Zebrafish ambra1a and ambra1b Silencing Affect Heart Development.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy ambra1a and ambra1b, both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of ambra1a and ambra1b knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.

The epg5 knockout zebrafish line: a model to study Vici syndrome.

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Downregulation of lizard immuno-genes in the regenerating tail and myogenes in the scarring limb suggests that tail regeneration occurs in an immuno-privileged organ.

Amputated tails of lizards regenerate while limbs form scars which histological structure is very different from the original organs. Lizards provide useful information for regenerative medicine and some hypotheses on the loss of regeneration in terrestrial vertebrates. Analysis of tail and limb transcriptomes shows strong downregulation in the tail blastema for immunoglobulins and surface B and T receptors, cell function, and metabolism. In contrast, in the limb blastema genes for myogenesis, muscle and cell function, and extracellular matrix deposition but not immunity are variably downregulated. The upregulated genes show that the regenerating tail is an embryonic organ driven by the Wnt pathway and non-coding RNAs. The strong inflammation following amputation, the non-activation of the Wnt pathway, and the upregulation of inflammatory genes with no downregulation of immune genes indicate that the amputated limb does not activate an embryonic program. Intense inflammation in limbs influences in particular the activity of genes coding for muscle proteins, cell functions, and stimulates the deposition of dense extracellular matrix proteins resulting in scarring limb outgrowths devoid of muscles. The present study complements that on upregulated genes, and indicates that the regenerating tail requires immune suppression to maintain this embryonic organ connected to the rest of the tail without be rejected or turned into a scar. It is hypothesized that the evolution of the adaptive immune system determined scarring instead of organ regeneration in terrestrial vertebrates and that lizards evolved the process of tail regeneration through a mechanism of immuno-evasion.

Transcriptome analysis of the regenerating tail vs. the scarring limb in lizard reveals pathways leading to successful vs. unsuccessful organ regeneration in amniotes.

BACKGROUND: Lizards are amniotes regenerating the tail but not the limb, and no information on their different gene expression is available. RESULTS: Transcriptomes of regenerating tail and limb blastemas show differences in gene expression between the two organs. In tail blastemal, snoRNAs and Wnt signals appear up-regulated probably in association with the apical epidermal peg (AEP), an epithelial region that sustains tail regeneration but is absent in the limb. A balance between pro-oncogenes and tumor suppressors is likely present in tail blastema allowing a regulated proliferation. Small collagens, protease inhibitors, embryonic keratins are up-regulated in the regenerating tail blastema but not in the limb where Wnt inhibitors, inflammation-immune and extracellular matrix proteins depress cell growth. CONCLUSIONS: The AEP and the spinal cord in the tail maintains Wnt and fibroblast growth signaling that stimulate blastema cell proliferation and growth while these signals are absent in the limb as a consequence of the intense inflammation. Regeneration of amniote appendages requires a control of cell proliferation and inflammatory-immune reactions to form an apical epidermal cap. Genes that control cell proliferation and inflammation, addressing regeneration and not tumor formation in the tail and scarring in the limb are discussed for future studies. Developmental Dynamics 246:116-134, 2017. © 2016 Wiley Periodicals, Inc.

Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria.

Ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. Nowadays, Ambra1 has been characterized only in mammals and zebrafish. Through bioinformatics searches and targeted cloning, we report the identification of the complete Ambra1 transcript in a non-vertebrate chordate, the tunicate Botryllus schlosseri. Tunicata is the sister group of Vertebrata and the only chordate group possessing species that reproduce also by blastogenesis (asexual reproduction). B. schlosseri Ambra1 deduced amino acid sequence is shorter than vertebrate homologues but still contains the typical WD40 domain. qPCR analyses revealed that the level of B. schlosseri Ambra1 transcription is temporally regulated along the colonial blastogenetic cycle. By means of similarity searches we identified Wdr5 and Katnb1 as proteins evolutionarily associated to Ambra1. Phylogenetic analyses on Bilateria indicate that: (i) Wdr5 is the most related to Ambra1, so that they may derive from an ancestral gene, (ii) Ambra1 forms a group of ancient genes evolved before the radiation of the taxon, (iii) these orthologous Ambra1 share the two conserved WD40/YVTN repeat-like-containing domains, and (iv) they are characterized by ancient duplications of WD40 repeats within the N-terminal domain.

Beneficial bacteria affect Danio rerio development by the modulation of maternal factors involved in autophagic, apoptotic and dorsalizing processes.

BACKGROUND/AIMS: Probiotic strains have been recognized to exert important roles in many biological systems, including immune response, growth, development and reproduction. However, to date, no studies have focused either on the relation among probiotics and maternal factors or on probiotics' ability to qualitatively and/or quantitatively modulate maternal transcripts. METHODS: In this study, the effects of Lactobacillus rhamnosus administered to parental fish on the control of maternal factors involved in autophagic, apoptotic and dorsalizing processes during zebrafish embryo development were assessed through q-PCRs, WMISH and TUNEL assay. RESULTS: The results we obtained show that probiotic induced significant changes in both maternal and zygotic mRNA levels involved in embryo development. The maternal autophagy-regulating genes herein investigated--ambra1a, ambra1b, beclin, lc3-, as well as those involved in the apoptotic process--caspase3, bcl2, bax--were modulated in disfavor and favor of the treated group, respectively. Also, the key transcripts ruling the dorsalizing process--goosecoid and chordin--were subject to a significant regulation of their gene expression. CONCLUSION: The results we acquired demonstrated that parentally administered Lactobacillus rhamnosus is able to modulate important physiological processes involved in zebrafish embryo development.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation.

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

Zebrafish ambra1a and ambra1b knockdown impairs skeletal muscle development.

The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle.

A living biosensor model to dynamically trace glucocorticoid transcriptional activity during development and adult life in zebrafish.

Glucocorticoids (GCs) modulate many cellular processes through the binding of the glucocorticoid receptor (GR) to specific responsive elements located upstream of the transcription starting site or within an intron of GC target genes. Here we describe a transgenic fish line harboring a construct with nine GC-responsive elements (GREs) upstream of a reporter (EGFP) coding sequence. Transgenic fish exhibit strong fluorescence in many known GC-responsive organs. Moreover, its enhanced sensitivity allowed the discovery of novel GC-responsive tissue compartments, such as fin, eyes, and otic vesicles. Long-term persistence of transgene expression is seen during adult stages in several organs. Pharmacological and genetic analysis demonstrates that the transgenic line is highly responsive to drug administration and molecular manipulation. Moreover, reporter expression is sensitively and dynamically modulated by the photoperiod, thus proving that these fish are an in vivo valuable platform to explore GC responsiveness to both endogenous and exogenous stimuli.

Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis.

AMBRA1 is a positive regulator of the BECN1-dependent program of autophagy recently identified in mouse. In this study, we cloned the full-length cDNAs of ambra1a and ambra1b zebrafish paralogous genes. As in mouse, both Ambra1 proteins contain the characteristic WD40 repeat region. The transcripts of both genes are present as maternal RNAs in the eggs and display a gradual decline until 8 hpf, being replaced by zygotic mRNAs from 12 hpf onwards. After 24 hpf, the transcripts are mainly localized in the head, suggesting a possible role in brain development. To check their developmental roles, we adopted morpholino knockdown to block either translation (ATGMOs) or splicing (SPLICMOs). Treatment with ATGMOs causes severe embryonic malformations, as prelarvae could survive for only 3 and 4 days in ambra1a and b morphants, respectively. Treatment with SPLICMOs led to developmental defects only at a late stage, indicating the importance of maternally supplied ambra1 transcripts. Analysis of the levels of Lc3-II, an autophagosome-specific marker, in the presence of lysosome inhibitors evidenced a reduction in the rate of autophagosome formation in both MOs-injected embryos at 48 hpf, more pronounced in the case of ambra1a gene. Although some defects, such as body growth delay, curved shape and hemorrhagic pericardial cavity were present in both morphants, the occurrence of specific phenotypes, such as major abnormalities of brain development in ambra1a morphants, suggests the possible acquisition of specific functions by the two paralogous genes that are both required during development and do not compensate each other following knockdown.