Cell sorting in development.

During the development of multicellular organisms, cell fate specification is followed by the sorting of different cell types into distinct domains from where the different tissues and organs are formed. Cell sorting involves both the segregation of a mixed population of cells with different fates and properties into distinct domains, and the active maintenance of their segregated state. Because of its biological importance and apparent resemblance to fluid segregation in physics, cell sorting was extensively studied by both biologists and physicists over the last decades. Different theories were developed that try to explain cell sorting on the basis of the physical properties of the constituent cells. However, only recently the molecular and cellular mechanisms that control the physical properties driving cell sorting, have begun to be unraveled. In this review, we will provide an overview of different cell-sorting processes in development and discuss how these processes can be explained by the different sorting theories, and how these theories in turn can be connected to the molecular and cellular mechanisms driving these processes.

Identifying same-cell contours in image stacks: a key step in making 3D reconstructions.

Identification of contours belonging to the same cell is a crucial step in the analysis of confocal stacks and other image sets in which cell outlines are visible, and it is central to the making of 3D cell reconstructions. When the cells are close packed, the contour grouping problem is more complex than that found in medical imaging, for example, because there are multiple regions of interest, the regions are not separable from each other by an identifiable background and regions cannot be distinguished by intensity differences. Here, we present an algorithm that uses three primary metrics-overlap of contour areas in adjacent images, co-linearity of the centroids of these areas across three images in a stack, and cell taper-to assign cells to groups. Decreasing thresholds are used to successively assign contours whose membership is less obvious. In a final step, remaining contours are assigned to existing groups by setting all thresholds to zero and groups having strong hour-glass shapes are partitioned. When applied to synthetic data from isotropic model aggregates, a curved model epithelium in which the long axes of the cells lie at all possible angles to the transection plane, and a confocal image stack, algorithm assignments were between 97 and 100% accurate in sets having at least four contours per cell. The algorithm is not particularly sensitive to the thresholds used, and a single set of parameters was used for all of the tests. The algorithm, which could be extended to time-lapse data, solves a key problem in the translation of image data into cell information.

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation.

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.

ERK1 and ERK2 MAPK are key regulators of distinct gene sets in zebrafish embryogenesis.

BACKGROUND: The MAPK signaling proteins are involved in many eukaryotic cellular processes and signaling networks. However, specific functions of most of these proteins in vertebrate development remain elusive because of potential redundancies. For instance, the upstream activation pathways for ERK1 and ERK2 are highly similar, and also many of their known downstream targets are common. In contrast, mice and zebrafish studies indicate distinct roles for both ERKs in cellular proliferation, oncogenic transformation and development. A major bottleneck for further studies is that relatively little is known of in vivo downstream signaling specific for these kinases. RESULTS: Microarray based gene expression profiling of ERK1 and ERK2 knockdown zebrafish embryos at various stages of early embryogenesis resulted in specific gene expression signature sets that showed pronounced differences in gene ontology analyses. In order to predict functions of these genes, zebrafish specific in silico signaling pathways involved in early embryogenesis were constructed using the GenMAPP program. The obtained transcriptome signatures were analyzed in the BMP, FGF, Nodal and Wnt pathways. Predicted downstream effects of ERK1 and ERK2 knockdown treatments on key pathways responsible for mesendoderm development were confirmed by whole mount in situ hybridization experiments. CONCLUSION: The gene ontology analyses showed that ERK1 and ERK2 target common and distinct gene sets, confirming the difference in knockdown phenotypes and diverse roles for these kinases during embryogenesis. For ERK1 we identified specific genes involved in dorsal-ventral patterning and subsequent embryonic cell migration. For ERK2 we identified genes involved in cell-migration, mesendoderm differentiation and patterning. The specific function of ERK2 in the initiation, maintenance and patterning of mesoderm and endoderm formation was biologically confirmed.

Functions of the MAPK family in vertebrate-development.

The mitogen activated protein kinase (MAPK) family, consisting of the extracellular signal regulated protein kinase, c-Jun amino terminal MAPK and p38 subfamilies, is conserved in evolution throughout the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, migration, proliferation, differentiation, survival and development. Gene-targeting approaches in mice, chickens, frogs and zebrafish revealed crucial roles of MAPK in vertebrate development. Gene-disruption or -silencing often lead to lethal effects, therefore the zebrafish ex utero development provides an excellent in vivo model to study the function of MAPK in early embryogenesis. In this review, we summarize the current understanding of the MAPK family function in vertebrate-development and place this into the perspective of possibilities for future research.

Characterization and expression patterns of the MAPK family in zebrafish.

The mitogen activated protein kinases (MAPK) family pathway is conserved in evolution through the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, proliferation, differentiation, survival and development. In this study we annotated and cloned members of the zebrafish MAPK gene-family, containing the ERK, JNK and p38 subfamilies. Their sequences were compared to orthologs of other vertebrates (human, mouse and rat) and the temporal and spatial expression levels of the zebrafish mapk genes were determined during early zebrafish development. Semi-quantitative reverse transcriptase-PCR analysis revealed that most mapk genes are expressed throughout zebrafish development. Erk2,3 and p38a were expressed at a constant level throughout zebrafish embryogenesis, whereas erk1,4,5,6,7 and p38b showed specific temporal expression patterns. The spatial expression patterns were obtained by whole mount in situ hybridization at 24 h post fertilization (hpf) and 48 hpf embryos. The expression patterns were localized in specific regions at both stages and were tightly regulated during embryogenesis. For p38b, no staining was detected at 24 and 48 hpf. However, its expression was demonstrated at blastula-stage. Together, we identified the zebrafish orthologs of the zebrafish MAPK gene family and determined their specific spatial and temporal expression and distribution patterns during zebrafish embryogenesis.

Genetic and transcriptome characterization of model zebrafish cell lines.

Compared with the increasing use of zebrafish as a model organism in many laboratories, zebrafish cell lines are still unexploited and limited in application, partly due to their unknown genetic and physiological properties. We characterize two zebrafish embryonic fibroblast cell lines, ZF4 and PAC2. We demonstrate the genetic stability of these two zebrafish cell lines and achieved genetic manipulation by either lipid-mediated transfection or an electroporation- based nucleofection method. Data from zebrafish chip analysis (Affymetrix) demonstrate unique characteristics of these two cell lines in gene expression levels, showing that different zebrafish cell lines can be classified by their transcriptome profile. Their transcriptional responses to serum growth factor exposure suggest that zebrafish fibroblast cell lines may be used to study processes related to wound-healing or cancer.