealed that fin vessel remodeling critically depends on Apela peptide. We found that Apela selectively accumulated in newly formed zebrafish fin tissue and vessels. The temporal expression of Apela, Apln, and their receptor Aplnr is different during the regenerative process. While morpholino-mediated knockdown of Apela (Mo-Apela) prevented vessel remodeling, exogenous Apela peptide mediated plexus repression and the development of arteries in regenerated fins. In contrast, Apela enhanced subintestinal venous plexus formation (SIVP). The use of sunitinib completely inhibited vascular plexus formation in zebrafish, which was not prevented by exogenous application. Furthermore, Apela regulates the expression of vessel remolding-related genes including VWF, IGFPB3, ESM1, VEGFR2, Apln, and Aplnr, thereby linking Apela to the vascular plexus factor network as generated by the STRING online database. Together, our findings reveal a new role for Apela in vessel regeneration and remodeling in fin zebrafish and provide a framework for further understanding the cellular and molecular mechanisms involved in vessel regeneration.

Insulin-like growth factor receptor / mTOR signaling elevates global translation to accelerate zebrafish fin regenerative outgrowth.

Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR)/mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.

A regeneration-triggered metabolic adaptation is necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration.

Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a modulation in the metabolic profile at early stages of regeneration to enhance glycolysis at the expense of mitochondrial oxidation. This metabolic adaptation mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.

Understanding the complexity of epimorphic regeneration in zebrafish caudal fin tissue: A transcriptomic and proteomic approach.

The complex epimorphic regeneration of zebrafish caudal fin tissue is hasty and absolute. This study was executed to understand the role of various genes/proteins involved in the regeneration of zebrafish caudal fin tissue through differential transcriptomics and proteomics analysis. Based on our study 1408 genes and 661 proteins were found differentially regulated in the regenerating caudal fin tissue for having at least 1-log fold change. Interleukin, Solute carrier, Protein arginine methyltransferase, Homeobox, Neurotransmitter and several novel genes were found to be associated with regeneration for its differential regulation during the mechanism. Based on the network and pathway analysis the differentially regulated genes and proteins were found allied with activation of cell proliferation, cell viability, cell survival & cell movement and inactivation of organismal death, morbidity, necrosis, death of embryo & cell death. This study has mapped a detailed insight of the genes/proteins expression associated with the epimorphic regeneration more profoundly.

Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives.

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M-1s-1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

The role of Gdf5 in the development of the zebrafish fin endoskeleton.

BACKGROUND: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota. RESULTS: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. CONCLUSIONS: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

MicroRNA roles in regeneration: Multiple lessons from zebrafish.

MicroRNAs (miRNAs) are small noncoding RNAs with pivotal roles in the control of gene expression. By comparing the miRNA profiles of uninjured vs. regenerating tissues and structures, several studies have found that miRNAs are potentially involved in the regenerative process. By inducing miRNA overexpression or inhibition, elegant experiments have directed regenerative responses validating relevant miRNA-to-target interactions. The zebrafish (Danio rerio) has been the epicenter of regenerative research because of its exceptional capability to self-repair damaged tissues and body structures. In this review, we discuss recent discoveries that have improved our understanding of the impact of gene regulation mediated by miRNAs in the context of the regeneration of fins, heart, retina, and nervous tissue in zebrafish. We compiled what is known about the miRNA control of regeneration in these tissues and investigated the links among up-regulated and down-regulated miRNAs, their putative or validated targets, and the regenerative process. Finally, we briefly discuss the forthcoming prospects, highlighting directions and the potential for further development of this field.

Evolved Bmp6 enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks.

Mutations in enhancers have been shown to often underlie natural variation but the evolved differences in enhancer activity can be difficult to identify in vivo. Threespine sticklebacks (Gasterosteus aculeatus) are a robust system for studying enhancer evolution due to abundant natural genetic variation, a diversity of evolved phenotypes between ancestral marine and derived freshwater forms, and the tractability of transgenic techniques. Previous work identified a series of polymorphisms within an intronic enhancer of the Bone morphogenetic protein 6 (Bmp6) gene that are associated with evolved tooth gain, a derived increase in freshwater tooth number that arises late in development. Here, we use a bicistronic reporter construct containing a genetic insulator and a pair of reciprocal two-color transgenic reporter lines to compare enhancer activity of marine and freshwater alleles of this enhancer. In older fish, the two alleles drive partially overlapping expression in both mesenchyme and epithelium of developing teeth, but the freshwater enhancer drives a reduced mesenchymal domain and a larger epithelial domain relative to the marine enhancer. In younger fish, these spatial shifts in enhancer activity are less pronounced. Comparing Bmp6 expression by in situ hybridization in developing teeth of marine and freshwater fish reveals similar evolved spatial shifts in gene expression. Together, these data support a model in which the polymorphisms within this enhancer underlie evolved tooth gain by shifting the spatial expression of Bmp6 during tooth development, and provide a general strategy to identify spatial differences in enhancer activity in vivo.

NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration.

Fish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

Collagen fibers provide guidance cues for capillary regrowth during regenerative angiogenesis in zebrafish.

Although well investigated, the importance of collagen fibers in supporting angiogenesis is not well understood. In this study, we demonstrate that extracellular collagen fibers provide guidance cues for endothelial cell migration during regenerative angiogenesis in the caudal zebrafish fin. Inhibition of collagen cross-linking by ß-Aminopropionitrile results in a 70% shorter regeneration area with 50% reduced vessel growth and disintegrated collagen fibers. The disrupted collagen scaffold impedes endothelial cell migration and induces formation of abnormal angioma-like blood vessels. Treatment of the Fli//colRN zebrafish line with the prodrug Nifurpirinol, which selectively damages the active collagen-producing 1α2 cells, reduced the regeneration area and vascular growth by 50% with wider, but less inter-connected, capillary segments. The regenerated area contained larger vessels partially covered by endothelial cells embedded in atypical extracellular matrix containing cell debris and apoptotic bodies, macrophages and granulocytes. Similar experiments performed in early embryonic zebrafish suggested that collagens are important also during embryonic angiogenesis. In vitro assays revealed that collagen I allows for the most efficient endothelial cell migration, followed by collagen IV relative to the complete absence of exogenous matrix support. Our data demonstrates severe vascular defects and restricted fin regeneration when collagens are impaired. Collagen I therefore, provides support and guidance for endothelial cell migration while collagen IV is responsible for proper lumen formation and vascular integrity.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Spiny and soft-rayed fin domains in acanthomorph fish are established through a BMP-gremlin-shh signaling network.

With over 18,000 species, the Acanthomorpha, or spiny-rayed fishes, form the largest and arguably most diverse radiation of vertebrates. One of the key novelties that contributed to their evolutionary success are the spiny rays in their fins that serve as a defense mechanism. We investigated the patterning mechanisms underlying the differentiation of median fin Anlagen into discrete spiny and soft-rayed domains during the ontogeny of the direct-developing cichlid fish Astatotilapia burtoni Distinct transcription factor signatures characterize these two fin domains, whereby mutually exclusive expression of hoxa13a/b with alx4a/b and tbx2b marks the spine to soft-ray boundary. The soft-ray domain is established by BMP inhibition via gremlin1b, which synergizes in the posterior fin with shh secreted from a zone of polarizing activity. Modulation of BMP signaling by chemical inhibition or gremlin1b CRISPR/Cas9 knockout induces homeotic transformations of spines into soft rays and vice versa. The expression of spine and soft-ray genes in nonacanthomorph fins indicates that a combination of exaptation and posterior expansion of an ancestral developmental program for the anterior fin margin allowed the evolution of robustly individuated spiny and soft-rayed domains. We propose that a repeated exaptation of such pattern might underly the convergent evolution of anterior spiny-fin elements across fishes.

A show of Hands: Novel and conserved expression patterns of teleost hand paralogs during craniofacial, heart, fin, peripheral nervous system and gut development.

BACKGROUND: Hand genes are required for the development of the vertebrate jaw, heart, peripheral nervous system, limb, gut, placenta, and decidua. Two Hand paralogues, Hand1 and Hand2, are present in most vertebrates, where they mediate different functions yet overlap in expression. In ray-finned fishes, Hand gene expression and function is only known for the zebrafish, which represents the rare condition of having a single Hand gene, hand2. Here we describe the developmental expression of hand1 and hand2 in the cichlid Copadichromis azureus. RESULTS: hand1 and hand2 are expressed in the cichlid heart, paired fins, pharyngeal arches, peripheral nervous system, gut, and lateral plate mesoderm with different degrees of overlap. CONCLUSIONS: Hand gene expression in the gut, peripheral nervous system, and pharyngeal arches may have already been fixed in the lobe- and ray-finned fish common ancestor. In other embryonic regions, such as paired appendages, hand2 expression was fixed, while hand1 expression diverged in lobe- and ray-finned fish lineages. In the lateral plate mesoderm and arch associated catecholaminergic cells, hand1 and hand2 swapped expression between divergent lineages. Distinct expression of cichlid hand1 and hand2 in the epicardium and myocardium of the developing heart may represent the ancestral pattern for bony fishes.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins.

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage.

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.

A calcineurin-mediated scaling mechanism that controls a K+-leak channel to regulate morphogen and growth factor transcription.

The increase in activity of the two-pore potassium-leak channel Kcnk5b maintains allometric juvenile growth of adult zebrafish appendages. However, it remains unknown how this channel maintains allometric growth and how its bioelectric activity is regulated to scale these anatomical structures. We show the activation of Kcnk5b is sufficient to activate several genes that are part of important development programs. We provide in vivo transplantation evidence that the activation of gene transcription is cell autonomous. We also show that Kcnk5b will induce the expression of different subsets of the tested developmental genes in different cultured mammalian cell lines, which may explain how one electrophysiological stimulus can coordinately regulate the allometric growth of diverse populations of cells in the fin that use different developmental signals. We also provide evidence that the post-translational modification of serine 345 in Kcnk5b by calcineurin regulates channel activity to scale the fin. Thus, we show how an endogenous bioelectric mechanism can be regulated to promote coordinated developmental signaling to generate and scale a vertebrate appendage.

Organs, limbs, fins and tails are made of multiple tissues whose growth is controlled by specific signals and genetic programmes. All these different cell populations must work together during development or regeneration to form a complete structure that is the right size in relation to the rest of the body. Growing evidence suggests that this synchronicity might be down to electric signals, which are created by movements of charged particles in and out of cells. In particular, previous work has identified two factors that control the development of fins in fish: the Kcnk5b potassium-leak channel, which allows positive ions to cross the cell membrane; and an enzyme called calcineurin, which can modify the activity of proteins. Kcnk5b and calcineurin seem to play similar roles in the proportional growth of the fins in relation to the body, but exactly how was unknown. To investigate this question, Yi et al. used genetically modified zebrafish to show how the Kcnk5b channel could control genes responsible for appendage growth. However, their tests on different cell types revealed that potassium movement through the Kcnk5b channel leads to different sets of developmental genes being turned on, depending on the tissue type of the cell. This could explain how one type of signal (in this case, movement of ions) can coordinate the growth of a wide range of tissues that use different combinations of developmental genes to form. Kcnk5b therefore appears to coordinate the regulation of the various combinations of genes needed for different fin tissues to develop, so that every component grows in a proportional, synchronized manner. Yi et al. also showed that calcineurin can modify the Kcnk5b channel to control its activity. In turn, this affects the movement of potassium ions across the membrane, changing electrical activity and, as a consequence, the proportional growth of the fin. Further work should explore how Kcnk5b and calcineurin link to other signals that regulate the size of fins and limbs. Ultimately, a finer understanding of the molecules controlling the growth of body parts will be useful in fields such as regenerative medicine or stem cell biology, which attempt to build organs for clinical therapies.

Heparan sulfate proteoglycan expression in the regenerating zebrafish fin.

BACKGROUND: Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)-anchored proteins, and transmembrane-anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown. RESULTS: Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions. CONCLUSIONS: HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration.