Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we ...established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (gr
). Homozygous gr
larvae are morphologically inconspicuous and, in contrast to GR
knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr
are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr
line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.
One of the biggest challenges in tumour research is the possibility to reprogram cancer cells towards less aggressive phenotypes. In this study, we reprogrammed primary Glioblastoma multiforme ...(GBM)-derived cells towards a more differentiated and less oncogenic phenotype by activating the Wnt pathway in a hypoxic microenvironment. Hypoxia usually correlates with malignant behaviours in cancer cells, but it has been recently involved, together with Wnt signalling, in the differentiation of embryonic and neural stem cells. Here, we demonstrate that treatment with Wnt ligands, or overexpression of β-catenin, mediate neuronal differentiation and halt proliferation in primary GBM cells. An hypoxic environment cooperates with Wnt-induced differentiation, in line with our finding that hypoxia inducible factor-1α (HIF-1α) is instrumental and required to sustain the expression of β-catenin transcriptional partners TCF-1 and LEF-1. In addition, we also found that Wnt-induced GBM cell differentiation inhibits Notch signalling, and thus gain of Wnt and loss of Notch cooperate in the activation of a pro-neuronal differentiation program. Intriguingly, the GBM sub-population enriched of cancer stem cells (CD133(+) fraction) is the primary target of the pro-differentiating effects mediated by the crosstalk between HIF-1α, Wnt, and Notch signalling. By using zebrafish transgenics and mutants as model systems to visualize and manipulate in vivo the Wnt pathway, we confirm that Wnt pathway activation is able to promote neuronal differentiation and inhibit Notch signalling of primary human GBM cells also in this in vivo set-up. In conclusion, these findings shed light on an unsuspected crosstalk between hypoxia, Wnt and Notch signalling in GBM, and suggest the potential to manipulate these microenvironmental signals to blunt GBM malignancy.
Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening ...of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.
Oxygen is a central molecule in the development of multicellular life, allowing efficient energy generation. Inadequate oxygen supply requires rapid adaptations to prevent cellular damage and the ...hypoxia-inducible factor (HIF) pathway plays a central role in this adaptation. Numerous diseases and disease processes are influenced by hypoxia and the HIF pathway. One component, von Hippel Lindau (VHL), is a well-known tumor suppressor, which acts at least in part via regulating HIF signaling. The zebrafish has become a central vertebrate model organism in which developmental and disease processes can be studied. In this review, we have tried to bring together knowledge on the HIF/hypoxic signaling pathway in zebrafish, including what is known on VHL functions.
To begin to understand pancreas development and the control of endocrine lineage formation in zebrafish, we have examined the expression pattern of several genes shown to act in vertebrate pancreatic ...development: pdx-1, insulin (W. M. Milewski et al., 1998, Endocrinology 139, 1440–1449), glucagon, somatostatin (F. Argenton et al., 1999, Mech. Dev. 87, 217–221), islet-1 (Korzh et al., 1993, Development 118, 417–425), nkx2.2 (Barth and Wilson, 1995, Development 121, 1755–1768), and pax6.2 (Nornes et al., 1998, Mech. Dev. 77, 185–196). To determine the spatial relationship between the exocrine and the endocrine compartments, we have cloned the zebrafish trypsin gene, a digestive enzyme expressed in differentiated pancreatic exocrine cells. We found expression of all these genes in the developing pancreas throughout organogenesis. Endocrine cells first appear in a scattered fashion in two bilateral rows close to the midline during mid-somitogenesis and converge during late-somitogenesis to form a single islet dorsal to the nascent duodenum. We have examined development of the endocrine lineage in a number of previously described zebrafish mutations. Deletion of chordamesoderm in floating head (Xnot homolog) mutants reduces islet formation to small remnants, but does not delete the pancreas, indicating that notochord is involved in proper pancreas development, but not required for differentiation of pancreatic cell fates. In the absence of knypek gene function, which is involved in convergence movements, the bilateral endocrine primordia do not merge. Presence of trunk paraxial mesoderm also appears to be instrumental for convergence since the bilateral endocrine primordia do not merge in spadetail mutants. We discuss our findings on zebrafish pancreatogenesis in the light of evolution of the pancreas in chordates.
The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive ...enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of
Delta and
Jagged/
Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different
delta and
jagged genes. Specifically,
deltaA knock-down embryos lack alpha cells, similarly to
mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely,
jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of
jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.
Objective: Arrhythmogenic cardiomyopathy (AC) is a heritable form of cardiomyopathy characterized by fibrofatty replacement which leads to right ventricular failure, arrhythmias, and sudden cardiac ...death, particularly in young patients and athletes. In spite of the recent discovery of genes whose mutations cause ACs. early molecular events leading to cell death and arrhythmias remain elusive. In the present study, we evaluate in vivo the pathogenic mechanisms of Desmoplakin (DSP) dysfunction, linked to the AC8 form, using zebrafish as a promising model for this life-threatening arrhythmic disorder. Purpose: The aim of the study is the generation of transient ACS zebrafish models, using an antisense knock-down strategy, and a stable zebrafish Dsp mutant, for subsequent structural and functional characterization. In addition, by exploiting zebrafish pathway reporter lines, we aim to study cell signaling alterations potentially involved in AC8 pathogenesis. The final goal is the assessment of our zebrafish AC8 models as a suitable tool for pathway-directed drug screening. Methods: A morpholino (MO)-based antisense strategy was used to obtain the knockdown of zebrafish dspa and dspb genes, both orthologous to human DSP. Moreover, we have analyzed a zebrafish Desmoplakin a (dspa) mutant line (sal 3356). obtained by ENU-induced mutagenesis. AC8 zebrafish models were morphologically characterized and. subsequently, functionally tested for alterations in different signaling pathways. Results: Knock-down of both dspa and dspb and homozygous dspa -/- mutant embryos show a general delay in the development, microcephaly, bradycardia, arrhythmias, pericardial effusion and altered heart rate. During early adulthood, dspa mutant fish exhibit mild bradycardia, cardiomegaly and peripheral effusion. TEM analysis of zebrafish tissues shows highly reduced and disorganized desmosomes, resembling "pale" desmosomes identified in endomyocardial biopsies from AC patients. Moreover, the analysis of signaling pathways detects a cardiac-specific reduction of Wnt signaling responsiveness in all AC8 models, confirming previous evidences that DSP suppression leads to a reduction of canonical Wnt signaling. Conclusions: Our AC8 transient and stable zebrafish models are able to recapitulate some AC features, pointing to zebrafish as a suitable model for the in vivo screening of molecularly-targeted drugs. Moreover, confirmation of the reduction in canonical Wnt signaling due to DSP mutations suggests that this pathway could be a general mechanism involved in the pathogenesis of desmosomal-associated AC forms, and, thus, a promising target for AC therapeutic intervention.
We have characterized and mapped the zebrafish
ptf1a gene, analyzed its embryonic expression, and studied its role in pancreas development. In situ hybridization experiments show that from the ...12-somite stage to 48 hpf,
ptf1a is dynamically expressed in the spinal cord, hindbrain, cerebellum, retina, and pancreas of zebrafish embryos. Within the endoderm,
ptf1a is initially expressed at 32 hpf in the ventral portion of the
pdx1 expression domain;
ptf1a is expressed in a subset of cells located on the left side of the embryo posteriorly to the liver primordium and anteriorly to the endocrine islet that arises from the posterodorsal pancreatic anlage. Then the
ptf1a expression domain buds giving rise to the anteroventral pancreatic anlage that grows posteriorly to eventually engulf the endocrine islet. By 72 hpf,
ptf1a continues to be expressed in the exocrine compartment derived from the anteroventral anlage. Morpholino-induced
ptf1a loss of function suppresses the expression of the exocrine markers, while the endocrine markers in the islet are unaffected. In
mind bomb (
mib) mutants, in which
delta-mediated
notch signalling is defective Dev. Cell 4 (2003) 67,
ptf1a is normally expressed. In addition, the
slow-muscle-omitted (
smu) mutants that lack expression of endocrine markers because of a defective
hedgehog signalling Curr. Biol. 11(2001) 1358 exhibit normal levels of
ptf1a. This indicates that
hedgehog signaling plays a different genetic role in the specification of the anteroventral (mostly exocrine) and posterodorsal (endocrine) pancreatic anlagen.
PDF
