Hendrik Korswagen

Hubrecht Institute

High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of Caenorhabditis elegans research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hand...

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that contr...

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that contr...

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that contr...

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that contr...

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that contr...

Polyploid cells contain more than 2 copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either 1 nucleus (mononucleation) or 2 or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different types of p...

RNA tomography or tomo-seq combines mRNA sequencing and cryo-sectioning to spatially resolve gene expression. We have adapted this method for the nematode Caenorhabditis elegans to generate anteroposterior gene expression maps at near-cellular resolution. Here, we provide a detailed overview of the method and present two approaches: one that includ...

Significance Cell migration is a central process in development, homeostasis, and disease. Among the extracellular signaling molecules that control this process are members of the Wnt family of secreted glycoproteins, which trigger distinct canonical and noncanonical pathways in responding cells. Studies in vertebrates have shown that these pathway...

Polyploid cells contain more than two copies of the genome and are found in many plant and animal tissues. Different types of polyploidy exist, in which the genome is confined to either one nucleus (mononucleation) or two or more nuclei (multinucleation). Despite the widespread occurrence of polyploidy, the functional significance of different type...

Divergence of gene function and expression during development can give rise to phenotypic differences at the level of cells, tissues, organs, and ultimately whole organisms. To gain insights into the evolution of gene expression and novel genes at spatial resolution, we compared the spatially-resolved transcriptomes of two distantly related nematod...

Cellular behaviors such as migration, division, and differentiation rely on precise timing, and yet the molecular events that govern these behaviors are highly stochastic. We investigate regulatory strategies that decrease the timing noise of molecular events. Autoregulatory feedback increases noise. Yet we find that in the presence of regulation b...

While measurement advances now allow extensive surveys of gene activity (large numbers of genes across many samples), interpretation of these data is often confounded by noise — expression counts can differ strongly across samples due to variation of both biological and experimental origin. Complimentary to perturbation approaches, we extract funct...

Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans, combined with an extensive genetic toolbox and the...

Cellular behaviors such as migration, division, and differentiation rely on precise timing, and yet the molecular events that govern these behaviors are highly stochastic. We investigate regulatory strategies that decrease the timing noise of molecular events. Autoregulatory feedback increases noise. Yet, we find that in the presence of regulation...

Directional migration of neurons and neuronal precursor cells is a central process in nervous system development. In the nematode Caenorhabditis elegans, the two Q neuroblasts polarize and migrate in opposite directions along the anteroposterior body axis. Several key regulators of Q cell polarization have been identified, including MIG-21, DPY-19/...

Appropriate Wnt morphogen secretion is required to control animal development and homeostasis. Although correct Wnt globular structure is essential for secretion, proteins that directly mediate Wnt folding and maturation remain uncharacterized. Here, we report that protein disulfide isomerase-1 (PDI-1), a protein-folding catalyst and chaperone, con...

Van Gogh-like (Vangl) and Prickle (Pk) are core components of the non-canonical Wnt planar cell polarity pathway that controls epithelial polarity and cell migration. Studies in vertebrate model systems have suggested that Vangl and Pk may also inhibit signaling through the canonical Wnt/β-catenin pathway, but the functional significance of this po...

Expression of mab-5/Hox mRNA is lost in the QL lineage in sfrp-1/SFRP; vang-1/Vangl; bar-1/β-catenin triple mutants. Displayed are the mab-5 mRNA counts in QL (after completion of initial migration) and QL.p. (TIF)

Expression of endogenous dlg-1::mNG [41]. DLG-1 is expressed in the seam (V) cells, but no expression is visible in the QL neuroblast (position marked by arrow). Scale bar = 10 μm. (TIF)

Live confocal imaging of sfrp-1(gk554); vang-1(tm1422) double mutants. Conditions as in S1 Movie. Note that QR.p stays in the posterior. (AVI)

Generation of Vangl1 and Vangl2 double mutant HEK293 cells. (A) Sequences of the guide RNAs and the mutations in the double mutant (DKO) cell line. (B) Western blot analysis of Vangl1 and Vangl2 expression in wild type and Vangl1/2 DKO cells. (C) Western blot analysis of Vangl1 and Vangl2 expression in control, Vangl1/2 DKO and DKO cells expressing...

Live time-lapse confocal imaging of wild type animals expressing plasma membrane and nuclear localized GFP in QR, QR.a (anterior daughter cell), QR.p (posterior daughter cell) and the seam (V) cells (transgene heIs63). Anterior is left. Images were acquired every 15 minutes for a total duration of 7 hours. Movies are played at 2 frames per second....

vang-1/Vangl and prkl-1/Pk are expressed in both the QL and QR lineage and show opposite expression dynamics during initial polarization and migration. Transcription dynamics of vang-1 (left panels) and prkl-1 (right panels) as quantified in QL (grey), QL.p (red), QR (blue) and QR.p (green), n>35 for both genes. The number of mRNA spots per cell is...

vang-1/Vangl acts upstream of bar-1/β-catenin and pop-1/TCF, and loss of prkl-1/Pk and cam-1/Ror do not affect canonical Wnt signaling. (A-D) Final positions of the QL.pax (A, B) and the QR.pax (C, D) with respect to the seam cells V1.a to V6.p (lower brackets indicate Vn.a (left) and Vn.p (right) daughters of Vn cells). Values listed are the cumul...

Numerical data of graphs presented in main and supplementary figures. (XLSX)

To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used spatial transcriptomics to generate high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, we...

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report th...

To advance our understanding of the genetic programs that drive cell and tissue specialization, it is necessary to obtain a comprehensive overview of gene expression patterns. Here, we have used RNA tomography to generate the first high-resolution, anteroposterior gene expression maps of C. elegans males and hermaphrodites. To explore these maps, w...

Important cellular processes such as migration, differentiation, and development often rely on precise timing. Yet, the molecular machinery that regulates timing is inherently noisy. How do cells achieve precise timing with noisy components? We investigate this question using a first-passage-time approach, for an event triggered by a molecule that...

Invertebrate and vertebrate nervous systems generate different types of dopaminergic neurons in distinct parts of the brain. We have taken a genetic approach to understand how the four functionally related, but lineally unrelated, classes of dopaminergic neurons of the nematode Caenorhabditis elegans, located in distinct parts of its nervous system...

Important cellular processes such as migration, differentiation, and development often rely on precise timing. Yet, the molecular machinery that regulates timing is inherently noisy. How do cells achieve precise timing with noisy components? We investigate this question using a first-passage-time approach, for an event triggered by a molecule that...

During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this review, we discuss the C. el...

The aging of eukaryotes depends on a number of genetic and environmental factors. One of the best-studied pathways controlling lifespan is the highly conserved insulin/IGF-1 pathway. Within this pathway is the well-known daf-2 mutation in the insulin receptor, which in Caenorhabditis elegans results in a 2- to 3-fold extension of life (Kenyon, 2010...

One of the key steps in determining the biological function of a gene of interest is to examine its expression pattern. Traditionally, gene expression in C. elegans has mostly been studied through reporter transgenes in which upstream regulatory sequence is fused to marker genes such as green fluorescent protein (GFP) or lacZ. In addition, expressi...

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a...

During the first stage of larval development, the Q neuroblasts and their descendants migrate to well-defined positions along the anteroposterior body axis, where they differentiate into sensory neurons and interneurons. The two Q neuroblasts are initially present at similar positions on the left and right lateral side, but this symmetry is broken...

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled b...

Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/β-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/β-catenin pathw...

This Podcast features an interview with Vitezslav Bryja, Hendrik Korswagen, and Reinoud de Groot about a Research Article that appears in the 18 March 2014 issue of Science Signaling

This chapter briefly discusses the lipid and sugar modification of Wnt proteins and focuses on the specialized secretion machinery that mediates the release of Wnt from producing cells and the mechanisms that facilitate and control the spreading of Wnt in morphogen gradient formation. Wnts undergo two major types of posttranslational modification,...

Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate spec...

Wnt proteins are secreted lipid-modified glycoproteins that control many aspects of development in organisms ranging from sponges to vertebrates. Wnt proteins are also important regulators of C. elegans development, with functions in processes as diverse as cell fate specification, asymmetric cell division, cell migration and synapse formation. In...

Variability in gene expression contributes to phenotypic heterogeneity even in isogenic populations. Here, we used the stereotyped, Wnt signaling-dependent development of the Caenorhabditis elegans Q neuroblast to probe endogenous mechanisms that control gene expression variability. We found that the key Hox gene that orients Q neuroblast migration...

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian in...

Secretion of Wnt proteins is mediated by the Wnt sorting receptor Wls, which transports Wnt from the Golgi to the cell surface for release. To maintain efficient Wnt secretion, Wls is recycled back to the trans-Golgi network (TGN) through a retromer dependent endosome to TGN retrieval pathway. It has recently been shown that this is mediated by an...

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative prot...

Supplementary Tables I, II, and III describing the ORA and GSEA results for this study.

Overview of lifespan data of the tested strains (A) and upon siRNA mediated targetting (B)

Overview of lifespan data in daf-16(mu86) background (A) and upon siRNA mediated targetting (B)

Supplementary Figures S1–11

Overview of performed brood size experiments of indicated strains (A) and upon siRNA mediated targeting (B)

The proteomics data set used for this study

Three Tales of Wnt Signaling The Wnt signaling pathway has important roles in regulating many biological processes during development and is also implicated in the behavior of some cancer cells (see the Perspective by Berndt and Moon ). Cruciat et al. (p. 1436 , published online 14 February) describe the mechanism of action of a protein found in a...

Forkhead box O (FOXO; DAF-16 in worms) transcription factors, which are of vital importance in cell-cycle control, stress resistance, tumor suppression, and organismal lifespan, are largely regulated through nucleo-cytoplasmic shuttling. Insulin signaling keeps FOXO/DAF-16 cytoplasmic, and hence transcriptionally inactive. Conversely, as in loss of...

Establishment of cell polarity is crucial for many biological processes including cell migration and asymmetric cell division. The establishment of cell polarity consists of two sequential processes: an external gradient is first sensed and then the resulting signal is amplified and maintained by intracellular signaling networks usually using posit...

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive and linear in vivo quantification of endogenous oxidative damage in t...

Coordinated movement depends on the creation of synapses between specific neurons in the motor circuit. In C. elegans, this important decision is regulated by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion because VA motor neurons are mis-wired with inputs normally reserved for their VB sisters. We have propo...

Sorting nexins are a large family of evolutionarily conserved phosphoinositide-binding proteins that have fundamental roles in orchestrating cargo sorting through the membranous maze that is the endosomal network. One ancient group of complexes that contain sorting nexins is the retromer. Here we discuss how retromer complexes regulate endosomal so...

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. Although several Wnt signal transduction mechanisms have been described in detail, it is still largely unknown how cells are specified to adopt such different Wnt signaling responses. Here, we have used the stereotypic migration of th...

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-...

The migration of neuroblasts along the anteroposterior body axis of C. elegans is controlled by multiple Wnts that act partially redundantly to guide cells to their precisely defined final destinations. How positional information is specified by this system is, however, still largely unknown. Here, we used a novel fluorescent in situ hybridization...

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases (HMTs) across species. Mammals contain seven H3K4 HMTs, Set1A, Set1B, and MLL1-MLL5. The activity of SET1 and MLL proteins relies on protein-protein interactions within larg...

Studies using chemical inhibitors have suggested that p38 MAP kinase is a key regulator of Wnt/β-catenin signaling. In this issue, Verkaar et al. (2011) show that cross-reactivity of p38 inhibitors with casein kinase Iδ/ɛ is responsible for Wnt/β-catenin pathway inhibition.

The Wnt family of signalling proteins has essential functions in development and adult tissue homoeostasis throughout the animal kingdom. Although signalling cascades triggered by Wnt proteins have been extensively studied, much remains to be learned about how Wnts are produced and secreted. Over the past few years, it has become clear that the sec...