Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration

Macrophages and neutrophils are the pivotal immune phagocytes that enter the wound after tissue injury to remove the cell debris and invaded microorganisms, which presumably facilitate the regrowth of injured tissues. Taking advantage of the regeneration abilities of zebrafish and the newly generated leukocyte-specific zebrafish lines with labeling of both leukocyte lineages, we assessed the behaviors and functions of neutrophils and macrophages during tail fin regeneration. Live imaging showed that within 6 hours post amputation, the inflammatory stage, neutrophils were the primary cells scavenging apoptotic bodies and small cell debris, although they had limited phagocytic capacity and quickly underwent apoptosis. From 6 hours post amputation on, the resolution and regeneration stage, macrophages became the dominant scavengers, efficiently resolving inflammation and facilitating tissue remodeling and regrowth. Ablation of macrophages but not neutrophils severely impaired the inflammatory resolution and tissue regeneration, resulting in the formation of large vacuoles in the regenerated fins. In contrast, removal of neutrophils slightly accelerates the regrowth of injured fin. Our study documents the differing behaviors and functions of macrophages and neutrophils during tissue regeneration.     

TALEN-mediated mutagenesis in zebrafish reveals a role for r-spondin 2 in fin ray and vertebral development

R-spondin (Rspo) encodes a multi-domain protein that modulates the Wnt-signaling pathway. Two distinct rspo2 zebrafish mutants were generated by TALEN-mediated mutagenesis: a null mutant, rspo2^null, lacking all functional domains, and a hypomorphic mutant, rspo2^tsp, lacking the two N-terminal domains. Mutants were analyzed mainly for abnormalities in the skeletal system. Fin ray skeletons were formed normally in the rspo2^tsp mutants, but were absent from the rspo2^null mutants. Hypoplasia of the neural/hemal arches and ribs was observed in both mutants. Thus, the two rspo2 mutants help to identify the functions of Rspo2 in skeletogenesis, as well as functional differences among multiple Rspo2 domains.     

Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.     

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.     

Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration

In zebrafish, mutations in the gap junction gene connexin43 lead to short bony fin ray segments that give rise to the short fin phenotype. The sof^b123 mutant exhibits fins that are half the length of wild-type fins and have reduced levels of cx43 mRNA. We find that sof^b123 regenerating fins exhibit reduced levels of cell proliferation. Interestingly, the number of dividing cells per unit length of fin growth is similar between wild-type and mutant fins, suggesting that the number of cells that enter the cell cycle is specifically affected in sof^b123. Expression of cx43 is identified in mitotic cells, which further suggests that Cx43 may contribute to establishing or maintaining the population of dividing cells. Indeed, missense alleles exhibiting high or low levels of gap junctional communication reveal a correlation between defects in direct cell-cell communication, cell proliferation, and segment length. Finally, targeted gene knockdown of cx43 in adult regenerating fins recapitulates the sof^b123 phenotype, revealing that the loss of Cx43 is sufficient to reduce both cell proliferation and segment length. We hypothesize that the level of gap junctional intercellular communication among dividing cells regulates the level of cell proliferation and ultimately regulates bone growth.     

The chemokine SDF-1 regulates blastema formation during zebrafish fin regeneration

The work presented in this study focuses on blastema formation in epimorphic regeneration. We describe the expression pattern of Sdf1a and Sdf1b (the chemokines stromal-cell-derived factor-1a and 1b) and their two receptors Cxcr4a and Cxcr4b during zebrafish fin regeneration. We demonstrate that Sdf1a/Cxcr4a plays a critical role in fin regeneration and more precisely in epidermal cell proliferation, an important process for blastema formation. In mammals, a single cxcr4 gene is involved both in chemotaxis and cell proliferation and survival; we discuss in this study a possible functional division of the two cxcr4 zebrafish genes.     

Expression patterns of dnmt3aa,dnmt3ab,and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.     

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.     

Position dependence of hemiray morphogenesis during tail fin regeneration in Danio rerio

The fins of actinopterygian can regenerate following amputation. Classical papers have shown that the ray, a structural unit of these fins, might regenerate independent of this appendage. Each fin ray is formed by two apposed contralateral hemirays. A hemiray may autonomously regenerate and segmentate in a position-independent manner. This is observed when heterotopically grafted into an interray space, after amputation following extirpation of the contralateral hemiray or when simply ablated. During this process, a proliferating hemiblastema is formed, as shown by bromodeoxyuridine incorporation, from which the complete structure will regenerate. This hemiblastema shows a patterning of gene expression domain similar to half ray blastema. Interactions between contralateral hemiblastema have been studied by recombinant rays composed of hemirays from different origins on the proximo-distal or dorso-ventral axis of the caudal fin. Dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocianine perchlorate labeling of grafted tissues was used as tissular marker. Our results suggest both that there are contralateral interactions between hemiblastema of each ray, and that hemiblastema may vary its morphogenesis, always differentiating as their host region. These non-autonomous, position-dependent interactions control coordinated bifurcations, segment joints and ray length independently. A morphological study of the developing and regenerating fin of another long fin mutant zebrafish suggests that contralateral hemiblastema interactions are perturbed in this mutant.     

Ndrg4 is required for normal myocyte proliferation during early cardiac development in zebrafish

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Ndrg4 knockdown in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

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.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

Fin-mutant female zebrafish (Danio rerio) exhibit differences in association preferences for male fin length

Females often choose to associate with males that have exaggerated traits. In fishes, this may reflect an overall preference for larger size in a potential mate. Female zebrafish (Danio rerio) prefer males with larger bodies but not longer fins. The availability of mutant and transgenic strains of zebrafish make this a unique model system in which to study the role of phenotypic variation in social and sexual behavior. We used mutant strains of zebrafish with truncated (short fin) and exaggerated (long fin) fins to further examine female preferences for fin length in dichotomous association tests. Wild type females showed no preferences between wild type males and short fin mutant males or between wild type males and long fin mutant males. short fin females also showed no preference for short fin males or wild type males while long fin females preferred to associate with long fin males over wild type males. These results suggest that the single gene long fin mutation that results in altered fin morphological may also be involved in a related female association preference.     

meis1 regulates the development of endothelial cells in zebrafish

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.