Search

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. Agency for Science, Technology and Research (A*STAR) Ministry of Education (MOE) Published version This work was funded by the Industry Aligned Fund (IAF) Agency for Science, Technology and Research (grant to T.J.C. and K.-W.T.); Ministry of Education (MoE) Tier 3 (grant 2016-T3-1-005 to T.J.C., C.W. and H.M.); Ministry of Education (MoE) Tier 1 (grant 2016-T1-001-055 to T.J.C. and C.Z.); Ministry of Education (MoE) Tier 2 (grant MOE-T2EP30221-0008 to C.W.); the Company of Biologists (travelling fellowship to M.J.T.); the National Science Foundation (grants IOS-1853949 to M.C.D. and 2203311 to C.M.); the Swiss National Science Foundation Sinergia (grant CRSII5_180345 to C.M.); the Swiss Bridge Foundation (C.M.); Additional Ventures Single Ventricle Research Fund (SVRF) (grant 1048003 to C.M.); the University of Colorado School of Medicine Anschutz Medical Campus and the Children’s Hospital Colorado Foundation (C.M.); the National Institutes of Health (NIH), National Institute of General Medical Sciences (grants 1T32GM141742-01 to H.R.M. and 3T32GM121742-02S1 to H.R.M.); Australian Research Council (discovery grant DP200103219 to F.J.T. and P.D.C); National Health and Medical Research Council (senior principal research fellow APP1136567 to P.D.C.); and the NIH (grant R35NS111564 to J.S. and M.E.B.).

Jensen et al . ( 1 ) question evidence presented of a chambered heart within placoderms, citing its small size and apparently ventral atrium. However, they fail to note the belly-up orientation of the placoderm within one nodule, and the variability of heart morphology within extant taxa. Thus, we remain confident in our interpretation of the mineralized organ as the heart.

The cells of the innate immune system are the sentinels of tissue homeostasis, acting as ‘first responders’ to cellular damage and infection. Although the complex interplay of different immune cells during the initial inflammatory phases of infection and repair has been documented over many decades, recent studies have begun to define a more direct role for specific immune cells in the modulation of tissue repair. One particular cell of the innate immune system, the macrophage, has emerged as a central integrator of the complex molecular processes that drive tissue repair and, in some cases, the development of specific cell types. Although macrophages display directed orchestration of stem cell activities, bidirectional cellular crosstalk mechanisms allow stem cells to regulate macrophage behaviour within their niche, thus increasing the complexity of niche regulation and control. In this Review, we characterize the roles of macrophage subtypes in individual regenerative and developmental processes and illustrate the surprisingly direct role for immune cells in coordinating stem cell formation and activation.

Purpose: Mutations in TCP-1 ring complex (TRiC) have been associated with Leber Congenital Amaurosis (LCA). TRiC is involved in protein folding and has 8 essential subunits including CCT5. Herein, we studied the retina of TRiC mutant zebrafish to evaluate the possible role of impaired actin and tubulin folding in LCA. Methods: The cct5tf 212b retina was histologically studied using Toluidine Blue staining as well as TUNEL, BrdU-labeling, and Phalloidin assays. Retinal organisation was assessed by quantification of the cellularity utilising DAPI. Results: Laminar organization of cct5tf 212b retinas was intact. Enhanced apoptosis throughout the cct5tf 212b retina was not compensated by higher proliferation rates, leaving the cct5tf 212b retina smaller in size. Quantification of retinal layer cellularity demonstrated that specifically the numbers of the amacrine and the retinal ganglion cells were depleted, suggesting that the cct5tf 212b retina was not uniformly affected by the reduced actin folding. Conclusion: Whereas the current literature suggests that LCA is predominantly affecting retinal photoreceptor cells and the retinal pigment epithelium, cct5tf 212b analyses demonstrated the important role of folding of actin by TRiC, suggesting that cct5

Mucopolysaccharidoses are lysosomal storage diseases that collectively represent a major cause of lethal, treatment-refractory childhood dementias 1–7 Clinically-useful interventions are hampered due to an incomplete understanding of their neuropathological origins. Using the zebrafish sgsh model of mucopolysaccharidosis IIIA 8 (MPS IIIA, Sanfilippo syndrome A), we conducted several ‘omics-based analyses, and developed and benchmarked a novel bioinformatic feature classification and ranking model for high-throughput datasets – ExIR – to prioritise important features in the progression of neurological manifestations of the disease. We find that the massive endolysosomal burden resulting from increased lysosomal storage of heparan sulfate and other secondarily accumulating substrates, such as sphingolipids, induces abnormal microtubule organisation and vesicle trafficking in neurons. This results in a gradual impairment of synaptic vesicle localisation at the presynaptic terminal and consequently impaired neuronal activity. Importantly, the endolysosomal phenotype in MPS IIIA zebrafish well-precedes the onset of neural pathology, though the larval MPS IIIA brain was found to be more susceptible to perturbation than wild type siblings. Collectively, these analyses demonstrate the presence of a progressive ‘functional neurodegenerative’ phenotype underpinning neurological disease in MPS IIIA. Our findings provide direct mechanistic evidence linking the well-described lysosomal storage basis for MPS IIIA to its disproportionately severe neural clinical involvement, enabling development and refinement of future therapeutic interventions for this currently untreatable disorder.HighlightsMPS IIIA represents one of the most common causes of broadly fatal childhood dementia, but the mechanisms underlying disease progression are poorly understood.The first systems-level analyses of disease state and progression in the CNS of an MPS IIIA animal model were performed.Experimental data-based Integrative Ranking (ExIR) was developed to provide unbiased prioritisation and classification of biological data as drivers, biomarkers and mediators of biological processes from high-throughput data at a systems level.Application of ExIR to a transcriptomic and proteomic analyses of a zebrafish model of MPS IIIA implies progressive deficiencies in synaptic activity as a key driver of disease progression correlating with progressive neuronal endolysosomal burden and secondary storage diseases.A novel unifying explanation of pathobiology and progression of MPS IIIA facilitates identification of clinically targetable features and may be generalised to other neuronopathic storage disorders.

SARS-CoV-2 primarily infects the respiratory tract, but pulmonary and cardiac complications occur in severe COVID-19. To elucidate molecular mechanisms in the lung and heart, we conducted paired experiments in human stem cell-derived lung alveolar type II (AT2) epithelial cell and cardiac cultures infected with SARS-CoV-2. With CRISPR- Cas9 mediated knock-out of ACE2, we demonstrated that angiotensin converting enzyme 2 (ACE2) was essential for SARS-CoV-2 infection of both cell types but further processing in lung cells required TMPRSS2 while cardiac cells required the endosomal pathway. Host responses were significantly different; transcriptome profiling and phosphoproteomics responses depended strongly on the cell type. We identified several antiviral compounds with distinct antiviral and toxicity profiles in lung AT2 and cardiac cells, highlighting the importance of using several relevant cell types for evaluation of antiviral drugs. Our data provide new insights into rational drug combinations for effective treatment of a virus that affects multiple organ systems.One-sentence summaryRational treatment strategies for SARS-CoV-2 derived from human PSC models

The origin and early diversification of jawed vertebrates involved major changes to skeletal and soft anatomy. Skeletal transformations can be examined directly by studying fossil stem gnathostomes; however, preservation of soft anatomy is rare. We describe the only known example of a three-dimensionally mineralized heart, thick-walled stomach, and bilobed liver from arthrodire placoderms, stem gnathostomes from the Late Devonian Gogo Formation in Western Australia. The application of synchrotron and neutron microtomography to this material shows evidence of a flat S-shaped heart, which is well separated from the liver and other abdominal organs, and the absence of lungs. Arthrodires thus show the earliest phylogenetic evidence for repositioning of the gnathostome heart associated with the evolution of the complex neck region in jawed vertebrates.

The muscular dystrophies encompass a broad range of pathologies with varied clinical outcomes. In the case of patients carrying defects in fukutin-related protein (FKRP), these diverse pathologies arise from mutations within the same gene. This is surprising as FKRP is a glycosyltransferase, whose only identified function is to transfer ribitol-5-phosphate to α-dystroglycan (α-DG). Although this modification is critical for extracellular matrix attachment, α-DG’s glycosylation status relates poorly to disease severity, suggesting the existence of unidentified FKRP targets. Here we reveal that FKRP directs sialylation of fibronectin, a process essential for collagen recruitment to the muscle basement membrane. Thus, our results reveal that FKRP simultaneously regulates the two major muscle-ECM linkages essential for fibre survival, and establishes a new disease axis for the muscular dystrophies., FKRP mutations cause muscular dystrophies with varied clinical presentations. The target of FKRP is α-dystroglycan, but here the authors show that FKRP also directs sialylation of fibronectin, a process that is essential for recruitment o collagen to the muscle basement membrane.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Duchenne muscular dystrophy is a severe muscle wasting disease caused by mutations in the dystrophin gene (dmd). Ataluren has been approved by the European Medicines Agency for treatment of Duchenne muscular dystrophy. Ataluren has been reported to promote ribosomal read‐through of premature stop codons, leading to restoration of full‐length dystrophin protein. However, the mechanism of Ataluren action has not been fully described. To evaluate the efficacy of Ataluren on all three premature stop codons featuring different termination strengths (UAA > UAG > UGA), novel dystrophin‐deficient zebrafish were generated. Pathological assessment of the muscle by birefringence quantification, a tool to directly measure muscle integrity, did not reveal a significant effect of Ataluren on any of the analysed dystrophin‐deficient mutants at 3 days after fertilization. Functional analysis of the musculature at 6 days after fertilization by direct measurement of the generated force revealed a significant improvement by Ataluren only for the UAA‐carrying mutant dmdta222a. Interestingly however, all other analysed dystrophin‐deficient mutants were not affected by Ataluren, including the dmdpc3 and dmdpc2 mutants that harbour weaker premature stop codons UAG and UGA, respectively. These in vivo results contradict reported in vitro data on Ataluren efficacy, suggesting that Ataluren might not promote read‐through of premature stop codons. In addition, Ataluren had no effect on dystrophin transcript levels, but mild adverse effects on wild‐type larvae were identified. Further assessment of N‐terminally truncated dystrophin opened the possibility of Ataluren promoting alternative translation codons within dystrophin, thereby potentially shifting the patient cohort applicable for Ataluren.

Myofibrils of the skeletal muscle are comprised of sarcomeres that generate force by contraction when myosin-rich thick filaments slide past actin-based thin filaments. Surprisingly little is known about the molecular processes that guide sarcomere assembly in vivo, despite deficits within this process being a major cause of human disease. To overcome this knowledge gap, we undertook a forward genetic screen coupled with reverse genetics to identify genes required for vertebrate sarcomere assembly. In this screen, we identified a zebrafish mutant with a nonsense mutation in mob4. In Drosophila, mob4 has been reported to play a role in spindle focusing as well as neurite branching and in planarians mob4 was implemented in body size regulation. In contrast, zebrafish mob4geh mutants are characterised by an impaired actin biogenesis resulting in sarcomere defects. Whereas loss of mob4 leads to a reduction in the amount of myofibril, transgenic expression of mob4 triggers an increase. Further genetic analysis revealed the interaction of Mob4 with the actin-folding chaperonin TRiC, suggesting that Mob4 impacts on TRiC to control actin biogenesis and thus myofibril growth. Additionally, mob4geh features a defective microtubule network, which is in-line with tubulin being the second main folding substrate of TRiC. We also detected similar characteristics for strn3-deficient mutants, which confirmed Mob4 as a core component of STRIPAK and surprisingly implicates a role of the STRIPAK complex in sarcomerogenesis.

In addition to conventional immunoglobulin, camelids and cartilaginous fish express a special class of antibody that consists only of heavy (H) chain (HCAbs). In the holocephalan elephantfish, there are two HCAb classes, one of which has evolved surprising features. The H-chain genes in cartilaginous fish are organized as 20-200 minigenes, or clusters, each consisting of VH, 1-3 DH, JH gene segments with one set of constant region exons. We report that HHC2 (holocephalan H-chain antibody 2) evolved from IgM H-chain clusters, but its DH gene segments have diverged considerably. The three DH in HHC2 clusters are A-rich, so that one to three potential reading frames for each DH encode lysine and arginine. All three are incorporated into the rearranged VDJ, ensuring that the ligand-binding site carries multiple basic residues, as cDNA sequences demonstrate. The electropositive character in HHC2 CDR3 is accompanied by a paucity of aromatic amino acids, the latter feature at variance to the established, interactive role of tyrosine not only in ligand-binding but generally at interfaces of protein complexes. The selection for these divergent HHC2 features challenges currently accepted ideas on what determines antibody reactivity and molecular recognition.

The fin-to-limb transition has long fascinated evolutionary biologists, but a unifying theory as to its developmental origins has remained elusive. New work by Hawkins and colleagues demonstrates the surprising potential of teleost fins to exhibit a Hox-regulated limb-like skeletal pattern, shedding new light on the evolution of proximo-distal patterning processes.

The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

The Zebrafish Embryo Genotyper (ZEG) device provides a promising tool for genotyping live embryos. Although the gross morphology and survival of embryos after the use of ZEG are unaffected, the cellular and molecular effects of the ZEG protocol remain unknown. To address this, we have examined the integrity of specific tissues, and evaluated the expression of stress-responsive genes to determine the impact of the ZEG protocol. Our analyses reveal that although ZEG results in a low-level acute stress response, no long-lasting effects are evident, supporting its utilization for a variety of downstream assays.

Animal models of human disease provide an in vivo system that can reveal molecular mechanisms by which mutations cause pathology, and, moreover, have the potential to provide a valuable tool for drug development. Here, we have developed a zebrafish model of Parkinson's disease (PD) together with a novel method to screen for movement disorders in adult fish, pioneering a more efficient drug-testing route. Mutation of the PARK7 gene (which encodes DJ-1) is known to cause monogenic autosomal recessive PD in humans, and, using CRISPR/Cas9 gene editing, we generated a Dj-1 loss-of-function zebrafish with molecular hallmarks of PD. To establish whether there is a human-relevant parkinsonian phenotype in our model, we adapted proven tools used to diagnose PD in clinics and developed a novel and unbiased computational method to classify movement disorders in adult zebrafish. Using high-resolution video capture and machine learning, we extracted novel features of movement from continuous data streams and used an evolutionary algorithm to classify parkinsonian fish. This method will be widely applicable for assessing zebrafish models of human motor diseases and provide a valuable asset for the therapeutics pipeline. In addition, interrogation of RNA-seq data indicate metabolic reprogramming of brains in the absence of Dj-1, adding to growing evidence that disruption of bioenergetics is a key feature of neurodegeneration. This article has an associated First Person interview with the first author of the paper., Summary: Using computational analyses to harness artificial intelligence, we have tested a genetic model of Parkinson's disease and reveal a distinct movement phenotype in adult zebrafish lacking Dj-1.

Skeletal muscle is paradigmatic of a regenerative tissue that repairs itself via the activation of a resident stem cell1. Termed the satellite cell, these normally quiescent cells are induced to proliferate by ill-defined wound-derived signals2. Identifying the source and nature of these pro-regenerative cues has been hampered by an inability to visualise the complex cellular interactions that occur within the wound environment. We therefore developed a zebrafish muscle injury model to systematically capture satellite cell interactions within the injury site, in real time, throughout the repair process. This analysis identified that a specific subset of macrophages ‘dwells’ within the injury, establishing a transient but obligate stem cell niche required for stem cell proliferation. Single cell profiling identified specific signals secreted from dwelling macrophages that include the cytokine, Nicotinamide phosphoribosyltransferase (NAMPT/Visfatin/PBEF). Here we show that NAMPT secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (CCR5) expressed on muscle stem cells. This analysis reveals that along with their well-described ability to modulate the pro-inflammatory and anti-inflammatory phases of wound repair, specific macrophage populations also provide a transient stem cell-activating niche, directly supplying pro-proliferative cues that govern the timing and rate of muscle stem cell-mediated repair processes.

Biological systems are composed of highly complex networks and decoding the functional significance of individual network components is critical for understanding healthy and diseased states. Several algorithms have been designed to identify the most influential regulatory points, or hub nodes, within a network. However, none of the current methods correct for inherent positional biases which limits their applicability. In addition, none of the currently available hub detection algorithms effectively combine network centrality measures together. To overcome this computational deficit, we undertook a statistical assessment of 200 real-world and simulated networks to decipher associations between centrality measures, and developed a novel algorithm termed “integrated hubness score” (IHS), which integrates the most important and commonly used network centrality measures, namely degree centrality, betweenness centrality and neighbourhood connectivity, in an unbiased way. When compared against the four most commonly used hub identification methods on four independent validated biological networks, the IHS algorithm outperformed all other assessed methods. Using this novel and universal method, researchers of any discipline can now identify the most influential network nodes.

The recently uncovered role of Fukutin-related protein (FKRP) in fibronectin glycosylation has challenged our understanding of the basis of disease pathogenesis in the muscular dystrophies. FKRP is a Golgi-resident glycosyltransferase implicated in a broad spectrum of muscular dystrophy (MD) pathologies that are not fully attributable to the well-described α-Dystroglycan hypoglycosylation. By revealing a new role for FKRP in the glycosylation of fibronectin, a modification critical for the development of the muscle basement membrane (MBM) and its associated muscle linkages, new possibilities for understanding clinical phenotype arise. This modification involves an interaction between FKRP and myosin-10, a protein involved in the Golgi organization and function. These observations suggest a FKRP nexus exists that controls two critical aspects to muscle fibre integrity, both fibre stability at the MBM and its elastic properties. This review explores the new potential disease axis in the context of our current knowledge of muscular dystrophies.

Myofibrils within skeletal muscle are composed of sarcomeres that generate force by contraction when their myosin-rich thick filaments slide past actin-based thin filaments. Although mutations in components of the sarcomere are a major cause of human disease, the highly complex process of sarcomere assembly is not fully understood. Current models of thin filament assembly highlight a central role for filament capping proteins, which can be divided into three protein families, each ascribed with separate roles in thin filament assembly. CapZ proteins have been shown to bind the Z-disc protein α-actinin to form an anchoring complex for thin filaments and actin polymerisation. Subsequent thin filaments extension dynamics are thought to be facilitated by Leiomodins (Lmods) and thin filament assembly is concluded by Tropomodulins (Tmods) that specifically cap the pointed end of thin filaments. To study thin filament assembly in vivo, single and compound loss-of-function zebrafish mutants within distinct classes of capping proteins were analysed. The generated lmod3- and capza1b-deficient zebrafish exhibited aspects of the pathology caused by variations in their human orthologs. Although loss of the analysed main capping proteins of the skeletal muscle, capza1b, capza1a, lmod3 and tmod4, resulted in sarcomere defects, residual organised sarcomeres were formed within the assessed mutants, indicating that these proteins are not essential for the initial myofibril assembly. Furthermore, detected similarity and location of myofibril defects, apparent at the peripheral ends of myofibres of both Lmod3- and CapZα-deficient mutants, suggest a function in longitudinal myofibril growth for both proteins, which is molecularly distinct to the function of Tmod4.

Muscle fiber detachment from myoseptal boundaries is a common finding in zebrafish models of muscular dystrophies. In some instances, there is a weakening of the interaction between muscle fiber and myosepta, which is yet to manifest as a fiber detachment phenotype. Therefore, to push the fiber detachment of muscle, mutant fish but not their wild-type siblings, beyond their binding threshold, a series of small electrical pulses can be applied to the larvae to create a maximal force contraction and ultimately fiber detachment. To do this, we built a digital pulse generator which delivers four 8 ms 30 V pulses in quick succession, and it has the advantage over older analog approaches to pulse generation because it improves accuracy and is appreciably less expensive. Our pulse generator significantly increases fiber detachment in the laminin-α2 deficient, congenital muscular dystrophy type 1a (MDC1a) model lama2

The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud.

Background Regeneration of neurons in the central nervous system is poor in humans. In other vertebrates neural regeneration does occur efficiently and involves reactivation of developmental processes. Within the neural retina of zebrafish, Müller glia are the main stem cell source and are capable of generating progenitors to replace lost neurons after injury. However, it remains largely unknown to what extent Müller glia and neuron differentiation mirror development. Methods Following neural ablation in the zebrafish retina, dividing cells were tracked using a prolonged labelling technique. We investigated to what extent extrinsic feedback influences fate choices in two injury models, and whether fate specification follows the histogenic order observed in development. Results By comparing two injury paradigms that affect different subpopulations of neurons, we found a dynamic adaptability of fate choices during regeneration. Both injuries followed a similar time course of cell death, and activated Müller glia proliferation. However, these newly generated cells were initially biased towards replacing specifically the ablated cell types, and subsequently generating all cell types as the appropriate neuron proportions became re-established. This dynamic behaviour has implications for shaping regenerative processes and ensuring restoration of appropriate proportions of neuron types regardless of injury or cell type lost. Conclusions Our findings suggest that regenerative fate processes are more flexible than development processes. Compared to development fate specification we observed a disruption in stereotypical birth order of neurons during regeneration Understanding such feedback systems can allow us to direct regenerative fate specification in injury and diseases to regenerate specific neuron types in vivo.

The muscle is separated from tendons by a specialised basement membrane that acts as the structural interface of the myotendinous junction (MTJ). In zebrafish, the larval MTJ forms at the vertical myosepta, which separate the individual myomeres that arise during somitogenesis. In this review, we examine the formation of the vertical myosepta in zebrafish. We then describe insights this gains us in the context of muscle basement membrane failure, the mechanistic basis of the inherited muscle wasting condition muscular dystrophy (MD). We examine recent manuscripts that investigate how a well-orchestrated integration of MTJ components is needed during vertical myosepta development. We find the process can be divided into three stereotypical stages of its development based on specific structural properties of the developing basement membrane. This review highlights insights that have been gleaned from vertical myosepta formation in zebrafish that maybe of value in developing therapeutic strategies for MD.

The Nuclear receptor subfamily 4 group A member 2 (Nr4a2) is crucial for the formation or maintenance of dopaminergic neurons in the central nervous system including the retina, where dopaminergic amacrine cells contribute to visual function. Little is known about which cells express Nr4a2 at which developmental stage. Furthermore, whether Nr4a2 functions in combination with other genes is poorly understood. Thus, we generated a novel transgenic to visualize Nr4a2 expression in vivo during zebrafish retinogenesis. A 4.1 kb fragment of the nr4a2a promoter was used to drive green fluorescent protein expression in this Tg(nr4a2a:eGFP) line. In situ hybridization showed that transgene expression follows endogenous RNA expression at a cellular level. Temporal expression and lineages were quantified using in vivo time-lapse imaging in embryos. Nr4a2 expressing retinal subtypes were characterized immunohistochemically. Nr4a2a:eGFP labeled multiple neuron subtypes including 24.5% of all amacrine interneurons. Nr4a2a:eGFP labels all tyrosine hydroxylase labeled dopaminergic amacrine cells, and other nondopaminergic GABAergic amacrine populations. Nr4a2a:eGFP is confined to a specific progenitor lineage identified by sequential expression of the bhlh transcription factor Atonal7 (Atoh7) and Pancreas transcription factor 1a (Ptf1a), and labels postmitotic postmigratory amacrine cells. Thus, developmental Nr4a2a expression indicates a role during late differentiation of specific amacrine interneurons. Tg(nr4a2a:eGFP) is an early marker of distinct neurons including dopaminergic amacrine cells. It can be utilized to assess consequences of gene manipulations and understand whether Nr4a2 only carries out its role in the presence of specific coexpressed genes. This will allow Nr4a2 use to be refined for regenerative approaches.

Skeletal muscle is a contractile, postmitotic tissue that retains the capacity to grow and regenerate throughout life in amniotes and teleost. Both muscle growth and regeneration are regulated by obligate tissue resident muscle stem cells. Given that considerable knowledge exists on the myogenic process, recent studies have focused on examining the molecular markers of muscle stem cells, and on the intrinsic and extrinsic signals regulating their function. From this, two themes emerge: firstly, muscle stem cells display remarkable heterogeneity not only with regards to their gene expression profile, but also with respect to their behavior and function; and secondly, the stem cell niche is a critical regulator of muscle stem cell function during growth and regeneration. Here, we will address the current understanding of these emerging themes with emphasis on the distinct processes used by amniotes and teleost, and discuss the challenges and opportunities in the muscle growth and regeneration fields. This article is characterized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Early Embryonic Development > Development to the Basic Body Plan Vertebrate Organogenesis > Musculoskeletal and Vascular.

Laminins comprise structural components of basement membranes, critical in the regulation of differentiation, survival and migration of a diverse range of cell types, including skeletal muscle. Mutations in one muscle enriched Laminin isoform, Laminin alpha2 (Lama2), results in the most common form of congenital muscular dystrophy, congenital muscular dystrophy type 1A (MDC1A). However, the exact cellular mechanism by which Laminin loss results in the pathological spectrum associated with MDC1A remains elusive. Here we show, via live tracking of individual muscle fibres, that dystrophic myofibres in the zebrafish model of MDC1A maintain sarcolemmal integrity and undergo dynamic remodelling behaviours post detachment, including focal sarcolemmal reattachment, cell extension and hyper-fusion with surrounding myoblasts. These observations imply the existence of a window of therapeutic opportunity, where detached cells may be “re-functionalised” prior to their delayed entry into the cell death program, a process we show can be achieved by muscle specific or systemic Laminin delivery. We further reveal that Laminin also acts as a pro-regenerative factor that stimulates muscle stem cell-mediated repair in lama2-deficient animals in vivo. The potential multi-mode of action of Laminin replacement therapy suggests it may provide a potent therapeutic axis for the treatment for MDC1A.

The development and growth of vertebrate axial muscle have been studied for decades at both the descriptive and molecular level. The zebrafish has provided an attractive model system for investigating both muscle patterning and growth due to its simple axial musculature with spatially separated fibre types, which contrasts to complex muscle groups often deployed in amniotes. In recent years, new findings have reshaped previous concepts that define how final teleost muscle form is established and maintained. Here, we summarise recent findings in zebrafish embryonic myogenesis with a focus on fibre type specification, followed by an examination of the molecular mechanisms that control muscle growth with emphasis on the role of the dermomyotome-like external cell layer. We also consider these data sets in a comparative context to gain insight into the evolution of axial myogenic patterning systems within the vertebrate lineage.

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

During neurogenesis, progenitors balance proliferation and cell cycle exit together with expression of fate determinant genes to ensure that the correct number of each of these neuron types is generated. Although intrinsic gene expression acting cell autonomously within each progenitor drives these processes, the final number of neurons generated is also influenced by extrinsic cues, representing a potential avenue to direct neurogenesis in developmental disorders or regenerative settings without the requirement to change intrinsic gene expression. Thus, it is important to understand which of these stages of neurogenesis are amenable to such extrinsic influences. Additionally, all types of neurons are specified in a highly conserved histogenic order, although its significance is unknown. This study makes use of conserved patterns of neurogenesis in the relatively simple yet highly organized zebrafish retina model, in which such histogenic birth order is well characterized. We directly visualize and quantify birth dates and cell fate determinant expression in WT vs. environments lacking different neuronal populations. This study shows that extrinsic feedback from developing retinal neurons is important for the temporal expression of intrinsic fate determinants but not for the timing of birth dates. We found no changes in cell cycle exit timing but did find a significant delay in the expression of genes driving the generation only of later- but not earlier-born cells, suggesting that the robustness of this process depends on continuous feedback from earlier-formed cell types. Thus, extrinsic cues selectively influence cell fate determinant progression, which may explain the function of the retinal histogenic order observed. J. Comp. Neurol. 524:2553-2566, 2016. © 2016 Wiley Periodicals, Inc.