Regeneration of amputated zebrafish fin rays from de novo osteoblasts

Determining the cellular source of new skeletal elements is critical for understanding appendage regeneration in amphibians and fish. Recent lineage-tracing studies indicated that zebrafish fin ray bone regenerates through the dedifferentiation and proliferation of spared osteoblasts, with limited if any contribution from other cell types. Here, we examined the requirement for this mechanism by using genetic ablation techniques to destroy virtually all skeletal osteoblasts in adult zebrafish fins. Animals survived this injury and restored the osteoblast population within 2 weeks. Moreover, amputated fins depleted of osteoblasts regenerated new fin ray structures at rates indistinguishable from fins possessing a resident osteoblast population. Inducible genetic fate mapping confirmed that new bone cells do not arise from dedifferentiated osteoblasts under these conditions. Our findings demonstrate diversity in the cellular origins of appendage bone and reveal that de novo osteoblasts can fully support the regeneration of amputated zebrafish fins.     

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.     

Morphogen-based simulation model of ray growth and joint patterning during fin development and regeneration

The fact that some organisms are able to regenerate organs of the correct shape and size following amputation is particularly fascinating, but the mechanism by which this occurs remains poorly understood. The zebrafish (Danio rerio) caudal fin has emerged as a model system for the study of bone development and regeneration. The fin comprises 16 to 18 bony rays, each containing multiple joints along its proximodistal axis that give rise to segments. Experimental observations on fin ray growth, regeneration and joint formation have been described, but no unified theory has yet been put forward to explain how growth and joint patterns are controlled. We present a model for the control of fin ray growth during development and regeneration, integrated with a model for joint pattern formation, which is in agreement with published, as well as new, experimental data. We propose that fin ray growth and joint patterning are coordinated through the interaction of three morphogens. When the model is extended to incorporate multiple rays across the fin, it also accounts for how the caudal fin acquires its shape during development, and regains its correct size and shape following amputation.     

Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins

Mechanisms that regulate the size and shape of bony structures are largely unknown. The molecular identification of the fin length mutant short fin (sof), which causes defects in the length of bony fin ray segments, may provide insights regarding the regulation of bone growth. In this report, we demonstrate that the sof phenotype is caused by mutations in the connexin43 (cx43) gene. This conclusion is supported by genetic mapping, reduced expression of cx43 in the original sof allele (sofb123), identification of missense mutations in three ENU-induced alleles, and by demonstration of partially abrogated cx43 function in sofb123 embryos. Expression of cx43 was identified in cells flanking the germinal region of newly growing segments as well as in the osteoblasts at segment boundaries. This pattern of cx43 expression in cells lateral to new segment growth is consistent with a model where cx43-expressing cells represent a biological ruler that measures segment size. This report identifies the first gene identification for a fin length mutation (sof) as well as the first connexin mutations in zebrafish, and therefore reveals a critical role for local cell-cell communication in the regulation of bone size and growth.     

Saltatory control of isometric growth in the zebrafish caudal fin is disrupted in long fin and rapunzel mutants

Zebrafish fins grow by sequentially adding new segments of bone to the distal end of each fin ray. In wild type zebrafish, segment addition is regulated such that an isometric relationship is maintained between fin length and body length over the lifespan of the growing fish. Using a novel, surrogate marker for fin growth in conjunction with cell proliferation assays, we demonstrate here that segment addition is not continuous, but rather proceeds by saltation. Saltation is a fundamental growth mechanism shared by disparate vertebrates, including humans. We further demonstrate that segment addition proceeds in conjunction with cyclic bursts of cell proliferation in the distal fin ray mesenchyme. In contrast, cells in the distal fin epidermis proliferate at a constant rate throughout the fin ray growth cycle. Finally, we show that two separate fin overgrowth mutants, long fin and rapunzel, bypass the stasis phase of the fin ray growth cycle to develop asymmetrical and symmetrical fin overgrowth, respectively.     

Conserved mechanisms regulate outgrowth in zebrafish fins

Regulation of size is one of the fundamental problems in biology. One general strategy has been to identify molecules required for cell growth and cell proliferation within an organ. This has been particularly revealing, identifying cell-autonomous pathways involved in cell growth, survival and proliferation. In order to identify pathways regulating overall limb growth and morphology, experiments have evaluated gene expression, transplanted and removed tissues, and knocked out genes. This work has provided a vast amount of information identifying molecular mechanisms regulating limb axis formation, outgrowth, and pattern formation. Using the zebrafish fin, genetic, cellular and molecular strategies have also been employed to follow both normal patterns of fin growth and growth in fin mutants. This review will focus on cellular and molecular regulation of the outgrowth and patterning of the zebrafish caudal fin during regeneration, and will emphasize similarities to other systems. Future perspectives describe opportunities using the zebrafish fin to reveal mechanisms underlying the regulation of final size.     

Hedgehog-dependent proliferation drives modular growth during morphogenesis of a dermal bone

In the developing skeleton, dermal bone morphogenesis includes the balanced proliferation, recruitment and differentiation of osteoblast precursors, yet how bones acquire unique morphologies is unknown. We show that Hedgehog (Hh) signaling mediates bone shaping during early morphogenesis of the opercle (Op), a well characterized dermal bone of the zebrafish craniofacial skeleton. ihha is specifically expressed in a local population of active osteoblasts along the principal growing edge of the bone. Mutational studies show that Hh signaling by this osteoblast population is both necessary and sufficient for full recruitment of pre-osteoblasts into the signaling population. Loss of ihha function results in locally reduced proliferation of pre-osteoblasts and consequent reductions in recruitment into the osteoblast pool, reduced bone edge length and reduced outgrowth. Conversely, hyperactive Hh signaling in ptch1 mutants causes opposite defects in proliferation and growth. Time-lapse microscopy of early Op morphogenesis using transgenically labeled osteoblasts demonstrates that ihha-dependent bone development is not only region specific, but also begins exactly at the onset of a second phase of morphogenesis, when the early bone begins to reshape into a more complex form. These features strongly support a hypothesis that dermal bone development is modular, with different gene sets functioning at specific times and locations to pattern growth. The Hh-dependent module is not limited to this second phase of bone growth: during later larval development, the Op is fused along the dysmorphic edge to adjacent dermal bones. Hence, patterning within a module may include adjacent regions of functionally related bones and might require that signaling pathways function over an extended period of development.     

F-spondin/spon1b expression patterns in developing and adult zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.     

Mir24-2-5p suppresses the osteogenic differentiation with Gnai3 inhibition presenting a direct target via inactivating JNK-p38 MAPK signaling axis

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3''UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects.Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish.Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway.Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.     

An amputation resets positional information to a proximal identity in the regenerating zebrafish caudal fin

ABSTRACT: BACKGROUND: Zebrafish has emerged as a powerful model organism to study the process of regeneration. This teleost fish has the ability to regenerate various tissues and organs like the heart, spinal cord, retina and fins. In this study, we took advantage of the existence of an excellent morphological reference in the zebrafish caudal fin, the bony ray bifurcations, as a model to study positional information upon amputation. We investigated the existence of positional information for bifurcation formation by performing repeated amputations at different proximal-distal places along the fin. RESULTS: We show that, while amputations performed at a long distance from the bifurcation do not change its final proximal-distal position in the regenerated fin, consecutive amputations done at 1 segment proximal to the bifurcation (near the bifurcation) induce a positional reset and progressively shift its position distally. Furthermore, we investigated the potential role of Shh and Fgf signalling pathways in the determination of the bifurcation position and observed that they do not seem to be involved in this process. CONCLUSIONS: Our results reveal that, an amputation near the bifurcation inhibits the formation of the regenerated bifurcation in the pre-amputation position, inducing a distalization of this structure. This shows that the positional memory for bony ray bifurcations depends on the proximal-distal level of the amputation.     

The smooth muscle microRNA miR-145 regulates gut epithelial development via a paracrine mechanism

MicroRNAs are potent modulators of cellular differentiation. miR-145 is expressed in, and promotes the differentiation of vascular and visceral smooth muscle cells (SMCs). Interestingly, we have observed that miR-145 also promotes differentiation of the gut epithelium in the developing zebrafish, a cell type where it is not expressed. Here we identify that a paracrine pathway involving the morphogens Sonic hedgehog (Shh) in epithelium and bone morphogenic protein 4 (Bmp4) in SMCs is modulated by miR-145. We show that expression of miR-145 in visceral SMCs normally represses the expression of the morphogen bmp4, as loss of miR-145 leads to upregulation of bmp4 in SMCs. We show that bmp4 in turn controls expression of Shh in the visceral epithelium. Conversely, in miR-145 morphants where bmp4 expression is increased, expression of sonic hedgehog a (shha) is strongly increased in gut epithelium. We show that expression of bmp4 is modulated by the miR-145 direct target gata6 but not a second potential direct target, klf5a. Thus although miR-145 is a tissue-restricted microRNA, it plays an essential role in promoting the patterning of both gut layers during gut development via a paracrine mechanism.     

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development

Background  

Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.     

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.     

A comparison of pectoral fin ray morphology and its impact on fin ray flexural stiffness in labriform swimmers

The organization of tissues in appendages often affects their mechanical properties and function. In the fish family Labridae, swimming behavior is associated with pectoral fin flexural stiffness and morphology, where fins range on a continuum from stiff to relatively flexible fins. Across this diversity, pectoral fin flexural stiffness decreases exponentially along the length of any given fin ray, and ray stiffness decreases along the chord of the fin from the leading to trailing edge. In this study, we examine the morphological properties of fin rays, including the effective modulus in bending (E), second moment of area (I), segmentation, and branching patterns, and their impact on fin ray stiffness. We quantify intrinsic pectoral fin ray stiffness in similarly sized fins of two closely related species that employ fins of divergent mechanics, the flapping Gomphosus varius and the rowing Halichoeres bivittatus. While segmentation patterns and E were similar between species, measurements of I and the number of fin ray branch nodes were greater in G. varius than in H. bivittatus. A multiple regression model found that of these variables, I was always significantly correlated with fin ray flexural stiffness and that variation in I always explained the majority of the variation in flexural stiffness. Thus, while most of the morphological variables quantified in this study correlate with fin ray flexural stiffness, second moment of area is the greatest factor contributing to variation in flexural stiffness. Further, interspecific variation in fin ray branching pattern could be used as a means of tuning the effective stiffness of the fin webbing to differences in swimming behavior and hydrodynamics. The comparison of these results to other systems begins to unveil fundamental morphological features of biological beams and yields insight into the role of mechanical properties in fin deformation for aquatic locomotion.     

Fate restriction in the growing and regenerating zebrafish fin

We use transposon-based clonal analysis to identify the lineage classes that make the adult zebrafish caudal fin. We identify nine distinct lineage classes, including epidermis, melanocyte/xanthophore, iridophore, intraray glia, lateral line, osteoblast, dermal fibroblast, vascular endothelium, and resident blood. These lineage classes argue for distinct progenitors, or organ founding stem cells (FSCs), for each lineage, which retain fate restriction throughout growth of the fin. Thus, distinct FSCs exist for the four neuroectoderm lineages, and dermal fibroblasts are not progenitors for fin ray osteoblasts; however, artery and vein cells derive from a shared lineage in the fin. Transdifferentiation of cells or lineages in the regeneration blastema is often postulated. However, our studies of single progenitors or FSCs reveal no transfating or transdifferentiation between these lineages in the regenerating fin. This result shows that, the same as in growth, lineages retain fate restriction when passed through the regeneration blastema.     

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.