Cytoskeletal dynamics of the teleostean fin ray during fin epimorphic regeneration

Teleost fishes can regenerate their fins by epimorphic regeneration, a process that involves the transition of the formerly quiescent tissues of the stump to an active, growing state. This involves dynamic modifications of cell phenotype and behavior that must rely on alterations of the cytoskeleton. We have studied the spatial and temporal distribution of three main components of the cytoskeleton (actin, keratin and vimentin) in the regenerating fin, in order to establish putative relationships between cell cytoskeleton and cell behavior. According to our results, the massive rearrangement undergone by the epidermis right after injury, which takes place by cell migration, correlates with a transient down-regulation of keratin and a strong up-regulation of actin in the epidermal cells. During the subsequent epidermal growth, based on cell proliferation, keratin normal pattern is recovered while actin is down-regulated, although not to normal (quiescent) levels. The epidermal basal layer in contact with the blastema displays a particular cytoskeletal profile, different to that of the rest of the epidermal cells, which reflects its special features. In the connective tissue compartment, somatic cells do not contain vimentin, but keratin, as intermediate filament. Proliferative and migrative activation of these cells after injury correlates with actin up-regulation. Although this initial activation does not involve keratin down-regulation, blastemal cells were later observed to lack keratin, suggesting that such cytoskeletal modification might be needed for connective tissue cells to dedifferentiate and form the blastema. Cell differentiation in the newly formed, regenerated ray is accompanied by actin down-regulation and keratin up-regulation.     

Laser ablation of the sonic hedgehog-a-expressing cells during fin regeneration affects ray branching morphogenesis

The zebrafish fin is an excellent system to study the mechanisms of dermal bone patterning. Fin rays are segmented structures that form successive bifurcations both during ontogenesis and regeneration. Previous studies showed that sonic hedgehog (shha) may regulate regenerative bone patterning based on its expression pattern and functional analysis. The present study investigates the role of the shha-expressing cells in the patterning of fin ray branches. The shha expression domain in the basal epidermis of each fin ray splits into two prior to ray bifurcation. In addition, the osteoblast proliferation profile follows the dynamic expression pattern of shha. A zebrafish transgenic line, 2.4shh:gfpABC#15, in which GFP expression recapitulates the endogenous expression of shha, was used to specifically ablate shha-expressing cells with a laser beam. Such ablations lead to a delay in the sequence of events leading to ray bifurcation without affecting the overall growth of the fin ray. These results suggest that shha-expressing cells direct localized osteoblast proliferation and thus regulate branching morphogenesis. This study reveals the fin ray as a new accessible system to investigate epithelial-mesenchymal interactions leading to organ branching.     

Fgf signaling instructs position-dependent growth rate during zebrafish fin regeneration

During appendage regeneration in urodeles and teleosts, tissue replacement is precisely regulated such that only the appropriate structures are recovered, a phenomenon referred to as positional memory. It is believed that there exists, or is quickly established after amputation, a dynamic gradient of positional information along the proximodistal (PD) axis of the appendage that assigns region-specific instructions to injured tissue. These instructions specify the amount of tissue to regenerate, as well as the rate at which regenerative growth is to occur. A striking theme among many species is that the rate of regeneration is more rapid in proximally amputated appendages compared with distal amputations. However, the underlying molecular regulation is unclear. Here, we identify position-dependent differences in the rate of growth during zebrafish caudal fin regeneration. These growth rates correlate with position-dependent differences in blastemal length, mitotic index and expression of the Fgf target genes mkp3, sef and spry4. To address whether PD differences in amounts of Fgf signaling are responsible for position-dependent blastemal function, we have generated transgenic fish in which Fgf receptor activity can be experimentally manipulated. We find that the level of Fgf signaling exhibits strict control over target gene expression, blastemal proliferation and regenerative growth rate. Our results demonstrate that Fgf signaling defines position-dependent blastemal properties and growth rates for the regenerating zebrafish appendage.     

A dynamic simulation model of tissue growth and cell patterning

The distributions of cells in tissues of experimental chimaeras and mosaics can serve as tests of mechanisms and rules by which single cells organize themselves into complex, multicellular structures during embryogenesis. We have devised a dynamic, computer simulation model of tissue growth and cell patterning which is directly applicable to the analysis of chimaeras and mosaics. In the model, schematized cells possess a small behavioral repertoire and simple rules for the carrying out of these behaviors. Populations of such cells evolve tissue patterns in real-time that are very similar to those seen in experimental animals. In particular, we have modeled the major pattern features seen in amphibian and mammalian eye chimaeras and mosaics. We have demonstrated that cell mixing can be a passive concomitant of interstitial cell division, a result which alleviates the need to postulate active cell mixing in such mammalian systems. We expect this approach to be a valuable addition to methods of pattern analysis in development.     

Proximodistal patterning during limb regeneration

Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of the limb is precisely replaced--amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.     

Ray-interray interactions during fin regeneration of Danio rerio

Teleost fin ray bifurcations are characteristic of each ray in each fin of the fishes. Control of the positioning of such morphological markers is not well understood. We present evidence suggesting that the interray blastema is necessary for a proper bifurcation of each ray during regeneration in Danio rerio (Hamilton-Buchanan) (Cyprinidae, Teleostei). We performed single ray ablations, heterotopical graftings of ray fragments and small holes in lateral rays which do not normally bifurcate, to generate recombinants in which the lateral rays are surrounded with ectopic interrays originating from different positions within the tail fin. These ray-interray recombinants do now bifurcate. Furthermore, we show that the interray tissue and surrounding epidermis can modulate the length of the ray. These results stress the role of the interray in inducing bifurcations of the ray blastema as well as modulating ray morphogenesis in general. In addition, gene expression analysis under these experimental conditions suggests that msxA and msxD expression in the ray and interray epidermis is controlled by the ray blastema and that bmp4 could be a candidate signal involved in these inductions.     

Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration

Fin regeneration in adult zebrafish is accompanied by re-establishment of the pigment stripes. To understand the mechanisms underlying fin stripe regeneration and regulation of normal melanocyte stripe morphology, we investigated the origins of melanocytes in the regenerating fin and their requirement for the kit receptor tyrosine kinase. Using pre-existing melanin as a lineage tracer, we show that most fin regeneration melanocytes develop from undifferentiated precursors, rather than from differentiated melanocytes. Mutational analysis reveals two distinct classes of regeneration melanocytes. First, an early regeneration class develops dependent on kit function. In the absence of kit function and kit-dependent melanocytes, a second class of melanocytes develops at later stages of regeneration. This late kit-independent class of regeneration melanocytes has little or no role in wild-type fin stripe development, thus revealing a secondary mode for regulation of fin stripes. Expression of melanocyte markers in regenerating kit mutant fins suggests that kit normally acts after mitf and before dct to promote development of the primary kit-dependent melanocytes. kit-dependent and kit-independent melanocytes are also present during fin stripe ontogeny in patterns similar to those observed during regeneration.     

A developmental transition in growth control during zebrafish caudal fin development

A long-standing question in developmental biology is how do growing and developing animals achieve form and then maintain it. We have revealed a critical transition in growth control during zebrafish caudal fin development, wherein a switch from allometric to isometric growth occurs. This morphological transition led us to hypothesize additional physiological changes in growth control pathways. To test this, we fasted juvenile and adult zebrafish. Juvenile fins continued allometric growth until development of the mature bi-lobed shape was completed. In contrast, the isometric growth of mature adult fins arrested within days of initiating a fast. We explored the biochemical basis of this difference in physiology between the two phases by assessing the sensitivity to rapamycin, a drug that blocks a nutrient-sensing pathway. We show that the nutrition-independent, allometric growth phase is resistant to rapamycin at 10-fold higher concentrations than are effective at arresting growth in the nutrition-dependent, isometric growth phase. We thus link a morphological transition in growth control between allometric and isometric growth mechanisms to different physiological responses to nutritional state of the animal and finally to different pharmacological responses to a drug (rapamycin) that affects the nutrition-sensing mechanism described from yeast to human.     

Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration

Unlike humans, some vertebrate animals are able to completely regenerate damaged appendages and other organs. For example, adult zebrafish will regenerate the complex structure of an amputated caudal fin to a degree that the original and replacement fins are indistinguishable. The blastema, a mass of cells that uniquely forms following appendage amputation in regenerating animals, is the major source of regenerated tissue. However, the cell lineage(s) that contribute to the blastema and their ultimate contribution(s) to the regenerated fin have not been definitively characterized. It has been suggested that cells near the amputation site dedifferentiate forming multipotent progenitors that populate the blastema and then give rise to multiple cell types of the regenerated fin. Other studies propose that blastema cells are non-uniform populations that remain restricted in their potential to contribute to different cell lineages. We tested these models by using inducible Cre-lox technology to generate adult zebrafish with distinct, isolated groups of genetically labeled cells within the caudal fin. We then tracked populations of several cell types over the entire course of fin regeneration in individual animals. We found no evidence for the existence of multipotent progenitors. Instead, multiple cell types, including epidermal cells, intra-ray fibroblasts, and osteoblasts, contribute to the newly regenerated tissue while remaining highly restricted with respect to their developmental identity. Our studies further demonstrate that the regenerating fin consists of many repeating blastema "units" dedicated to each fin ray. These blastemas each have an organized structure of lineage restricted, dedifferentiated cells that cooperate to regenerate the caudal fin.     

The Cx43-like connexin protein Cx40.8 is differentially localized during fin ontogeny and fin regeneration

Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.     

Posterior<Emphasis Type="Italic"> hoxa</Emphasis> genes expression during zebrafish bony fin ray development and regeneration suggests their involvement in scleroblast differentiation

Expression of two zebrafish developmental posterior hoxa genes, hoxa11b and hoxa13b, was studied by in situ hybridization during pectoral and caudal fin development and regeneration. Expression was restricted to cells of the bony rays region. During fin development, molecular cytological analysis revealed that a subpopulation of mesenchymal cells expressed these two hoxa genes during their early differentiation in the subapical region of the developing ray. These cells were identified as differentiating dermal bone making cells (scleroblasts). During fin regeneration, hoxa11b and hoxa13b genes are both induced in undifferentiated cells of the distalmost blastema region (DMB) and the proliferating zone (PZ) and later in differentiating bone-forming cells. In addition, the transient regionalization of the hoxa13b expression pattern in differentiated bone-forming cells along the proximodistal axis of the regenerating ray suggests that hoxa13b could participate in ray patterning. This study is the first to establish a correlation between hoxa gene expression and dermal bone cell differentiation.     

Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration

A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9420-9) contains supplementary material, which is available to authorized users.     

The Drosophila tumor suppressor expanded regulates growth, apoptosis, and patterning during development

The Drosophila expanded (ex) gene encodes a protein thought to play a role in signaling at apical junctions of epithelial cells. Previous studies have characterized this gene as a tumor suppressor involved in regulating the growth of a subset of Drosophila imaginal discs (Boedigheimer, M., Laughon, A., 1993. expanded: a gene involved in the control of cell proliferation in imaginal discs, Development 118, 1291-1301); although ex negatively regulates cell proliferation in the developing wing, it appeared to have a conflicting role in the eye. In contrast, our analysis of the loss-of-function phenotype indicates that ex does, in fact, regulate growth in the eye. We also show that this gene plays a role in patterning of the eye, mainly at the level of planar polarity. Our studies further demonstrate that, contrary to what was expected based on loss-of-function data, the tissue reduction phenotypes resulting from Ex overexpression are attributable to the induction of apoptotic cell death. Taken together, our data suggest that Ex is a versatile molecule that plays a role in most of the processes that govern disc development.     

A simulation model of mire patterning – revisited

The development of regular, oriented hummock-hollow or string-flark patterns in boreal peatlands can be explained by a simple spatially explicit model in which hummocks have a lower hydraulic conductivity than hollows and are more likely to occur at lower water levels. Revisiting a previously published model, the function used to simulate water flow is shown to be faulty. Nevertheless, the corrected model confirms the general conclusions of the original paper, showing the robustness of the approach.
Five phases can be discerned in the development of the pattern. Once the hummock strings reach from left to right across the grid, there is an increase in the volume of water stored in the system. The number of hummocks and hollows differs substantially from what would be expected based on the mean water level, showing that their arrangement is more important than the actual number of hummocks.
Parameter settings at which a pattern develops are sharply delineated, indicating there is a positive feedback mechanism that enhances initial patterning. Like in natural mire systems, the patterns anastomose and merge. In contrast to what is generally assumed for natural systems, the patterns show a distinct downward movement.     

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration

The origin of cells that generate the blastema following appendage amputation has been a long-standing question in epimorphic regeneration studies. The blastema is thought to originate from either stem (or progenitor) cells or differentiated cells of various tissues that undergo dedifferentiation. Here, we investigate the origin of cells that contribute to the regeneration of zebrafish caudal fin skeletal elements. We provide evidence that the process of lepidotrichia (bony rays) regeneration is initiated as early as 24 hours post-amputation and that differentiated scleroblasts acquire a proliferative state, detach from the lepidotrichia surface, migrate distally, integrate into the blastema and dedifferentiate. These findings provide novel insights into the origin of cells in epimorphic appendage regeneration in zebrafish and suggest conservation of regeneration mechanisms between fish and amphibians.