Sequestosome-1 (SQSTM1/p62) as a target in dopamine catabolite-mediated cellular dyshomeostasis.

Alterations in the dopamine catabolic pathway are known to contribute to the degeneration of nigrostriatal neurons in Parkinson's disease (PD). The progressive cellular buildup of the highly reactive intermediate 3,4-dihydroxyphenylacetaldehye (DOPAL) generates protein cross-linking, oligomerization of the PD-linked αSynuclein (αSyn) and imbalance in protein quality control. In this scenario, the autophagic cargo sequestome-1 (SQSTM1/p62) emerges as a target of DOPAL-dependent oligomerization and accumulation in cytosolic clusters. Although DOPAL-induced oxidative stress and activation of the Nrf2 pathway promote p62 expression, p62 oligomerization rather seems to be a consequence of direct DOPAL modification. DOPAL-induced p62 clusters are positive for ubiquitin and accumulate within lysosomal-related structures, likely affecting the autophagy-lysosomal functionality. Finally, p62 oligomerization and clustering is synergistically augmented by DOPAL-induced αSyn buildup. Hence, the substantial impact on p62 proteostasis caused by DOPAL appears of relevance for dopaminergic neurodegeneration, in which the progressive failure of degradative pathways and the deposition of proteins like αSyn, ubiquitin and p62 in inclusion bodies represent a major trait of PD pathology.

Microbial Polysaccharides Extracted from Different Mature Muds of the Euganean Thermal District Show Similar Anti-Inflammatory Activity In Vivo.

The Euganean Thermal District, situated in North-East Italy, is one of Europe's largest and oldest thermal centres. The topical application of its therapeutic thermal muds is recognised by the Italian Health System as a beneficial treatment for patients suffering from arthro-rheumatic diseases. Polysaccharides produced by the mud microbiota have been recently identified as anti-inflammatory bioactive molecules. In this paper we analysed the efficacy of Microbial-Polysaccharides (M-PS) derived from mature muds obtained at different maturation temperatures, both within and outside the codified traditional mud maturation range. M-PSs were extracted from six mature muds produced by five spas of the Euganean Thermal District and investigated for their chemical properties, monosaccharide composition and in vivo anti-inflammatory potential, using the zebrafish model organism. Additionally, mature muds were characterized for their microbiota composition using Next-Generation Sequencing. The results showed that all M-PSs exhibit similar anti-inflammatory potential, referable to their comparable chemical composition. This consistency was observed despite changes in cyanobacteria populations, suggesting a possible role of the entire microbial community in shaping the properties of these biomolecules. These findings highlight the importance of scientific research in untangling the origins of the therapeutic efficacy of Euganean Thermal muds in the treatment of chronic inflammatory conditions.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

A novel DSP zebrafish model reveals training- and drug-induced modulation of arrhythmogenic cardiomyopathy phenotypes.

Arrhythmogenic cardiomyopathy (AC) is an inherited disorder characterized by progressive loss of the ventricular myocardium causing life-threatening ventricular arrhythmias, syncope and sudden cardiac death in young and athletes. About 40% of AC cases carry one or more mutations in genes encoding for desmosomal proteins, including Desmoplakin (Dsp). We present here the first stable Dsp knock-out (KO) zebrafish line able to model cardiac alterations and cell signalling dysregulation, characteristic of the AC disease, on which environmental factors and candidate drugs can be tested. Our stable Dsp knock-out (KO) zebrafish line was characterized by cardiac alterations, oedema and bradycardia at larval stages. Histological analysis of mutated adult hearts showed reduced contractile structures and abnormal shape of the ventricle, with thinning of the myocardial layer, vessels dilation and presence of adipocytes within the myocardium. Moreover, TEM analysis revealed "pale", disorganized and delocalized desmosomes. Intensive physical training protocol caused a global worsening of the cardiac phenotype, accelerating the progression of the disease. Of note, we detected a decrease of Wnt/ß-catenin signalling, recently associated with AC pathogenesis, as well as Hippo/YAP-TAZ and TGF-ß pathway dysregulation. Pharmacological treatment of mutated larvae with SB216763, a Wnt/ß-catenin agonist, rescued pathway expression and cardiac abnormalities, stabilizing the heart rhythm. Overall, our Dsp KO zebrafish line recapitulates many AC features observed in human patients, pointing at zebrafish as a suitable system for in vivo analysis of environmental modulators, such as the physical exercise, and the screening of pathway-targeted drugs, especially related to the Wnt/ß-catenin signalling cascade.

Sex and Brain: The Role of Sex Chromosomes and Hormones in Brain Development and Parkinson's Disease.

Sex hormones and genes on the sex chromosomes are not only key factors in the regulation of sexual differentiation and reproduction but they are also deeply involved in brain homeostasis. Their action is crucial for the development of the brain, which presents different characteristics depending on the sex of individuals. The role of these players in the brain is fundamental in the maintenance of brain function during adulthood as well, thus being important also with respect to age-related neurodegenerative diseases. In this review, we explore the role of biological sex in the development of the brain and analyze its impact on the predisposition toward and the progression of neurodegenerative diseases. In particular, we focus on Parkinson's disease, a neurodegenerative disorder that has a higher incidence in the male population. We report how sex hormones and genes encoded by the sex chromosomes could protect from the disease or alternatively predispose toward its development. We finally underline the importance of considering sex when studying brain physiology and pathology in cellular and animal models in order to better understand disease etiology and develop novel tailored therapeutic strategies.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

A zebrafish HCT116 xenograft model to predict anandamide outcomes on colorectal cancer.

Colon cancer is one of the leading causes of death worldwide. In recent years, cannabinoids have been extensively studied for their potential anticancer effects and symptom management. Several in vitro studies reported anandamide's (AEA) ability to block cancer cell proliferation and migration, but evidence from in vivo studies is still lacking. Thus, in this study, the effects of AEA exposure in zebrafish embryos transplanted with HCT116 cells were evaluated. Totally, 48 hpf xenografts were exposed to 10 nM AEA, 10 nM AM251, one of the cannabinoid 1 receptor (CB1) antagonist/inverse agonists, and to AEA + AM251, to verify the specific effect of AEA treatment. AEA efficacy was evaluated by confocal microscopy, which demonstrated that these xenografts presented a smaller tumor size, reduced tumor angiogenesis, and lacked micrometastasis formation. To gain deeper evidence into AEA action, microscopic observations were completed by molecular analyses. RNA seq performed on zebrafish transcriptome reported the downregulation of genes involved in cell proliferation, angiogenesis, and the immune system. Conversely, HCT116 cell transcripts resulted not affected by AEA treatment. In vitro HCT116 culture, in fact, confirmed that AEA exposure did not affect cell proliferation and viability, thus suggesting that the reduced tumor size mainly depends on direct effects on the fish rather than on the transplanted cancer cells. AEA reduced cell proliferation and tumor angiogenesis, as suggested by socs3 and pcnp mRNAs and Vegfc protein levels, and exerted anti-inflammatory activity, as indicated by the reduction of il-11a, mhc1uba, and csf3b mRNA. Of note, are the results obtained in groups exposed to AM251, which presence nullifies AEA's beneficial effects. In conclusion, this study promotes the efficacy of AEA in personalized cancer therapy, as suggested by its ability to drive tumor growth and metastasis, and strongly supports the use of zebrafish xenograft as an emerging model platform for cancer studies.

epg5 knockout leads to the impairment of reproductive success and courtship behaviour in a zebrafish model of autophagy-related diseases.

BACKGROUND: Dysregulation of the autophagic flux is linked to a wide array of human diseases, and recent findings highlighted the central role of autophagy in reproduction, as well as an association between impairment of autophagy and behavioural disorders. Here we deepened on the possible multilevel link between impairment of the autophagic processes and reproduction at both the physiological and the behavioural level in a zebrafish mutant model. METHODS: Using a KO epg5 zebrafish line we analysed male breeding success, fertility rate, offspring survival, ejaculate quality, sperm and testes morphology, and courtship behaviour. To this aim physiological, histological, ultrastructural and behavioural analyses on epg5+/+ and mutant epg5-/- males coupled to WT females were applied. RESULTS: We observed an impairment of male reproductive performance in mutant epg5-/- males that showed a lower breeding success with a reduced mean number of eggs spawned by their WT female partners. The spermatogenesis and the ability to produce fertilising ejaculates were not drastically impaired in our mutant males, whereas we observed a reduction of their courtship behaviour that might contribute to explain their lower overall reproductive success. CONCLUSION: Collectively our findings corroborate the hypothesis of a multilevel link between the autophagic process and reproduction. Moreover, by giving a first glimpse on behavioural disorders associated to epg5 KO in model zebrafish, our results open the way to more extensive behavioural analyses, also beyond the reproductive events, that might serve as new tools for the molecular screening of autophagy-related multisystemic and neurodegenerative diseases.

Learning and visual discrimination in newly hatched zebrafish.

With the exception of humans, early cognitive development has been thoroughly investigated only in precocial species, well developed at birth and with a broad behavioral and cognitive repertoire. We investigated another highly altricial species, the zebrafish, Danio rerio, whose embryonic development is very rapid (< 72 h). The hatchlings' nervous system is poorly developed, and their cognitive capacities are largely unknown. Larvae trained at 8 days post fertilization rapidly learned to associate a visual pattern with a food reward, showing significant performance at 10 days post fertilization. We exploited this ability to study hatchlings' discrimination learning capacities. Larvae rapidly and accurately learned color and shape discriminations. They also discriminated a figure from its mirror image and from its 90°-rotated version, although with lower performance. Our study revealed impressive similarities in learning and visual discrimination capacities between newborn and adult zebrafish, despite their enormous differences in brain size and degree of development.

In vivo anti-inflammatory and antioxidant effects of microbial polysaccharides extracted from Euganean therapeutic muds.

Therapeutic thermal mud produced by spas of the Euganean Thermal District (Italy) is used as a treatment for arthro-rheumatic diseases. Its production involves the growth of a specific microbiota embedded in a polysaccharidic matrix. Polysaccharides (Microbial-PolySaccharides, M-PS) released in the mud by the resident microorganisms were extracted and analyzed. The monosaccharidic composition analysis showed the presence of galacturonic acid, mannose, xylose, ribose and glucose and a high percentage of sulfated groups in the polymers. To assess their involvement in the therapeutic efficacy of the mud, the M-PS were tested using the model organism zebrafish (Danio rerio). The anti-inflammatory and antioxidant activities were evaluated after confirming the lack of toxic effects during development. Inflammatory state was induced chemically with copper sulfate, or through tail fin amputation procedure and UVB exposure. Recovery from inflammatory condition after exposure to M-PS was always observed with specific morphometric analyses, and further supported by qPCR. Genes linked with the inflammatory and oxidative stress response were investigated confirming the M-PS treatment's efficacy.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

STW 5 Herbal Preparation Modulates Wnt3a and Claudin 1 Gene Expression in Zebrafish IBS-like Model.

AIM: Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by chronic abdominal pain and stool irregularities. STW 5 has proven clinical efficacy in functional gastrointestinal disorders, including IBS, targeting pathways that suppress inflammation and protect the mucosa. Wnt signaling is known to modulate NF-kß-dependent inflammatory cytokine production. This sparked the idea of evaluating the impact of STW 5 on the expression of inflammatory-response and Wnt/ß catenin-target genes in an IBS-like model. MAIN METHODS: We used zebrafish and dextran sodium sulfate (DSS) treatment to model IBS-like conditions in vivo and in vitro and examined the effects of subsequent STW 5 treatment on the intestines of DSS-treated fish and primary cultured intestinal and neuronal cells. Gross gut anatomy, histology, and the expression of Wnt-signaling and cytokine genes were analyzed in treated animals and/or cells, and in controls. KEY FINDINGS: DSS treatment up-regulated the expression of interleukin-8, tumor necrosis factor-α, wnt3a, and claudin-1 in explanted zebrafish gut. Subsequent STW 5 treatment abolished both the macroscopic signs of gut inflammation, DSS-induced mucosecretory phenotype, and normalized the DSS-induced upregulated expression of il10 and Wnt signaling genes, such as wnt3a and cldn1 in explanted zebrafish gut. Under inflammatory conditions, STW 5 downregulated the expression of the pro-inflammatory cytokine genes il1ß, il6, il8, and tnfα while it upregulated the expression of the anti-inflammatory genes il10 and wnt3a in enteric neuronal cells in vitro. SIGNIFICANCE: Wnt signaling could be a novel target for the anti-inflammatory and intestinal permeability-restoring effects of STW 5, possibly explaining its clinical efficacy in IBS.

Knockout of the hsd11b2 Gene Extends the Cortisol Stress Response in Both Zebrafish Larvae and Adults.

The Hsd11b2 enzyme converts cortisol into its inactive form, cortisone and regulates cortisol levels, in particular in response to stress. Taking advantage of CRISPR/Cas9 technology, we generated a hsd11b2 zebrafish mutant line to evaluate the involvement of this gene in stress response regulation. The absence of a functional Hsd11b2 affects survival of zebrafish, although homozygous hsd11b2-/- mutants can reach adulthood. Reproductive capability of hsd11b2-/- homozygous adult males is almost completely abrogated, while that of females is reduced. Interestingly, basal cortisol levels and glucocorticoid-dependent transcriptional activities are not affected by the mutation. In agreement with basal cortisol results, we also demonstrated that basal response to light (LMR-L/D) or mechanical (VSRA) stimuli is not significantly different in wild-type (hsd11b2+/+) compared to mutant larvae. However, after exposure to an acute stressor, the cortisol temporal patterns of synthesis and release are prolonged in both 5 days post fertilization larvae and one-year-old adult hsd11b2-/- zebrafish compared to wild-type siblings, showing at the same time, at 5 dpf, a higher magnitude in the stress response at 10 min post stress. All in all, this new zebrafish model represents a good tool for studying response to different stressors and to identify mechanisms that are induced by cortisol during stress response.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Gene expression in regenerating and scarring tails of lizard evidences three main key genes (wnt2b, egfl6, and arhgap28) activated during the regulated process of tail regeneration.

We have analyzed the expression of key genes orchestrating tail regeneration in lizard under normal and scarring conditions after cauterization. At 1-day post-cauterization (1 dpc), the injured blastema contains degenerating epithelial and mesenchymal cells, numerous mast cells, and immune cells. At 3 and 7 dpc, a stratified wound epidermis is forming while fibrocytes give rise to a scarring connective tissue. Oncogenes such as wnt2b, egfl6, wnt6, and mycn and the tumor suppressor arhgap28 are much more expressed than other oncogenes (hmga2, rhov, fgf8, fgfr4, tert, shh) and tumor suppressors (apcdd1, p63, rb, fat2, bcl11b) in the normal blastema and at 7 dpc. Blastemas at 3 dpc feature the lowest upregulation of most genes, likely derived from damage after cauterization. Immunomodulator genes nfatc4 and lef1 are more expressed at 7 dpc than in normal blastema and 3 dpc suggesting the induction of immune response favoring scarring. Balanced over-expression of oncogenes, tumor suppressor genes, and immune modulator genes determines regulation of cell proliferation (anti-oncogenic), of movement (anti-metastatic), and immunosuppression in the normal blastema. Significant higher expression of oncogenes wnt2b and egfl6 in normal blastema and higher expression of the tumor suppressor arhgap28 in the 7 dpc blastema indicate that they are among the key/master genes that determine the regulated regeneration of the tail.

Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish.

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr-/-). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr-/-) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr-/- females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr-/- follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr-/- follicles are likely to contribute to the reduced fertilization rate observed in mutants.

Glucocorticoid receptor activities in the zebrafish model: a review.

Glucocorticoids (GCs) are steroid hormones that contribute to the regulation of many physiological processes, such as inflammation, metabolism and stress response, mainly through binding to their cognate receptor, GR, which works as a ligand-activated transcription factor. Due to their pleiotropy and the common medical use of these steroids to treat patients affected by different pathologies, the investigation of their mechanisms of action is extremely important in biology and clinical research. The evolutionary conservation of GC physiological function, biosynthesis pathways, as well as the sequence and structure of the GC nuclear receptors has stimulated, in the last 20 years, the use of zebrafish (a teleost of Cyprinidae family) as a reliable model organism to investigate this topic. In this review, we wanted to collect many of the most significant findings obtained by the the scientific community using zebrafish to study GCs and their receptors. The paper begins by describing the experiments with transient knockdown of zebrafish gr to gain insights, mainly during development, and continues with the discoveries provided by the generation of transgenic reporter lines. Finally, we discuss how the generation of mutant lines for either gr or the enzymes involved in GC synthesis has significantly advanced our knowledge on GC biology.

Measuring recognition memory in zebrafish larvae: issues and limitations.

Recognition memory is the capacity to recognize previously encountered objects, events or places. This ability is crucial for many fitness-related activities, and it appears very early in the development of several species. In the laboratory, recognition memory is most often investigated using the novel object recognition test (NORt), which exploits the tendency of most vertebrates to explore novel objects over familiar ones. Despite that the use of larval zebrafish is rapidly increasing in research on brain, cognition and neuropathologies, it is unknown whether larvae possess recognition memory and whether the NORt can be used to assess it. Here, we tested a NOR procedure in zebrafish larvae of 7-, 14- and 21-days post-fertilization (dpf) to investigate when recognition memory first appears during ontogeny. Overall, we found that larvae explored a novel stimulus longer than a familiar one. This response was fully significant only for 14-dpf larvae. A control experiment evidenced that larvae become neophobic at 21-dpf, which may explain the poor performance at this age. The preference for the novel stimulus was also affected by the type of stimulus, being significant with tri-dimensional objects varying in shape and bi-dimensional geometrical figures but not with objects differing in colour. Further analyses suggest that lack of effect for objects with different colours was due to spontaneous preference for one colour. This study highlights the presence of recognition memory in zebrafish larvae but also revealed non-cognitive factors that may hinder the application of NORt paradigms in the early developmental stages of zebrafish.

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.