Myogenic regulatory factors of the myod family (MRFs) are transcription factors essential for mammalian skeletal myogenesis. Here we show that a mutation in the zebrafish myod gene delays and reduces ...early somitic and pectoral fin myogenesis, reduces miR-206 expression, and leads to a persistent reduction in somite size until at least the independent feeding stage. A mutation in myog, encoding a second MRF, has little obvious phenotype at early stages, but exacerbates the loss of somitic muscle caused by lack of Myod. Mutation of both myod and myf5 ablates all skeletal muscle. Haploinsufficiency of myod leads to reduced embryonic somite muscle bulk. Lack of Myod causes a severe reduction in cranial musculature, ablating most muscles including the protractor pectoralis, a putative cucullaris homologue. This phenotype is accompanied by a severe dysmorphology of the cartilaginous skeleton and failure of maturation of several cranial bones, including the opercle. As myod expression is restricted to myogenic cells, the data show that myogenesis is essential for proper skeletogenesis in the head.
►Myod is a transcription factor that controls skeletal myogenesis. ►Myod is essential for most cranial myogenesis and viability in zebrafish. ►Lack of cranial muscle leads to cartilage and bone maturation defects in the head. ►Myod and Myf5 cooperate to drive all embryonic and larval myogenesis.
The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases ...including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (
Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene,
claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish
cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore,
cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus,
cldn15lb mutants provide a novel
in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.
► A novel gene,
claudin 15-like b, is identified in zebrafish and has 2 isoforms. ► Cldn15lb is dynamically expressed in hepatocytes and biliary epithelial cells. ► Disruption of
cldn15lb affects hepatocyte polarization and canaliculi formation. ► Biliary ducts in
cldn15lb mutants do not remodel and form a disorganized network. ► Cldn15lb regulates cell behaviors during biliary duct morphogenesis.
ABSTRACT
Disc1 is a schizophrenia risk gene that engages multiple signaling pathways during neurogenesis and brain development. Using the zebrafish as a tool, we analyze the function of zebrafish ...Disc1 (zDisc1) at the earliest stages of brain and body development. We define a “tool” as a biological system that gives insight into mechanisms underlying a human disorder, although the system does not phenocopy the disorder. A zDisc1 peptide binds to GSK3β, and zDisc1 directs early brain development and neurogenesis, by promoting β‐catenin‐mediated Wnt signaling and inhibiting GSK3β activity. zDisc1 loss‐of‐function embryos additionally display a convergence and extension phenotype, demonstrated by abnormal movement of dorsolateral cells during gastrulation, through changes in gene expression, and later through formation of abnormal, U‐shaped muscle segments, and a truncated tail. These phenotypes are caused by alterations in the noncanonical Wnt pathway, via Daam and Rho signaling. The convergence and extension phenotype can be rescued by a dominant negative GSK3β construct, suggesting that zDisc1 inhibits GSK3β activity during noncanonical Wnt signaling. This is the first demonstration that Disc1 modulates the noncanonical Wnt pathway and suggests a previously unconsidered mechanism by which Disc1 may contribute to the etiology of neuropsychiatric disorders.—De Rienzo, G., Bishop, J. A., Mao, Y., Pan, L., Ma, T. P., Moens, C. B., Tsai, L. H., Sive, H. Disc1 regulates both β‐catenin‐mediated and noncanonical Wnt signaling during vertebrate embryogenesis. FASEB J. 25, 4184–4197 (2011). www.fasebj.org
Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we ...characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis.
Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the “unfolded protein ...response” (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases.
Studies of the zebrafish epithalamus have provided recent insights into the development of left-right brain asymmetry, which is crucial to normal human brain function. The habenular nuclei of ...zebrafish are robustly asymmetric, with dense elaboration of neuropil only in the left lateral subnucleus. Because this feature is tightly correlated with asymmetric expression of K(+) channel tetramerization domain-containing proteins 12.1 and 12.2 (Kctd12.1/12.2), we screened for Kctd12.1-interacting proteins to identify molecular mechanisms leading to neuropil asymmetry, and uncovered a novel interaction between Kctd12.1 and Unc-51-like kinase 2 (Ulk2). We show here that knockdown of Ulk2 or overexpression of Kctd12 proteins reduces asymmetric neuropil elaboration. Conversely, overexpression of Ulk2 or mutation of kctd12 genes causes excess neuropil elaboration. We conclude that Ulk2 activity promotes neuropil elaboration while Kctd12 proteins limit Ulk2 activity asymmetrically. This work describes a regulatory mechanism for neuronal process extension that may be conserved in other developmental contexts in addition to the epithalamus.
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading ...pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
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•Small molecules LDC7559 and NA-11 inhibit the phagocytic oxidative burst•NA-11 dampens flux through the pentose phosphate pathway to limit cellular NADPH•Cryo-EM confirms agonism of PFKL through binding at the AMP/ADP allosteric pocket•PFKL is a regulatory node for NOX2-dependent NETosis and tissue damage
The small molecule LDC7559 and its more potent analog, NA-11, suppress excessive NOX2-dependent oxidative burst and NETosis, as well as subsequent tissue damage and inflammation, without compromising basal ROS production. They selectively activate the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) to suppress glycolytic flux through the pentose phosphate pathway that leads to NOX2-dependent outputs.
Reverse genetics in zebrafish by TILLING Moens, Cecilia B; Donn, Thomas M; Wolf-Saxon, Emma R ...
Briefings in functional genomics & proteomics,
11/2008, Volume:
7, Issue:
6
Journal Article
Open access
TILLING, for Targeting Induced Local Lesions in Genomes, is a reverse genetics strategy that identifies mutations in specific genes of interest in chemically mutagenized populations. First described ...in 2000 for mutation detection in Arabidopsis, TILLING is now used in a wide range of plants including soybean, rice, barley and maize as well as for animal model systems, including Arabidopsis, Drosophila, Caenorhabditis elegans, rat, medaka and zebrafish and for the discovery of naturally occurring polymorphisms in humans. This review summarizes current TILLING methodologies as they have been applied to the zebrafish, ongoing TILLING projects and resources in the zebrafish community, and the future of zebrafish TILLING.
The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug ...activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.
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