Thomas Thumberger

CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool.

Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites.

The evolution and conservation of left-right patterning mechanisms

Morphological asymmetry is a common feature of animal body plans, from shell coiling in snails to organ placement in humans. The signaling protein Nodal is key for determining this laterality. Many vertebrates, including humans, use cilia for breaking symmetry during embryonic development: rotating cilia produce a leftward flow of extracellular fluids that induces the asymmetric expression of Nodal. By contrast, Nodal asymmetry can be induced flow-independently in invertebrates. Here, we ask when and why flow evolved. We propose that flow was present at the base of the deuterostomes and that it is required to maintain organ asymmetry in otherwise perfectly bilaterally symmetrical vertebrates.

Serotonin Signaling Is Required for Wnt-Dependent GRP Specification and Leftward Flow in Xenopus

In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus.

Quantitative Analysis of Embryogenesis: A Perspective for Light Sheet Microscopy

It is a challenge in developmental biology to understand how an embryos genes, proteins, and cells function and interact to govern morphogenesis, cell fate specification, and patterning. These processes span very different spatial and temporal scales. Despite much progress, simultaneous observation of such vastly differing scales has been beyond the scope of conventional microscopy. Light sheet microscopy fills this gap and is increasingly used for long-term, high-speed recordings of large specimens with high contrast and up to subcellular spatial resolution. We provide an overview of applications of light sheet microscopy in developmental biology and discuss future perspectives in this field.

A novel serotonin-secreting cell type regulates ciliary motility in the mucociliary epidermis of Xenopus tadpoles

The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.

Ciliogenesis and cerebrospinal fluid flow in the developing Xenopus brain are regulated by foxj1

BackgroundCirculation of cerebrospinal fluid (CSF) through the ventricular system is driven by motile cilia on ependymal cells of the brain. Disturbed ciliary motility induces the formation of hydrocephalus, a pathological accumulation of CSF resulting in ventricle dilatation and increased intracranial pressure. The mechanism by which loss of motile cilia causes hydrocephalus has not been elucidated. The aim of this study was: (1) to provide a detailed account of the development of ciliation in the brain of the African clawed frog Xenopus laevis; and (2) to analyze the relevance of ependymal cilia motility for CSF circulation and brain ventricle morphogenesis in Xenopus.MethodsGene expression analysis of foxj1, the bona fide marker for motile cilia, was used to identify potentially ciliated regions in the developing central nervous system (CNS) of the tadpole. Scanning electron microscopy (SEM) was used to reveal the distribution of mono- and multiciliated cells during successive stages of brain morphogenesis, which was functionally assessed by bead injection and video microscopy of ventricular CSF flow. An antisense morpholino oligonucleotide (MO)-mediated gene knock-down that targeted foxj1 in the CNS was applied to assess the role of motile cilia in the ventricles.ResultsRNA transcripts of foxj1 in the CNS were found from neurula stages onwards. Following neural tube closure, foxj1 expression was seen in distinct ventricular regions such as the zona limitans intrathalamica (ZLI), subcommissural organ (SCO), floor plate, choroid plexus (CP), and rhombomere boundaries. In all areas, expression of foxj1 preceded the outgrowth of monocilia and the subsequent switch to multiciliated ependymal cells. Cilia were absent in foxj1 morphants, causing impaired CSF flow and fourth ventricle hydrocephalus in tadpole-stage embryos.ConclusionsMotile ependymal cilia are important organelles in the Xenopus CNS, as they are essential for the circulation of CSF and maintenance of homeostatic fluid pressure. The Xenopus CNS ventricles might serve as a novel model system for the analysis of human ciliary genes whose deficiency cause hydrocephalus.

Linking early determinants and cilia-driven leftward flow in left-right axis specification of Xenopus laevis: a theoretical approach.

In vertebrates, laterality - the asymmetric placement of the viscera including organs of the gastrointestinal system, heart and lungs - is under the genetic control of a conserved signaling pathway in the left lateral plate mesoderm (LPM). A key feature of this pathway, shared by embryos of all non-avian vertebrate classes analyzed to date (e.g. fish, amphibia and mammals) is the formation of a transitory midline epithelial structure. Remarkably, the motility of cilia projecting from this epithelium produce a leftward-directed movement of extracellular liquid. This leftward flow precedes any sign of asymmetry in gene expression. Numerous analyses have shown that this leftward flow is not only necessary, but indeed sufficient to direct laterality. Interestingly, however, cilia-independent mechanisms acting much earlier in development in the frog Xenopus have been reported during the earliest cleavage stages, a period before any major zygotic gene transcription. The relationship between these two distinct mechanisms is not understood. In this review we present the conserved and critical steps of Xenopus LR axis formation. Next, we address the basic question of how an early asymmetric activity might contribute to, feed into, or regulate the conserved cilia-dependent pathway. Finally, we discuss the possibility that Spemanns organizer is itself polarized in the left-right dimension. In attempting to reconcile the sufficiency of the cilia-dependent pathway with potential earlier-acting asymmetries, we offer a general practical experimental checklist for the Xenopus community working on the process of left-right determination. This approach indicates areas where work still needs to be done to clarify the relationship between early determinants and cilia-driven leftward flow.

Connexin26-mediated transfer of laterality cues in Xenopus.

Summary A cilia-driven leftward flow of extracellular fluid breaks bilateral symmetry in the dorsal midline of the neurula stage vertebrate embryo. The left-specific Nodal signaling cascade in the lateral plate mesoderm (LPM) is key to asymmetric morphogenesis and placement of organs during subsequent development. The nature of the initial asymmetric cue(s) as well as the transfer of information from the midline to the left side has remained elusive. Gap junctional communication has been previously involved in Xenopus left-right (LR) development, however a function at cleavage stages was inferred from inhibitor experiments. Here we show by heptanol-mediated block of connexin function that flow stages during neurulation represent the critical time window. Flow in Xenopus occurs at the gastrocoel roof plate (GRP), a ciliated sheath of cells of mesodermal fate transiently positioned within the dorsal epithelial lining of the forming archenteron. We reasoned that endodermal cells immediately adjacent to the GRP are important for transfer of asymmetry. A systematic screen identified two connexin genes, Cx26 and Cx32, which were co-expressed in these lateral endodermal cells. Gain- and loss-of-function experiments pinpointed Cx26 as the critical connexin for LR development, while Cx32 had no effect on laterality. Importantly, GRP morphology, ciliation and flow were not affected in Cx26 morphants. Our results demonstrate a decisive role of Cx26 in the transfer of laterality cues from the GRP to the left LPM, providing a novel access to the identification of the initial asymmetric signal generated by flow.

Cilia are required for asymmetric nodal induction in the sea urchin embryo.

BackgroundLeft-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.ResultsHere we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.ConclusionsBased on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

Dynamics of in vivo ASC speck formation

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.