Francesco Argenton - Academia.edu (original) (raw)

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Papers by Francesco Argenton

Molecular and cellular biology, 1996

The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is impli... more The E2A protein is a mammalian transcription factor of the helix-loop-helix family which is implicated in cell-specific gene expression in several cell lineages. Mouse E2A contains two independent transcription activation domains, ADI and ADII; whereas ADI functions effectively in a variety of cultured cell lines, ADII shows preferential activity in pancreatic beta cells. To analyze this preferential activity in an in vivo setting, we adapted a system involving transient gene expression in microinjected zebra fish embryos. Fertilized one- to four-cell embryos were coinjected with an expression plasmid and a reporter plasmid. The expression plasmids used encode the yeast Gal4 DNA-binding domain (DBD) alone, or Gal4 DBD fused to ADI, ADII, or VP16. The reporter plasmid includes the luciferase gene linked to a promoter containing repeats of UASg, the Gal4-binding site. Embryo extracts prepared 24 h after injection showed significant luciferase activity in response to each of the three ...

Disease models & mechanisms, 2014

Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tum... more Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRAS(G12D) during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRAS(G12D) expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRAS(G12D) line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor dev...

Cell Reports, 2013

Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeost... more Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis.

Cardiovascular Research, 2014

Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves de... more Valvular heart disease is responsible for considerable morbidity and mortality. Cardiac valves develop as the heart contracts, and they function throughout the lifetime of the organism to prevent retrograde blood flow. Their precise morphogenesis is crucial for cardiac function. Zebrafish is an ideal model to investigate cardiac valve development as it allows these studies to be carried out in vivo through non-invasive imaging. Accumulating evidence suggests a role for contractility and intracardiac flow dynamics in cardiac valve development. However, these two factors have proved difficult to uncouple, especially since altering myocardial function affects the intracardiac flow pattern. Here, we describe novel zebrafish models of developmental valve defects. We identified two mutant alleles of myosin heavy chain 6 that can be raised to adulthood despite having only one functional chamber-the ventricle. The adult mutant ventricle undergoes remodelling, and the atrioventricular (AV) valves fail to form four cuspids. In parallel, we characterized a novel mutant allele of southpaw, a nodal-related gene involved in the establishment of left-right asymmetry, which exhibits randomized heart and endoderm positioning. We first observed that in southpaw mutants the relative position of the two cardiac chambers is altered, affecting the geometry of the heart, while myocardial function appears unaffected. Mutant hearts that loop properly or exhibit situs inversus develop normally, whereas midline, unlooped hearts exhibit defects in their transvalvular flow pattern during AV valve development as well as defects in valve morphogenesis. Our data indicate that intracardiac flow dynamics regulate valve morphogenesis independently of myocardial contractility.

Development, 2014

Canonical β-catenin-dependent Wnt signal transduction is important for several biological phenome... more Canonical β-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of β-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating β-catenin nuclear localization are poorly understood. We show that Simplet/ Fam53B (Smp) is required for Wnt signaling by positively regulating β-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two β-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of β-catenin. Conversely, overexpression of smp increases β-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the β-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for β-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating β-catenin nuclear localization: the protein colocalizes with β-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces β-catenin nuclear localization and transcriptional activity; human SMP mediates β-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human β-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of β-catenindependent Wnt signal transduction.

International Journal of Developmental Biology, 2007

Spata2 (spermatogenesis-associated protein 2) was originally described as a novel gene involved i... more Spata2 (spermatogenesis-associated protein 2) was originally described as a novel gene involved in the spermatogenic process. In this study, we cloned a potential zebrafish spata2 orthologue. The consensus open reading frame (1650 bp) encodes a polypeptide of 550 amino acids which shares 37% identity with the human SPATA2. Bioinformatic analysis reveals a small pattern PW [KR] KE [YF][RK] which seems to be of particular interest in the light of its strong conservation between SPATA2 and the recently discovered TAMO protein of D. melanogaster. RT-PCR analysis in adult zebrafish tissues revealed that spata2 mRNA has a broad distribution. Whole-mount in situ hybridization demonstrated that spata2 transcripts are maternally derived and becomes strongly localized in the central nervous system at early developmental stages. From 5 dpf, spata2 expression becomes detectable in the gut and pronephric duct epithelium, suggesting a wide tissue function during vertebrate development.

ABSTRACT Introduction: Human AF9 (alias MLLT3) gene was initially identified as one of the most c... more ABSTRACT Introduction: Human AF9 (alias MLLT3) gene was initially identified as one of the most common translocation partners of MLL associated with acute myeloid leukemia. More recently it has been demonstrated that AF9 can be a positive regulator of early erythroid/megakaryocytic lineage decisions. However, many biological functions of AF9 and its role in leukemia remain unclear. Here, we aimed to identify the zebrafish homologue of human AF9 and to analyze its function using the zebrafish model system. Methods and Results: A single zebrafish af9 gene was identified by homology searching at zebrafish genome database (www.ncbi.nlm.nih.gov/). The putative af9 protein showed 60% identity and 73% similarity to human counterpart. These results from the perspective of phylogenetic and synteny analyses indicated that zebrafish af9 was the likely homologue of human AF9. We used whole-mount in situ hybridization to analyzed its spatial and temporal expression patterns during embryonic development. Af9 was expressed specifically in the posterior-lateral mesoderm at the 5-somite stage and in the intermediate cell mass (ICM, site of primitive hematopoiesis) between 18 to 24 hours post fertilization (hpf). In order to characterize the role of af9 in early hematopoietic development, we performed over-expression and loss-of- function experiments in zebrafish embryos, and then analyzed various hematopoietic markers by reverse transcriptase quantitative PCR (RT-qPCR). Inducing ectopic af9 expression by mRNA injection we found an increased expression of gata1 and hbae1 (erythroid markers) and reduced expression of runx1, scl, lmo2, c-myb (stem cell/precursor associated markers) as well as pu.1 and l-plastin (myeloid markers) at 22 hpf-old embryos. Conversely af9 loss-of-function obtained using anti-sense morpholinos showed decreased expression of gata1 and hbae1 mRNA, while pu.1 and l-plastin increased their expression in morphant embryos at 22 hpf. The same morphants did not show any appreciable change in stem cell/precursor marker expression. Conclusion: The finding that gata1 expression is controlled by af9 suggests that this gene might promote the erythroid development in zebrafish primitive hematopoiesis. As the genetic programs of AF9 are evolutionary conserved between human and zebrafish, these findings provide new insights and opening further

A recent theory on the evolution of sexuality, has hypothesized heritable variation in the functi... more A recent theory on the evolution of sexuality, has hypothesized heritable variation in the functional properties of centrosomes, leading to competition for the organization of the mitotic spindle when different centrosomes enter a common cytoplasm. We present here data on polyethylene glycol-induced polykaryocytes of cultured Chinese hamster fibroblasts indirectly supporting centrosome competition. On the assumption that sib centrosomes are similar and variation increases with cell generations, the frequencies of multipolar mitoses were compared in cultures fused under conditions favoring sib cell fusion or fusion of distantly related cells. Multipolar mitoses were considerably more frequent in the former, when the average difference between pairs of centrosomes was assumed to be too small for one centrosome to "outcompete" the other.

Annals of The New York Academy of Sciences, 2005

This study describes the developmental expression of zYb and zYc receptors in zebrafish. RT-PCR d... more This study describes the developmental expression of zYb and zYc receptors in zebrafish. RT-PCR demonstrated that both mRNAs are present from 24-hour postfertilization (hpf) and that their expression increased during larval development. Whole-mount in situ hybridization showed zYb mRNA expression in the epithalamus (24 hpf), telencephalon, hypothalamus, rhombencephalon (24-96 hpf), mesencephalon (48-96 hpf), hatching gland, and otic vesicle (48-64 hpf).

Biochemical Journal, 2007

PknL, a eukaryotic like serine threonine protein kinase from Mycobacterium tuberculosis, is predi... more PknL, a eukaryotic like serine threonine protein kinase from Mycobacterium tuberculosis, is predicted to be involved in transcriptional regulation and cell division. Attempts to clone and over-express the protein in Escherichia coli using pET43.1c as the vector were unsuccessful. The fusion protein was expressed as a truncated product and showed feeble autokinase activity. To overcome this technical glitch, the pknL ORF was cloned into a mycobacterial expression vector, pALACE and the purified His-tagged protein was evaluated for autokinase activity. Phosphorylation experiments with exogenous substrates like myelin basic protein (MBP) were performed. For the fast identification of protein phosphorylation sites, chromatographic methods of separating the [c-32 P]phosphate radio labeled amino acids using thin-layer chromatography (TLC) on cellulose sheets was carried out. Thus, the activity of PknL was demonstrated using autophosphorylation and substrate phosphorylation experiments. Phospho amino acid determinations revealed that PknL was phosphorylated predominantly on serine and also on threonine residues. A single amino acid substitution of lysine to methionine in the active site completely abolished enzymatic action, thereby confirming the authenticity of the kinase function of the expressed protein.