The creation of molecular tools able to unravel in vivo spatiotemporal activation of specific cell signaling events during cell migration, differentiation and morphogenesis is of great relevance to ...developmental cell biology. Here, we describe the generation, validation and applications of two transgenic reporter lines for Wnt/β-catenin signaling, named TCFsiam, and show that they are reliable and sensitive Wnt biosensors for in vivo studies. We demonstrate that these lines sensitively detect Wnt/β-catenin pathway activity in several cellular contexts, from sensory organs to cardiac valve patterning. We provide evidence that Wnt/β-catenin activity is involved in the formation and maintenance of the zebrafish CNS blood vessel network, on which sox10 neural crest-derived cells migrate and proliferate. We finally show that these transgenic lines allow for screening of Wnt signaling modifying compounds, tissue regeneration assessment as well as evaluation of potential Wnt/β-catenin genetic modulators.
► TCF responsive elements linked to a fluorescent reporter reveal Wnt/b-catenin activity in a whole zebrafish. ► Wnt reporter (7xTCFsiam) fish unveils novel tissues of wnt/b-catenin dynamic expression. ► Wnt activity can be followed in vivo in embryonic, larval and adult sensory organs and brain endothelia. ► Wnt signaling is revealed in vivo in regenerating tissues. ► Wnt reporter fish can be used both for drug screenings and wnt mutants phenotyping.
Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel ...cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na
/K
-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings
by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.
Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the ...WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo–pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke’s pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.
Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and ...lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown.
In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation.
Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.
•Zebrafish with mutations in tcf7l2 lose left-right asymmetries in habenular neurons•Tcf7l2 is expressed in both left and right-sided habenular neurons•Tcf7l2 enables neurons to respond to signals that differ between left and right
Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. This study in zebrafish by Hüsken et al. reveals that Tcf7l2 is essential for lateralized fate selection by neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.
Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The ...molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma.
Insulin signaling is essential for the maintenance of glucose homeostasis. ROCK2 has been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. However, the in vivo ...function of ROCK2 in muscle remains to be elucidated. To explore the physiological role of muscle ROCK2 in the regulation of whole-body glucose homeostasis and insulin sensitivity in vivo, mice lacking ROCK2 in skeletal muscle selectively (myogenic-Cre; ROCK2lox/lox) were studied. Here we show that muscle-specific ROCK1-deficient mice displayed insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of muscle-specific ROCK2. These effects were independent of changes in adiposity. To determine the mechanism(s) by which muscle-specific deletion of ROCK2 causes insulin resistance, we measured the ability of insulin to activate downstream distal pathways in skeletal muscle. Insulin-stimulated IRS-1 tyrosine phosphorylation in skeletal muscle was markedly reduced by ∼80% in myogenic-Cre; ROCK2lox/lox mice compared with control (ROCK2lox/lox) mice. Concurrently, impaired insulin-induced phosphorylation of Akt, AS160, GSK, and S6K was found when ROCK2 was specifically deleted in muscle. Of pathological significance, ROCK2 activity in skeletal muscle was markedly decreased in obese type 2 diabetic mice in response to insulin, along with an impairment of IRS-1 phosphorylation. These data suggest that ROCK2 deficiency results in systemic insulin resistance by impairing insulin signaling in skeletal muscle, at the level of IRS-1. Thus, our results identify ROCK2 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.
Disclosure
H. Kim: None. R.M. Pereira: None. A.G. Uner: None. H. Lee: None. Y. Kim: None.
Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of ...function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this,
mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.
The COVID-19 pandemic will likely take years to control globally, and constant epidemic surveillance will be required to limit the spread of SARS-CoV-2, especially considering the emergence of new ...variants that could hamper the effect of vaccination efforts. We developed a simple and robust –
Phone Screen Testing
(
PoST
) – method to detect SARS-CoV-2-positive individuals by RT-PCR testing of smartphone screen swab samples. We show that 81.3–100% of individuals with high-viral-load SARS-CoV-2 nasopharyngeal-positive samples also test positive for
PoST
, suggesting this method is effective in identifying COVID-19 contagious individuals. Furthermore, we successfully identified polymorphisms associated with SARS-CoV-2 Alpha, Beta, and Gamma variants, in SARS-CoV-2-positive
PoST
samples. Overall, we report that
PoST
is a new non-invasive, cost-effective, and easy-to-implement smartphone-based smart alternative for SARS-CoV-2 testing, which could help to contain COVID-19 outbreaks and identification of variants of concern in the years to come.
Hypothalamic and extra-hypothalamic neuronal populations play important roles in the regulation of energy balance and glucose homeostasis. However, the underlying mechanisms of this control remain ...unclear. We have recently reported that inhibition of hypothalamic pro-opiomelanocortin (POMC) neuronal activity leads to a reduction in blood glucose levels of normoglycemic POMC-cre mice. This effect is independent of energy intake. Since hypothalamic POMC neurons project to the paraventricular nucleus of the hypothalamus (PVH) and regulate energy metabolism via melanocortin-4 receptor (MC4R), we hypothesized that changes in the activity of MC4R-expressing neurons in the PVH could alter glycemic levels. To address this, we stereotaxically injected designer receptors exclusively activated by designer drug (DREADD) viruses into the PVH of MC4R-cre mice to chemogenetically and selectively activate or inhibit these neurons. We continuously activated or inhibited these neurons by injecting clozapine-N-oxide (CNO) every 8 hours for 5 days and monitored daily blood glucose levels and energy intake. Interestingly, inhibition of MC4R-expressing neurons in the PVH leads to a significant reduction (∼ 30%) in blood glucose levels despite of increased food intake. On the other hand, blood glucose levels are significantly increased, but food intake is greatly decreased when MC4R-expressing neurons are activated. We further determined the peripheral insulin response via MC4R-expressing neurons in the PVH. We found that CNO-induced inhibition or activation of MC4R-expressing neurons did not affect serum insulin levels. These data suggest that MC4R-expressing neurons in the PVH can regulate blood glucose levels and food intake via divergent cellular mechanisms, which is independent of circulating insulin. Thus, the inhibition of MC4R-expressing neurons may be a promising target for novel antidiabetic medicines.
Disclosure
A.G. Uner: None. H. Lee: None. J. Han: None. R.M. Pereira: None. X. Sun: None. H. Kim: None. Y. Kim: None.
Tcf7l2 mediates Wnt/β-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/β-Catenin signalling elicit such a diversity of ...biological outcomes are poorly understood. Here, we study the function of zebrafish
alternative splice variants and show that only variants that include exon five or an analogous human
variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking
function. Knockdown of exon five specific
variants in
mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of
can influence the transcriptional output of the Wnt pathway.
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