Marie-Andrée Akimenko

Old questions, new tools, and some answers to the mystery of fin regeneration†

Pluridisciplinary approaches led to the notion that fin regeneration is an intricate phenomenon involving epithelial–mesenchymal and reciprocal exchanges throughout the process as well as interactions between ray and interray tissue. The establishment of a blastema after fin amputation is the first event leading to the reconstruction of the missing part of the fin. Here, we review our knowledge on the origin of the blastema, its formation and growth, and of the mechanisms that control differentiation and patterning of the regenerate. Our current understanding results from studies of fin regeneration performed in various teleost fish over the past century. We also report the recent breakthroughs that have been made in the past decade with the arrival of a new model, the zebrafish, Danio rerio, which now offers the possibility to combine cytologic, molecular, and genetic analyses and open new perspectives in this field. Developmental Dynamics 226:190–201, 2003.

Bone patterning is altered in the regenerating zebrafish caudal fin after ectopic expression of sonic hedgehog and bmp2b or exposure to cyclopamine

Amputation of the zebrafish caudal fin stimulates regeneration of the dermal skeleton and reexpression of sonic hedgehog (shh)-signaling pathway genes. Expression patterns suggest a role for shh signaling in the secretion and patterning of the regenerating dermal bone, but a direct role has not been demonstrated. We established an in vivo method of gene transfection to express ectopically genes in the blastema of regenerating fins. Ectopic expression of shh or bmp2 in the blastema-induced excess bone deposition and altered patterning of the regenerate. The effects of shh ectopic expression could be antagonized by ectopic expression of chordin, an inhibitor of bone morphogenetic protein (bmp) signaling. We disrupted shh signaling in the regenerating fin by exposure to cyclopamine and found a dose-dependent inhibition of fin outgrowth, accumulation of melanocytes in the distal region of each fin ray, loss of actinotrichia, and reduction in cell proliferation in the mesenchyme. Morphological changes were accompanied by an expansion, followed by a reduction, in domains of shh expression and a rapid abolition of ptc1 expression. These results implicate shh and bmp2b signaling in the proliferation and/or differentiation of specialized bone-secreting cells in the blastema and suggest shh expression may be controlled by regulatory feedback mechanisms that define the region of bone secretion in the outgrowing fin.

Cell proliferation and movement during early fin regeneration in zebrafish

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di‐I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU‐injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di‐I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.

Characterization of two new zebrafish members of the hedgehog family : Atypical expression of a Zebrafish indian hedgehog gene in skeletal elements of both endochondral and dermal origins

We have characterized two new members of the Hedgehog (Hh) family in zebrafish, ihha and dhh, encoding for orthologues of the tetrapod Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) genes, respectively. Comparison of ihha and Type X collagen (col10a1) expression during skeletal development show that ihha transcripts are located in hypertrophic chondrocytes of cartilaginous elements of the craniofacial and fin endoskeleton. Surprisingly, col10a1 expression was also detected in cells forming intramembranous bones of the head and in flat cells surrounding cartilaginous structures. The expression of col10a1 in both endochondral and intramembranous bones reflects an atypical composition of the extracellular matrix of the zebrafish craniofacial skeleton. In addition, during fin ray regeneration, both ihha and col10a1 are detected in scleroblasts, osteoblast‐like cells secreting the matrix of the dermal bone fin ray. The presence of cartilage markers suggests that the dermal fin ray possesses an intermediate phenotype between cartilage and bone. Developmental Dynamics 235:478–489, 2006.

Loss of fish actinotrichia proteins and the fin-to-limb transition

The early development of teleost paired fins is strikingly similar to that of tetrapod limb buds and is controlled by similar mechanisms. One early morphological divergence between pectoral fins and limbs is in the fate of the apical ectodermal ridge (AER), the distal epidermis that rims the bud. Whereas the AER of tetrapods regresses after specification of the skeletal progenitors, the AER of teleost fishes forms a fold that elongates. Formation of the fin fold is accompanied by the synthesis of two rows of rigid, unmineralized fibrils called actinotrichia, which keep the fold straight and guide the migration of mesenchymal cells within the fold. The actinotrichia are made of elastoidin, the components of which, apart from collagen, are unknown. Here we show that two zebrafish proteins, which we name actinodin 1 and 2 (And1 and And2), are essential structural components of elastoidin. The presence of actinodin sequences in several teleost fishes and in the elephant shark (Callorhinchus milii, which occupies a basal phylogenetic position), but not in tetrapods, suggests that these genes have been lost during tetrapod species evolution. Double gene knockdown of and1 and and2 in zebrafish embryos results in the absence of actinotrichia and impaired fin folds. Gene expression profiles in embryos lacking and1 and and2 function are consistent with pectoral fin truncation and may offer a potential explanation for the polydactyly observed in early tetrapod fossils. We propose that the loss of both actinodins and actinotrichia during evolution may have led to the loss of lepidotrichia and may have contributed to the fin-to-limb transition.

Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos

Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause abnormal craniofacial cartilage development in other vertebrates, result in dose- and stage-dependent losses of dlx homeobox gene expression in several regions of the embryo. Dlx expression in neural crest cells migrating from the hindbrain and in the visceral arch primordia is particularly sensitive to RA treatment. The strongest effects are observed when RA is administered prior to or during crest cell migration but effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, our results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements.

Screen for genes differentially expressed during regeneration of the zebrafish caudal fin

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up‐regulated during “initiation” (1 day) and “outgrowth and differentiation” (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT‐PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT‐PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration. Developmental Dynamics 231:527–541, 2004.

Epidermal Expression of Apolipoprotein E Gene During Fin and Scale Development and Fin Regeneration in Zebrafish

Apolipoprotein E (apoE) plays an important role in systemic and local lipid homeostasis. We have examined the expression of apoE during morphogenesis and regeneration of paired and unpaired fins and during scale development in zebrafish (Danio rerio). In situ hybridization analysis revealed that, during embryogenesis, apoE is expressed in the epithelial cells of the median fin fold and of the pectoral fin buds. ApoE remains expressed in the elongating fin folds throughout development of the fins. During the larval to juvenile transition, apoE transcripts were present in the distal, interray and lateral epidermis of developing fins. Furthermore, as scale buds started to form, apoE was expressed in large scale domains which later, became restricted to the external posterior epidermal part of scales. A low level of transcripts could be observed at later developmental stages at these locations probably because fins and scales continue to grow throughout the animals life. During regeneration of both pectoral and caudal fins, a marked increase in apoE expression is observed as early as 12 hours after amputation in the wound epidermis. High levels of apoE transcripts are then localized primarily in the basal cell layer of the apical epidermis. The levels of apoE expression were maximum between the second to fourth days and then progressively declined to basal level by day 14. ApoE transcripts were also observed in putative macrophages infiltrated in the mesenchymal compartment of regenerating fins a few hours after amputation. In conclusion, apoE is highly expressed in the epidermis of developing fins and scales and during fin regeneration while no expression can be detected in the skin of the trunk. ApoE may play a specific role in fin and scale differentiation at sites where important epidermo‐dermal interactions occur for the elaboration of the dermal skeleton and/or for lipid uptake and redistribution within these rapidly growing structures. Dev Dyn 1999;214:207–215.

Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole-mount in situ hybridization method.

The caudal fin of adult zebrafish is used to study the molecular mechanisms that govern regeneration processes. Most reports of gene expression in regenerating caudal fins rely on in situ hybridization (ISH) on whole‐mount samples followed by sectioning of the samples. In such reports, expression is mostly confined to cells other than those located between the dense collagenous structures that are the actinotrichia and lepidotrichia. Here, we re‐examined the expression of genes by performing ISH directly on cryo‐sections of regenerates. We detected expression of some of these genes in cell types that appeared to be non‐expressing when ISH was performed on whole‐mount samples. These results demonstrate that ISH reagents have a limited capacity to penetrate between the regenerating skeletal matrices and suggest that ISH performed directly on fin sections is a preferable method to study gene expression in fin regenerates. Developmental Dynamics 237:417–425, 2008.

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.