Abstract:
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.The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.The zebrafish has become a widely used vertebrate model system for which a large tool-box of genetic and cell biological methods has been established [1,2]. Research using zebrafish is further supported by the zebrafish sequencing project, which has facilitated the generation of microarrays for large scale expression profiling. It has been proposed that proteomics should complement the genome-wide expression profiling [3]. However, a major obstacle in the application of proteomics has been the high proportion of yolk proteins in early embryos. Proteomic studies in zebrafish have therefore been limited to adult tissues [4]. One study targeted larval stages 48 or 72 hpf (hours post fertilization), when the yolk to cell mass ratio is already decreased [5], however, without identifying the proteins. Therefore, it remains unclear whether at this stage analysis without deyolking provides satisfactory information about cellular proteins. Thus, the development of a reliable method to remove the interfering yolk from cells on a large scale is required to apply proteomics to early embryos.Here, we provide detailed protocols for all zebrafish-specific steps of a proteomic experiment from dechorionation to mass spectrometry-based protein identification. As a key step, we present and validate a method for batch removal of the yolk from early embryos.In the early embryo, the cells forming the embry

Abstract:
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.

Abstract:
Epithelial spreading is a common and fundamental aspect of various developmental and disease-related processes such as epithelial closure and wound healing. A key challenge for epithelial tissues undergoing spreading is to increase their surface area without disrupting epithelial integrity. Here we show that orienting cell divisions by tension constitutes an efficient mechanism by which the Enveloping Cell Layer (EVL) releases anisotropic tension while undergoing spreading during zebrafish epiboly. The control of EVL cell-division orientation by tension involves cell elongation and requires myosin II activity to align the mitotic spindle with the main tension axis. We also found that in the absence of tension-oriented cell divisions and in the presence of increased tissue tension, EVL cells undergo ectopic fusions, suggesting that the reduction of tension anisotropy by oriented cell divisions is required to prevent EVL cells from fusing. We conclude that cell-division orientation by tension constitutes a key mechanism for limiting tension anisotropy and thus promoting tissue spreading during EVL epiboly.

Abstract:
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.

Abstract:
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.

Abstract:
We consider the following problem for various infinite time machines. If a real is computable relative to large set of oracles such as a set of full measure or just of positive measure, a comeager set, or a nonmeager Borel set, is it already computable? We show that the answer is independent from ZFC for ordinal time machines (OTMs) with and without ordinal parameters and give a positive answer for most other machines. For instance, we consider, infinite time Turing machines (ITTMs), unresetting and resetting infinite time register machines (wITRMs, ITRMs), and \alpha-Turing machines for countable admissible ordinals \alpha.

Abstract:
Stable numerical simulations for a hyperbolic system of conservation laws of relaxation type but not in divergence form are obtained by incorporating the physical entropy into the simulations. The entropy balance is utilized as an additional equation to eliminate the numerically critical terms with simple substitutions. The method has potential for a wider applicability than the particular example presented here.

Abstract:
We call a subset of an ordinal $\lambda$ recognizable if it is the unique subset $x$ of $\lambda$ for which some Turing machine with ordinal time and tape, which halts for all subsets of $\lambda$ as input, halts with the final state $0$. Equivalently, such a set is the unique subset $x$ which satisfies a given $\Sigma_1$ formula in $L[x]$. We prove several results about sets of ordinals recognizable from ordinal parameters by ordinal time Turing machines. Notably we show the following results from large cardinals. (1) Computable sets are elements of $L$, while recognizable objects with infinite time computations appear up to the level of Woodin cardinals. (2) A subset of a countable ordinal $\lambda$ is in the recognizable closure for subsets of $\lambda$ if and only if it is an element of $M^{\infty}$, where $M^{\infty}$ denotes the inner model obtained by iterating the least measure of $M_1$ through the ordinals, and where the recognizable closure for subsets of $\lambda$ is defined by closing under relative recognizability for subsets of $\lambda$.

Abstract:
A grammar-compressed ranked tree is represented with a linear space overhead so that a single traversal step, i.e., the move to the parent or the i-th child, can be carried out in constant time. Moreover, we extend our data structure such that equality of subtrees can be checked in constant time.