The Function ofsilberblickin the Positioning of the Eye Anlage in the Zebrafish Embryo

AbstractIn zebrafish, as in other vertebrates, an initially singular eye field within the neural plate has to split during morphogenesis to allow the development of two separated eyes. It has been suggested that anterior progression of midline tissue within the neural plate is involved in the bilateralization of the eye field. Mutations in the recently identifiedsilberblick(slb) gene cause an incomplete separation of the eyes. During gastrulation and early somitogenesis, the ventral midline of the central nervous system (CNS) together with the underlying axial mesendoderm is shortened and broadened inslbembryos. While in wild-type embryos the ventral CNS midline extends to the anterior limit of the neural plate at the end of gastrulation, there is a gap between the anterior tip of the ventral CNS midline and the anterior edge of the neural plate inslb.To investigate the cause for the shortening of the ventral CNS midline inslbwe determined the fate of labeled ventral CNS midline cells in wild-type andslbembryos at different stages of development. Inslb,anterior migration of ventral CNS midline cells is impaired, which indicates that migration of these cells is needed for elongation of the ventral CNS midline. The anterior shortening of the ventral CNS midline inslbleads to medial instead of bilateral induction of optic stalks followed by a partial fusion of the eyes at later developmental stages. The analysis of theslbphenotype indicates that anterior migration of midline cells within the neural plate is required for proper induction and subsequent bilateralization of an initially singular eye field. These findings may therefore provide a starting point in elucidating the role of neural plate morphogenesis in positioning of the eyes

Reassembling gastrulation

During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process

Tissue rheology in embryonic organization

Tissue morphogenesis in multicellular organisms is brought about by spatiotemporal coordination of mechanical and chemical signals. Extensive work on how mechanical forces together with the well‐established morphogen signalling pathways can actively shape living tissues has revealed evolutionary conserved mechanochemical features of embryonic development. More recently, attention has been drawn to the description of tissue material properties and how they can influence certain morphogenetic processes. Interestingly, besides the role of tissue material properties in determining how much tissues deform in response to force application, there is increasing theoretical and experimental evidence, suggesting that tissue material properties can abruptly and drastically change in development. These changes resemble phase transitions, pointing at the intriguing possibility that important morphogenetic processes in development, such as symmetry breaking and self‐organization, might be mediated by tissue phase transitions. In this review, we summarize recent findings on the regulation and role of tissue material properties in the context of the developing embryo. We posit that abrupt changes of tissue rheological properties may have important implications in maintaining the balance between robustness and adaptability during embryonic development

Proteomics of early zebrafish embryos

BACKGROUND: Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. RESULTS: As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins. CONCLUSION: The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis

The Medusa of Spatial Sorting: Topological Construction

We consider the simultaneous movement of finitely many colored points in space, calling it a spatial sorting process. The name suggests a purpose that drives the collection to a configuration of increased or decreased order. Mapping such a process to a subset of space-time, we use persistent homology measurements of the time function to characterize the process topologically

Defective neuroepithelial cell cohesion affects tangential branchiomotor neuron migration in the zebrafish neural tube

Facial branchiomotor neurons (FBMNs) in zebrafish and mouse embryonic hindbrain undergo a characteristic tangential migration from rhombomere (r) 4, where they are born, to r6/7. Cohesion among neuroepithelial cells (NCs) has been suggested to function in FBMN migration by inhibiting FBMNs positioned in the basal neuroepithelium such that they move apically between NCs towards the midline of the neuroepithelium instead of tangentially along the basal side of the neuroepithelium towards r6/7. However, direct experimental evaluation of this hypothesis is still lacking. Here, we have used a combination of biophysical cell adhesion measurements and high-resolution time-lapse microscopy to determine the role of NC cohesion in FBMN migration. We show that reducing NC cohesion by interfering with Cadherin 2 (Cdh2) activity results in FBMNs positioned at the basal side of the neuroepithelium moving apically towards the neural tube midline instead of tangentially towards r6/7. In embryos with strongly reduced NC cohesion, ectopic apical FBMN movement frequently results in fusion of the bilateral FBMN clusters over the apical midline of the neural tube. By contrast, reducing cohesion among FBMNs by interfering with Contactin 2 (Cntn2) expression in these cells has little effect on apical FBMN movement, but reduces the fusion of the bilateral FBMN clusters in embryos with strongly diminished NC cohesion. These data provide direct experimental evidence that NC cohesion functions in tangential FBMN migration by restricting their apical movement

Beobachten und Ertasten zellulärer Maschinen

Wie erledigen zelluläre Maschinen ihre Aufgaben? Wie funktionieren sie? Zur Beantwortung dieser Fragen werden in unserer wissenschaftlichen Arbeitsgruppe bionanotechnologische Methoden entwickelt, die es ermöglichen, diese nur wenige Nanometer großen Maschinen bei der Ausübung ihrer Arbeiten zu beobachten. Gleichzeitig detektieren diese Methoden molekulare Wechselwirkungsmechanismen, die die einzelnen Maschinen der Zelle steuern. Erste Beispiele zeigen den Einfluss pharmazeutischer Wirkstoffe zur Regulierung der Proteinfunktion im molekularen Detail. Hierdurch werden völlig neue Möglichkeiten geschaffen, um molekulare Schalter zellulärer Maschinen zu finden und zu betätigen.How do cellular machines function to fulfil their specific tasks so efficiently? How are they regulated? We apply and develop bio-nanotechnological approaches to directly observe these nanoscale cellular machines while they are at work. At the same time, we can gain insights into the working schedule of a cell, and can detect molecular mechanisms which drive and direct single cellular machines. With this unique possibility to reveal the switching mechanisms of the cellular machines, we could apply these switches to purposefully direct and regulate cellular processes. First examples follow the actions of pharmacological compounds on the function of a cellular machine at molecular resolution, and provide hitherto unexpected perspectives to develop and optimise such compounds to precisely target the function of cellular processes

Combined effect of cell geometry and polarity domains determines the orientation of unequal division

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s)

Zebrafish embryonic explants undergo genetically encoded self-assembly

Embryonic stem cell cultures are thought to self-organize into embryoid bodies, able to undergo symmetry-breaking, germ layer specification and even morphogenesis. Yet, it is unclear how to reconcile this remarkable self-organization capacity with classical experiments demonstrating key roles for extrinsic biases by maternal factors and/or extraembryonic tissues in embryogenesis. Here, we show that zebrafish embryonic tissue explants, prepared prior to germ layer induction and lacking extraembryonic tissues, can specify all germ layers and form a seemingly complete mesendoderm anlage. Importantly, explant organization requires polarized inheritance of maternal factors from dorsal-marginal regions of the blastoderm. Moreover, induction of endoderm and head-mesoderm, which require peak Nodal-signaling levels, is highly variable in explants, reminiscent of embryos with reduced Nodal signals from the extraembryonic tissues. Together, these data suggest that zebrafish explants do not undergo bona fide self-organization, but rather display features of genetically encoded self-assembly, where intrinsic genetic programs control the emergence of order