Modeling Human Muscular Dystrophies in Zebrafish: Mutant Lines, Transgenic Fluorescent Biosensors, and Phenotyping Assays

: Muscular dystrophies (MDs) are a heterogeneous group of myopathies characterized by progressive muscle weakness leading to death from heart or respiratory failure. MDs are caused by mutations in genes involved in both the development and organization of muscle fibers. Several animal models harboring mutations in MD-associated genes have been developed so far. Together with rodents, the zebrafish is one of the most popular animal models used to reproduce MDs because of the high level of sequence homology with the human genome and its genetic manipulability. This review describes the most important zebrafish mutant models of MD and the most advanced tools used to generate and characterize all these valuable transgenic lines. Zebrafish models of MDs have been generated by introducing mutations to muscle-specific genes with different genetic techniques, such as (i) N-ethyl-N-nitrosourea (ENU) treatment, (ii) the injection of specific morpholino, (iii) tol2-based transgenesis, (iv) TALEN, (v) and CRISPR/Cas9 technology. All these models are extensively used either to study muscle development and function or understand the pathogenetic mechanisms of MDs. Several tools have also been developed to characterize these zebrafish models by checking (i) motor behavior, (ii) muscle fiber structure, (iii) oxidative stress, and (iv) mitochondrial function and dynamics. Further, living biosensor models, based on the expression of fluorescent reporter proteins under the control of muscle-specific promoters or responsive elements, have been revealed to be powerful tools to follow molecular dynamics at the level of a single muscle fiber. Thus, zebrafish models of MDs can also be a powerful tool to search for new drugs or gene therapies able to block or slow down disease progression

A Smad3 transgenic reporter reveals TGF-beta control of zebrafish spinal cord development

TGF-beta (TGFβ) family mediated Smad signaling is involved in mesoderm and endoderm specification, left-right asymmetry formation and neural tube development. The TGFβ1/2/3 and Activin/Nodal signal transduction cascades culminate with activation of SMAD2 and/or SMAD3 transcription factors and their overactivation are involved in different pathologies with an inflammatory and/or uncontrolled cell proliferation basis, such as cancer and fibrosis. We have developed a transgenic zebrafish reporter line responsive to Smad3 activity. Through chemical, genetic and molecular approaches we have seen that this transgenic line consistently reproduces in vivo Smad3-mediated TGFβ signaling. Reporter fluorescence is activated in phospho-Smad3 positive cells and is responsive to both Smad3 isoforms, Smad3a and 3b. Moreover, Alk4 and Alk5 inhibitors strongly repress the reporter activity. In the CNS, Smad3 reporter activity is particularly high in the subpallium, tegumentum, cerebellar plate, medulla oblongata and the retina proliferative zone. In the spinal cord, the reporter is activated at the ventricular zone, where neuronal progenitor cells are located. Colocalization methods show in vivo that TGFβ signaling is particularly active in neuroD+ precursors. Using neuronal transgenic lines, we observed that TGFβ chemical inhibition leads to a decrease of differentiating cells and an increase of proliferation. Similarly, smad3a and 3b knock-down alter neural differentiation showing that both paralogues play a positive role in neural differentiation. EdU proliferation assay and pH3 staining confirmed that Smad3 is mainly active in post-mitotic, non-proliferating cells. In summary, we demonstrate that the Smad3 reporter line allows us to follow in vivo Smad3 transcriptional activity and that Smad3, by controlling neural differentiation, promotes the progenitor to precursor switch allowing neural progenitors to exit cell cycle and differentiate

Tcf7l2 plays pleiotropic roles in the control of glucose homeostasis, pancreas morphology, vascularization and regeneration

Type 2 diabetes (T2D) is a disease characterized by impaired insulin secretion. The Wnt signaling transcription factor Tcf7l2 is to date the T2D-associated gene with the largest effect on disease susceptibility. However, the mechanisms by which TCF7L2 variants affect insulin release from \u3b2-cells are not yet fully understood. By taking advantage of a tcf7l2 zebrafish mutant line, we first show that these animals are characterized by hyperglycemia and impaired islet development. Moreover, we demonstrate that the zebrafish tcf7l2 gene is highly expressed in the exocrine pancreas, suggesting potential bystander effects on \u3b2-cell growth, differentiation and regeneration. Finally, we describe a peculiar vascular phenotype in tcf7l2 mutant larvae, characterized by significant reduction in the average number and diameter of pancreatic islet capillaries. Overall, the zebrafish Tcf7l2 mutant, characterized by hyperglycemia, pancreatic and vascular defects, and reduced regeneration proves to be a suitable model to study the mechanism of action and the pleiotropic effects of Tcf7l2, the most relevant T2D GWAS hit in human populations

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src–mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL

The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance, and characterized by both a strong reduction in mtDNA content and severe mitochondrial defects in affected tissues. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been associated with hepatocerebral forms of MDS. The zebrafish mpv17 null mutant lacks the guanine-based reflective skin cells named iridophores and represents a promising model to clarify the role of Mpv17. In this study, we characterized the mitochondrial phenotype of mpv17-/- larvae and found early and severe ultrastructural alterations in liver mitochondria, as well as significant impairment of the respiratory chain, leading to activation of the mitochondrial quality control. Our results provide evidence for zebrafish Mpv17 being essential for maintaining mitochondrial structure and functionality, while its effects on mtDNA copy number seem to be subordinate. Considering that a role in nucleotide availability had already been postulated for MPV17, that embryos blocked in pyrimidine synthesis do phenocopy mpv17-/- knockouts (KOs) and that mpv17-/- KOs have impaired Dihydroorotate dehydrogenase activity, we provided mpv17 mutants with the pyrimidine precursor orotic acid (OA). Treatment with OA, an easily available food supplement, significantly increased both iridophore number and mtDNA content in mpv17-/- mutants, thus linking the loss of Mpv17 to pyrimidine de novo synthesis and opening a new simple therapeutic approach for MPV17-related MDS

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/\u3b2-catenin Signaling Regulation

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/\u3b2-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease

Treponema pallidum (syphilis) antigen TpF1 induces angiogenesis through the activation of the IL-8 pathway

Over 10 million people every year become infected by Treponema pallidum and develop syphilis, a disease with broad symptomatology that, due to the difficulty to eradicate the pathogen from the highly vascularized secondary sites of infection, is still treated with injections of penicillin. Unlike most other bacterial pathogens, T. pallidum infection produces indeed a strong angiogenic response whose mechanism of activation, however, remains unknown. Here, we report that one of the major antigen of T. pallidum, the TpF1 protein, has growth factor-like activity on primary cultures of human endothelial cells and activates specific T cells able to promote tissue factor production. The growth factor-like activity is mediated by the secretion of IL-8 but not of VEGF, two known angiogenic factors. The pathogen's factor signals IL-8 secretion through the activation of the CREB/NF-\u3baB signalling pathway. These findings are recapitulated in an animal model, zebrafish, where we observed that TpF1 injection stimulates angiogenesis and IL-8, but not VEGF, secretion. This study suggests that the angiogenic response observed during secondary syphilis is triggered by TpF1 and that pharmacological therapies directed to inhibit IL-8 response in patients should be explored to treat this disease

Emilin-1 controls arterial blood pressure by regulating contractility of vascular smooth muscle cells

Emilin-1 is a protein of the elastic extracellular matrix (ECM) expressed in interstitial connective tissue and in the cardiovascular system. Emilin1 null mice display hypotrophic remodeling of the wall of conductance arteries and increased blood pressure. The protein regulates the bioavailability of TGF-b by inhibiting proteolysis of the proTGF-b precursor to LAP/TGF-b, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin-1, the amount of active TGF-b is increased. As Emilin-1 is expressed in blood vessels starting from early stages of embryonic development to adulthood, a key question concerning the function of the protein is whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult. The conditional gene targeting procedure chosen to inactivate the Emilin1 gene in smooth muscle cells (SMCs) of adult mice included the use of floxed Emilin1 and CreERT2 (a tamoxifen inducible Cre recombinase) under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. Tamoxifen administration induced activity of Cre specifically in vascular and visceral SMCs, as revealed by X-gal staining of tissues from animals with the Rosa26R mutation. When Emilin1flox/flox mice carrying the Smmhc-CreERT2 transgene were given tamoxifen for 7 days, Emilin-1 disappeared completely in 10-12 days from start of treatment. In the same time, blood pressure increased of about 20 mmHg, a level that was stably maintained thereafter. The myogenic response of second branch meseteric arteries, evaluated using a pressure myograph, was found to be increased in Emilin1-/- mice. Additional experiments with aorta and mesenteric artery SMC cultures from control and mutant mice showed that lack of Emilin-1  enhanced phosphorylation of myosin light chain 20 when cells were stimulated with the a1-adrenergic receptor agonists phenylephrine or with angiotensin II. Moreover, basal cytosolic Ca2+ levels and calcium transients induced by stimulation with phenylephrine and angiotensin II were increased in SMCs from Emilin1-/- mutants. The data suggest that Emilin-1 expression is continuously required for regulation of blood pressure and that the increase of TGF-b activity induced by diminished Emilin-1  stimulates, likely through alteration of intracellular calcium homeostasis, contractility of vascular SMC to mechanical and chemical stimuli with ensuing hypertension

Conditional inactivation of Emilin1 and Col6a1 genes

Conditional gene expression methods are important approaches for examining the function of particular genes in development and disease. In particular, a lentiviral vector system for RNAi- mediated in vivo silencing of Col6a1 and a Cre-loxP based procedure for conditional-inducible knockout (KO) of Emilin1 were used in this work. The use of lentiviral vectors can accelerate the generation of animals with substantial suppression of gene expression in an inducible way. Some authors have observed limitations of the technique due to low efficiency of transgenesis and mosaicism in transgenic mice. We have generated several transgenic mouse lines expressing siRNAs that target the α1(VI) mRNA. Characterization of the different lines and comparison of the phenotypes with that of Col6a1 knockout mice have allowed a systematic evaluation of the different factors affecting silencing of Col6a1, a gene of the extracellular matrix with a complex pattern of tissue-specific expression. The results, obtained with vectors pLVTHM and pLVPT-rtTRKRAB, point out three parameters as major determinants of the efficiency of interference: the choice of interfering sequence, the number of proviral copies integrated into the mouse genome and the site of integration of the provirus. A lentiviral vector (pLVPT-rtTRKRAB) with doxycycline inducible production of shRNA was also tested. Control of expression by the drug was stringent in many tissues; however, in some tissues turning off of shRNA synthesis was not complete. The data support the application of the lentiviral vectors used here in transgenesis (Frka, Facchinello, et al., 2009). Emilin1 is a gene coding for a protein, Emilin-1, of the elastic extracellular matrix expressed in interstitial connective tissue and in the cardiovascular system starting form early stages of embryonic development to adulthood. Emilin1 null mice display reduced diameter of blood vessels and arterial hypertension. The protein regulates the bioavailability of TGF-β, a cytokine with major effects on the cardiovascular system. Specifically, it has been shown that Emilin1 inhibits proteolysis of the proTGF-β precursor to LAP/TGF-β, a complex from which the growth factor can be subsequently released for receptor binding. In the absence of Emilin1, the amount of active TGF-β is increased, reducing the proliferation rate of smooth muscle cells and the diameter of blood vessels. To establish whether the Emilin1-/- phenotype is the result of a developmental defect or the function of the protein is required for the regulation of blood pressure and arterial structure also in the adult, a conditional gene targeting procedure was used to inactivate Emilin1 in a tissue and time-specific manner. The genetic set up of the transgenic mouse model included the use of floxed Emilin1, CreERT2 (a tamoxifen inducible Cre recombinase) tissue-specific drivers and Rosa26R-lacZ, an inducible reporter for histological visualization of gene rearrangement. A targeting vector was synthesized in which exons 1-2 of the Emilin1 gene were flanked by loxP sites (floxed allele). The correct integration of the targeting construct in embryonic stem (ES) cell clones was confirmed by Southern blot and PCR analyses. Four of the ES cell clones were subsequently injected into blastocysts resulting in chimeric mice with 10-100% chimerism. The CreERT2 gene was driven by the Emilin1 or the smooth muscle myosin heavy chain (SMMHC) promoters in order to be expressed in cells active in Emilin1 synthesis or in smooth muscle cells respectively. After tamoxifen administration, activity of both promoters was evident in vascular and visceral smooth muscle cells, where SMMHC-CreERT2 induced recombination more strongly than Emilin1-CreERT2. PCR and RT-PCR analyses confirmed that the SMMHC-CreERT2 was expressed and efficiently excised the loxP flanked sequences in the Emilin1flox/wt locus sufficiently to reduce Emilin1 mRNA expression. Emilin1flox/flox mice appeared and bred normally and showed no difference in Emilin1 mRNA expression and in blood pressure levels as compared to controls, confirming that introduction of the loxP sites did not interfere with regulation of the gene. Moreover preliminary data showed that adult animals of the double transgenic lines Emilin1flox/flox and SMMHC-Cre-ERT2 treated with tamoxifen displayed increased blood pressure. This result suggests that hypertension in Emilin1flox/flox mice is not due to a developmental defect of blood vessels, but to the lack of a continuous effect of Emilin-1 on blood pressure regulation even in the adult.Tecniche che prevedono il controllo dell’espressione genica tramite inattivazione condizionale sono di particolare utilità nello studio della funzione di geni durante lo sviluppo e nel determinare patologie. In questo lavoro sono stati utilizzati un sistema di silenziamento in vivo del gene Col6a1 mediante RNAi utlizzando vettori lentivirali e un sistema “Cre/loxP” per generare un modello murino di knockout condizionale inducibile. L’uso di vettori lentivirali può accelerare la generazione di animali transgenici con una consistente riduzione dell’espressione genica e in modo inducibile, anche se in letteratura alla tecnica sono state attribuite alcune limitazioni dovute alla bassa efficienza di transgenesi e alla presenza di mosaicismo nei topi così ottenuti. In questo lavoro abbiamo prodotto diverse linee di animali transgenici con diminuiti livelli di messaggero per il gene Col6a1 grazie alla produzione di siRNA. La caratterizzazione delle varie linee e la comparazione del fenotipo con quello dei topi knockout per lo stesso gene ha permesso l’identificazione dei diversi fattori che sono in grado di influire sul processo di silenziamento di Col6a1, un gene codificante per una proteina della matrice extracellulare con un complesso pattern di espressione tessuto specifica. I risultati ottenuti con i vettori pLVTHM e pLVPT-rtTRKRAB, hanno permesso di definire tre importanti fattori come maggiori determinanti nell’efficienza dell’interferenza: la scelta della sequenza interferente, il numero di copie provirali integrate nel genoma e il sito di integrazione degli stessi. É stato inoltre utilizzato un vettore lentivirale (pLVPT-rtTRKRAB) per la produzione inducibile di shRNA mediante somministrazione di doxyciclina. Il controllo dell’espressione dopo doxycicilina è risultato essere stringente in molti tessuti, anche se in alcuni la inattivazione della produzione di shRNA non è stata completa. I risultati ottenuti dimostrano l’applicabilità di vettori lentivirali nella generazione di animali transgenici (Frka, Facchinello, et al., 2009). Emilina1 è una proteina della matrice extracellulare presente nei tessuti connettivi interstiziali e nel sistema cardiovascolare a partire da stadi precoci di sviluppo embrionale e nell’adulto. I topi mancanti di Emilina1 sono ipertesi e mostrano un diametro ridotto dei vasi. Emilina1 svolge la sua funzione regolando la biodisponibilità di TGF-β, una citochina che svolge importanti funzioni nel sistema cardiovascolare. In particolare, è stato dimostrato che Emilina1 inibisce la proteolisi del precursore pro-TGF-β a LAP/TGF-β, un complesso dal quale il fattore di crescita attivo deve essere successivamente rilasciato per permetterne il legame con il recettore. In assenza di Emilina1, la quantità di TGF-β attivo è aumentata, riducendo la proliferazione delle cellule muscolari lisce e quindi il diametro del vaso. Per stabilire se il fenotipo dei topi Emilina1-/- è il risultato di un’alterata morfogenesi dei vasi o se la funzione della proteina è richiesta nella regolazione della pressione e nella struttura del vaso anche nell’animale adulto, è stato necessario produrre un knockout condizionale del gene di Emilin1 in modo da indurre l’assenza della proteina con un preciso controllo temporale e spaziale. Il modello murino prevede la presenza di siti loxP posti nel gene di Emilin1 in modo da produrre l’inattivazione genica, una Cre-ERT2 (cioè una Cre ricombinasi inducibile tramite tamoxifen) e il locus Rosa26R-lacZ (un gene reporter inducibile per la rilevazione istologica delle cellule nelle quali si è avuto il riarrangiamento genetico). É stato preparato un costrutto dove l’esone 1 e 2 del gene di Emilina1 sono fiancheggiati da due siti loxP. Mediante Southern Blot e PCR, è stata verificata la corretta integrazione del costrutto nelle cellule embrionali staminali di topo. 4 cloni ricombinanti omologhi così identificati sono stati trasferiti in vivo mediante microiniezione in blastocisti ottenendo topi chimerici con un grado di chimerismo compreso tra il 10 e il 100%. L’espressione del gene Cre-ERT2 è stato posto sotto il controllo delle sequenze promotoriali proprie di Emilina1 o, alternativamente, del gene per la catena pesante della miosina di muscolo liscio, in modo da essere attivamente espressa dalle cellule che producono Emilina1 o da quelle muscolari lisce rispettivamente. Dopo la somministrazione di tamoxifen, l’attività di entrambi i promotori è evidente sia nelle cellule muscolare lisce vascolari e viscerali, dove SMMHC-CreERT2 induce una ricombinazione più efficiente rispetto al costrutto Emilina1-CreERT2. Le analisi mediante PCR e RT-PCR confermano che la cre ricombinasi sotto il promotore della miosina di muscolo liscio induce efficientemente la ricombinazione dei siti loxP nel locus di Emilina1, permettendo in questo modo una riduzione consistente dei livelli di messaggero. I topi Emilina1flox/flox generati sono fertili e presentano un fenotipo normale e inoltre comparati con degli animali di controllo non mostrano differenze per quanto riguarda l’espressione del messaggero di Emilina1 e nei livelli di pressione sanguigna, confermando che l’introduzione dei siti loxP non interferisce con la regolazione dell’espressione del gene. Risultati preliminari inoltre indicano un aumento della pressione sanguigna nelle doppie linee transgeniche Emilina1flox/flox e SMMHC-Cre-ERT2 in seguito a trattamento con tamoxifen. Questo risultato suggerisce che l’ipertensione non è dovuta ad un’alterata morfogenesi dei vasi sanguigni, ma al ruolo continuativo di Emilina-1 nella regolazione della pressione sanguigna anche nell’adulto