A median fin derived from the lateral plate mesoderm and the origin of paired fins.

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories1. Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication2. Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins.

A bi-modal function of Wnt signalling directs an FGF activity gradient to spatially regulate neuronal differentiation in the midbrain.

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. We have employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. We generated mathematical models to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. We tested Sprouty genes as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. We propose a model in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain.

Ret signalling integrates a craniofacial muscle module during development.

An appropriate organisation of muscles is crucial for their function, yet it is not known how functionally related muscles are coordinated with each other during development. In this study, we show that the development of a subset of functionally related head muscles in the zebrafish is regulated by Ret tyrosine kinase signalling. Three genes in the Ret pathway (gfra3, artemin2 and ret) are required specifically for the development of muscles attaching to the opercular bone (gill cover), but not other adjacent muscles. In animals lacking Ret or Gfra3 function, myogenic gene expression is reduced in forming opercular muscles, but not in non-opercular muscles derived from the same muscle anlagen. These animals have a normal skeleton with small or missing opercular muscles and tightly closed mouths. Myogenic defects correlate with a highly restricted expression of artn2, gfra3 and ret in mesenchymal cells in and around the forming opercular muscles. ret(+) cells become restricted to the forming opercular muscles and a loss of Ret signalling results in reductions of only these, but not adjacent, muscles, revealing a specific role of Ret in a subset of head muscles. We propose that Ret signalling regulates myogenesis in head muscles in a modular manner and that this is achieved by restricting Ret function to a subset of muscle precursors.

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.

Loss of function of parathyroid hormone receptor 1 induces Notch-dependent aortic defects during zebrafish vascular development.

OBJECTIVE: Coarctation of the aorta is rarely associated with known gene defects. Blomstrand chondrodysplasia, caused by mutations in the parathyroid hormone receptor 1 (PTHR1) is associated with coarctation of the aorta in some cases, although it is unclear whether PTHR1 deficiency causes coarctation of the aorta directly. The zebrafish allows the study of vascular development using approaches not possible in other models. We therefore examined the effect of loss of function of PTHR1 or its ligand parathyroid hormone-related peptide (PTHrP) on aortic formation in zebrafish. APPROACH AND RESULTS: Morpholino antisense oligonucleotide knockdown of either PTHR1 or PTHrP led to a localized occlusion of the mid-aorta in developing zebrafish. Confocal imaging of transgenic embryos showed that these defects were caused by loss of endothelium, rather than failure to lumenize. Using a Notch reporter transgenic ([CSL:Venus]qmc61), we found both PTHR1 and PTHrP knockdown-induced defective Notch signaling in the hypochord at the site of the aortic defect before onset of circulation, and the aortic occlusion was rescued by inducible Notch upregulation. CONCLUSIONS: Loss of function of either PTHR1 or PTHrP leads to a localized aortic defect that is Notch dependent. These findings may underlie the aortic defect seen in Blomstrand chondrodysplasia, and reveal a link between parathyroid hormone and Notch signaling during aortic development.

Regulation of neural crest cell fate by the retinoic acid and Pparg signalling pathways.

Although the regulation of osteoblast and adipocyte differentiation from mesenchymal stem cells has been studied for some time, very little is known about what regulates their appearance in discrete regions of the embryo. Here we show that, as in other vertebrates, zebrafish osteoblasts and adipocytes originate in part from cephalic neural crest (CNC) precursors. We investigated the roles that the retinoic acid (RA) and Peroxisome proliferator-activated receptor gamma (Pparg) pathways play in vivo and found that both pathways act on CNC to direct adipocyte differentiation at the expense of osteoblast formation. In addition, we identify two distinct roles for RA in the osteoblast lineage: an early role in blocking the recruitment of osteoblasts and a later role in mature osteoblasts to promote bone matrix synthesis. These findings might help to increase our understanding of skeletal and obesity-related diseases and aid in the development of stem cell-based regenerative therapies.

Tetraspanin Cd9b plays a role in fertility in zebrafish.

In mice, CD9 expression on the egg is required for efficient sperm-egg fusion and no effects on ovulation or male fertility are observed in CD9 null animals. Here we show that cd9b knockout zebrafish also appear to have fertility defects. In contrast to mice, fewer eggs were laid by cd9b knockout zebrafish pairs and, of the eggs laid, a lower percentage were fertilised. These effects could not be linked to primordial germ cell numbers or migration as these were not altered in the cd9b mutants. The decrease in egg numbers could be rescued by exchanging either cd9b knockout partner, male or female, for a wildtype partner. However, the fertilisation defect was only rescued by crossing a cd9b knockout female with a wildtype male. To exclude effects of mating behaviour we analysed clutch size and fertilisation using in vitro fertilisation techniques. Number of eggs and fertilisation rates were significantly reduced in the cd9b mutants suggesting the fertility defects are not solely due to courtship behaviours. Our results indicate that CD9 plays a more complex role in fish fertility than in mammals, with effects in both males and females.

Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/pinscher.

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak(-/-) cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic(-/-) chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.

Shop talk: Sugars, bones, and a disease called multiple hereditary exostoses.

On October 29, 2009, researchers and physicians gathered at the Sheraton Four Points Hotel in Boston for 4 days to discuss a disease called multiple hereditary exostoses (MHE). MHE is an autosomal dominant disease that is associated with mutations in two enzymes that are required for heparan sulfate (HS) synthesis. Children with the disease form numerous benign bone tumors (osteochondromas) and have >2% chance of developing chondrosarcoma. The aim of the meeting was to generate new ideas for the diagnoses, treatment, and cure of this disease. Discussions ranged from orthopedic surgical treatment and patients' personal experiences to fundamental questions in skeletal biology and the precise molecular role that HS plays in developmental signaling pathways.

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration.

Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

Clinical pathologies of bone fracture modelled in zebrafish.

Reduced bone quality or mineral density predict susceptibility to fracture and also attenuate subsequent repair. Bone regrowth is also compromised by bacterial infection, which exacerbates fracture site inflammation. Because of the cellular complexity of fracture repair, as well as genetic and environmental influences, there is a need for models that permit visualisation of the fracture repair process under clinically relevant conditions. To characterise the process of fracture repair in zebrafish, we employed a crush fracture of fin rays, coupled with histological and transgenic labelling of cellular responses; the results demonstrate a strong similarity to the phased response in humans. We applied our analysis to a zebrafish model of osteogenesis imperfecta (OI), which shows reduced bone quality, spontaneous fractures and propensity for non-unions. We found deficiencies in the formation of a bone callus during fracture repair in our OI model and showed that clinically employed antiresorptive bisphosphonates can reduce spontaneous fractures in OI fish and also measurably reduce fracture callus remodelling in wild-type fish. The csf1ra mutant, which has reduced osteoclast numbers, also showed reduced callus remodelling. Exposure to excessive bisphosphonate, however, disrupted callus repair. Intriguingly, neutrophils initially colonised the fracture site, but were later completely excluded. However, when fractures were infected with Staphylococcus aureus, neutrophils were retained and compromised repair. This work elevates the zebrafish bone fracture model and indicates its utility in assessing conditions of relevance to an orthopaedic setting with medium throughput.This article has an associated First Person interview with the first author of the paper.

Axon sorting in the optic tract requires HSPG synthesis by ext2 (dackel) and extl3 (boxer).

Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.

Mandibular arch muscle identity is regulated by a conserved molecular process during vertebrate development.

Vertebrate head muscles exhibit a highly conserved pattern of innervation and skeletal connectivity and yet it is unclear whether the molecular basis of their development is likewise conserved. Using the highly conserved expression of Engrailed 2 (En2) as a marker of identity in the dorsal mandibular muscles of zebrafish, we have investigated the molecular signals and tissues required for patterning these muscles. We show that muscle En2 expression is not dependent on signals from the adjacent neural tube, pharyngeal endoderm or axial mesoderm and that early identity of head muscles does not require bone morphogenetic pathway, Notch or Hedgehog (Hh) signalling. However, constrictor dorsalis En2 expression is completely lost after a loss of fibroblast growth factor (Fgf) signalling and we show that is true throughout head muscle development. These results suggest that head muscle identity is dependent on Fgf signalling. Data from experiments performed in chick suggest a similar regulation of En2 genes by Fgf signalling revealing a conserved mechanism for specifying head muscle identity. We present evidence that another key gene important in the development of mouse head muscles, Tbx1, is also critical for specification of mandibular arch muscle identity and that this is independent of Fgf signalling. These data imply that dorsal mandibular arch muscle identity in fish, chick and mouse is specified by a highly conserved molecular process despite differing functions of these muscles in different lineages.

Linking wound response and inflammation to regeneration in the zebrafish larval fin.

The study of regenerative biology aims to elucidate the innate ability of organisms to replace tissues or organs after they have been removed or damaged. The zebrafish is a powerful model for the analysis of intracellular signalling and cell behaviour and as such has made major contributions to our understanding of regenerative biology. The larval fin fold is an emerging model to understand how different signalling pathways interact to coordinate regeneration. Tissue damage causes the immediate release of signals that initiate wound closure and inflammation. Following this, regenerative cells proliferate and migrate to the damaged area. Each of these processes has been analysed using the larval fin fold model to provide a framework for how fin regeneration takes place. This review gives an overview of the current state of this field with particular emphasis on the different signalling networks that are required during fin fold regeneration.

Damage-induced reactive oxygen species enable zebrafish tail regeneration by repositioning of Hedgehog expressing cells.

Many aquatic vertebrates have a remarkable ability to regenerate limbs and tails after amputation. Previous studies indicate that reactive oxygen species (ROS) signalling initiates regeneration, but the mechanism by which this takes place is poorly understood. Developmental signalling pathways have been shown to have proregenerative roles in many systems. However, whether these are playing roles that are specific to regeneration, or are simply recapitulating their developmental functions is unclear. Here, we analyse zebrafish larval tail regeneration and find evidence that ROS released upon wounding cause repositioning of notochord cells to the damage site. These cells secrete Hedgehog ligands that are required for regeneration. Hedgehog signalling is not required for normal tail development suggesting that it has a regeneration-specific role. Our results provide a model for how ROS initiate tail regeneration, and indicate that developmental signalling pathways can play regenerative functions that are not directly related to their developmental roles.

Expression of osterix Is Regulated by FGF and Wnt/ß-Catenin Signalling during Osteoblast Differentiation.

Osteoblast differentiation from mesenchymal cells is regulated by multiple signalling pathways. Here we have analysed the roles of Fibroblast Growth Factor (FGF) and canonical Wingless-type MMTV integration site (Wnt/ß-Catenin) signalling pathways on zebrafish osteogenesis. We have used transgenic and chemical interference approaches to manipulate these pathways and have found that both pathways are required for osteoblast differentiation in vivo. Our analysis of bone markers suggests that these pathways act at the same stage of differentiation to initiate expression of the osteoblast master regulatory gene osterix (osx). We use two independent approaches that suggest that osx is a direct target of these pathways. Firstly, we manipulate signalling and show that osx gene expression responds with similar kinetics to that of known transcriptional targets of the FGF and Wnt pathways. Secondly, we have performed ChIP with transcription factors for both pathways and our data suggest that a genomic region in the first intron of osx mediates transcriptional activation. Based upon these data, we propose that FGF and Wnt/ß-Catenin pathways act in part by directing transcription of osx to promote osteoblast differentiation at sites of bone formation.

HSPG-deficient zebrafish uncovers dental aspect of multiple osteochondromas.

Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2⁻/⁻ fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2⁻/⁻ fish. Histological analysis reveals that ext2⁻/⁻ fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2⁻/⁻ fish have a single tooth at the end of the 5(th) pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2⁻/⁻ teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2⁺/⁻ adults. The tooth morphology in ext2⁻/⁻ was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.

Hedgehog signalling is required for perichondral osteoblast differentiation in zebrafish.

In tetrapod long bones, Hedgehog signalling is required for osteoblast differentiation in the perichondrium. In this work we analyse skeletogenesis in zebrafish larvae treated with the Hedgehog signalling inhibitor cyclopamine. We show that cyclopamine treatment leads to the loss of perichondral ossification of two bones in the head. We find that the Hedgehog co-receptors patched1 and patched2 are expressed in regions of the perichondrium that will form bone before the onset of ossification. We also show that cyclopamine treatment strongly reduces the expression of osteoblast markers in the perichondrium and that perichondral ossification is enhanced in patched1 mutant fish. This data suggests a conserved role for Hedgehog signalling in promoting perichondral osteoblast differentiation during vertebrate skeletal development. However, unlike what is seen during long bone development, we did not observe ectopic chondrocytes in the perichondrium when Hedgehog signalling is blocked. This result may point to subtle differences between the development of the skeleton in the skull and limb.

Regulation of zebrafish hatching by tetraspanin cd63.

Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

Discovery of 6-substituted indole-3-glyoxylamides as lead antiprion agents with enhanced cell line activity, improved microsomal stability and low toxicity.

A series of highly potent indole-3-glyoxylamide based antiprion agents was previously characterized, focusing on optimization of structure-activity relationship (SAR) at positions 1-3 of the indole system. New libraries interrogating the SAR at indole C-4 to C-7 now demonstrate that introducing electron-withdrawing substituents at C-6 may improve biological activity by up to an order of magnitude, and additionally confer higher metabolic stability. For the present screening libraries, both the degree of potency and trends in SAR were consistent across two cell line models of prion disease, and the large majority of compounds showed no evidence of toxic effects in zebrafish. The foregoing observations thus make the indole-3-glyoxylamides an attractive lead series for continuing development as potential therapeutic agents against prion disease.