Biology subjects

den Hertog, J.

Publications and source records attributed to den Hertog, J..

10 recordsLinked to original sources

bioRxiv2026

K-Ras controls asymmetric cell divisions from the primary cilium

The Ras-MAPK pathway drives central cellular processes, including cell proliferation and differentiation. How exactly Ras controls differentiation is however not understood. Supported by mathematical modelling and single-cell RNA sequencing we show that K-Ras4B sustains ciliation during differentiation thus restricting commitment of skeletal muscle stem and progenitor cells during asymmetric cell divisions. Modulation of K-Ras4B abundance or expression of oncogenic K-Ras4B-G12C perturb normal differentiation. K-Ras4B, but not N-Ras and H-Ras, localizes to the primary cilium and its abundance there depends on the ciliary trafficking chaperone PDE6D. The presence of B-Raf and active MEK at the base of and active ERK inside the cilium suggests that K-Ras4B is active there. Conditions that localize a K-Ras4B mutant only to the cilium are sufficient to sustain ciliation and normal differentiation. Finally, in vivo modulation of K-Ras4B activity during zebrafish embryogenesis perturbs ciliation-dependent heart-looping. Our results thus imply a novel fundamental role of K-Ras4B in controlling ciliation and differentiation and suggest an explanation for the phenotypic similarities between RASopathies and ciliopathies.

cell biology↗

bioRxiv2025

The protein-tyrosine phosphatase Shp2 is essential for lymphatic endothelial cell differentiation in zebrafish

Mutations in genes associated with the VEGFC/VEGFR3 signaling pathway are strongly linked to a range of developmental and functional defects in the lymphatic system. PTPN11 encodes SHP2, a protein-tyrosine phosphatase that plays a critical role in receptor protein-tyrosine kinase (RTK) signaling. In this study, we investigated lymphangiogenesis in mutant zebrafish embryos lacking functional Shp2 and demonstrate that Shp2 is essential for normal lymphatic development in the trunk and head regions of zebrafish embryos. Embryos lacking functional Shp2 failed to develop lymphatic vessels and lymphatic endothelial cells (LECs), whereas venous intersegmental vessel (vISV) formation remained unaffected. While endothelial cells sprouted normally from the posterior cardinal vein (PCV), they did not migrate to the horizontal myoseptum to form parachordal lymphangioblast (PL) cells. Rescue experiments using Shp2 mutants demonstrated that catalytic activity and signaling properties of Shp2 are required for restoration of normal lymphangiogenesis. These findings highlight a pivotal role for Shp2 signaling in the migration and differentiation of lymphatic endothelial cells without affecting venous angiogenesis.

developmental biology↗

bioRxiv2023

Establishment and characterization of a new Pseudomonas aeruginosa infection model using 2D airway organoids and dual RNA sequencing

Pseudomonas aeruginosa is a Gram-negative bacterium that is notorious for infections in the airway of cystic fibrosis (CF) subjects. Often, these infections become chronic, leading to higher morbidity and mortality rates. Bacterial quorum sensing (QS) coordinates the expression of virulence factors and the formation of biofilms at a population level. QS has become the focus of attention for development of alternatives to antimicrobials targeting P. aeruginosa infections. However, a better understanding of the bacteria-host interaction, and the role of QS in infection, is required. In this study, we set up a new P. aeruginosa infection model, using 2D airway organoids derived from healthy and CF individuals. Using dual RNA-sequencing, we dissected their interaction, focusing on the role of QS. As expected, P. aeruginosa induced epithelial inflammation. However, QS signaling did not affect the epithelial airway cells. The epithelium influenced several infection-related processes of P. aeruginosa, including metabolic changes, induction of type 3 and type 6 secretion systems (T3SS and T6SS), and increased expression of antibiotic resistance genes, including mexXY efflux pump and several porins. Interestingly, the epithelium influenced the regulation by QS of the type 2 (T2SS) and T6SS. Finally, we compared our model with in vivo P. aeruginosa transcriptomic datasets, from samples directly isolated from the airways of CF subjects. This shows that our model recapitulates important aspects of in vivo infection, like enhanced denitrification, betaine/choline metabolism, increased antibiotic resistance, as well as an overall decrease of motility-related genes. This relevant infection model is interesting for future investigations, helping to reduce the burden of P. aeruginosa infections in CF.

microbiology↗

bioRxiv2022

Paecilomycone inhibits quorum sensing in Gram-negative bacteria

Pseudomonas aeruginosa is an opportunistic pathogen that causes major healthcare concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are highly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as inhibitor of QS in both C. violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4- dihydroxy-2-heptylquinoline (PQS), but not 2-aminoacetophenone (2-AA). We suggest that paecilomycone affects QS in P. aeruginosa by targeting the PqsBC complex and alternative targets, or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone towards eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. ImportanceAntibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major healthcare concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, which inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone towards eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.

microbiology↗

bioRxiv2022

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae

PTPN6 encodes SHP1, a protein tyrosine phosphatase that has an essential role in immune cell function. SHP1 missense and splice site mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in mice that lack functional SHP1 partially. Complete lack of SHP1 function in mouse motheaten mutants leads to severe defects and lethality at 2-6 weeks after birth. Here, we investigated the function of Shp1 in developing zebrafish embryos. We generated a ptpn6 knockout zebrafish line lacking functional Shp1. Zebrafish develop ex utero and are transparent, which facilitates analysis of the function of Shp1 during early hematopoietic development. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development the myeloid lineage was affected, which resulted in a decrease in the number of neutrophils, and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, directional migration of neutrophils and macrophages was decreased in response to wounding and less macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impairs neutrophil and macrophage function and caused severe inflammation and lethality at the larval stage.

developmental biology↗

bioRxiv2021

Harzianic Acid has multi-target antimicrobial activity against Gram-Positive Bacteria

The thermophilic fungus Oidiodendron flavum is a saprobe that is commonly isolated from soil. Here, we identified a Gram-positive bacteria-selective antimicrobial secondary metabolite from this fungal species, harzianic acid (HA). Using Bacillus subtilis strain 168 combined with several assays, we found that HA targeted the cell membrane, though only at high concentrations. To further study the antimicrobial activity of HA, we isolated an HA-resistant strain, Bacillus subtilis strain M9015, and discovered that the mutant has more translucent colonies, has cross resistance to rifampin, and harbors five mutations in the coding region of four distinct genes. Further analysis of these genes indicated that the mutation in atpE might be responsible for the translucency of the colony, and mutation in yusO for resistance to both HA and rifampin. We conclude that HA is a multi-target antimicrobial agent against Gram-positive bacteria.

microbiology↗

bioRxiv2021

Classification of antimicrobial mechanism of action using bacterial time-lapse imaging

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. Using this method, we found that the poorly studied antimicrobial, harzianic acid, a secondary metabolite that we purified from the fungal culture of Oidiodendron flavum, targets the cell envelope. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

microbiology↗

bioRxiv2020

Inflammation potentiates JMML-like blood defects in Shp2 mutant Noonan syndrome

Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with Shp2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies and in vivo transplantability of HSPCs. Single cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies.

developmental biology↗

bioRxiv2020

Phosphatidylinositol-3 kinase activity controls survival and stemness of zebrafish hematopoietic stem/progenitor cells

Hematopoietic Stem and Progenitor Cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multi-potent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs. 2 Key pointsO_LILoss of Pten and inhibition of PI3K induced apoptosis of hematopoietic stem/progenitor cells upon endothelial to hematopoietic transition C_LIO_LISurviving hematopoietic stem/progenitor cells committed to all blood lineages but displayed reduced stemness C_LI

developmental biology↗

bioRxiv2020

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish

Zebrafish models are well established tools for investigating underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein (BMP) type I receptor kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin induced by cercosporamide was strikingly similar as the phenotypes caused by renowned small molecule BMP type I receptor kinase inhibitors and inactivating mutations in zebrafish BMP receptors. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of BMP type I receptors (also called activin receptor-like kinases (ALKs)). In mammalian cells, cercosporamide selectively inhibited constitutively active BMP type I receptor-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. Taken together, we believe cercosporamide may be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMP receptor signaling, including Fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.View Full Text

pharmacology and toxicology↗