Organogensis

VI.1 COORDINATORS
VI.2 PARTICIPANTS
VI.3 SUMMARY
VI.4 INTRODUCTION
VI.5 SPECIFIC AIMS
VI.6 BACKGROUND AND SIGNIFICANCE
VI.7 THEORETICAL FRAMEWORK
VI.8 PRELIMINARY RESULTS
VI.9 RESEARCH DESIGN AND METHODS

• VI.9.i Gastrulation
  • VI.9.i.a Overview
  • VI.9.i.b Experiments
    • VI.9.i.b.1 The role of chemotaxis in primitive streak formation in chick
      • VI.9.i.b.1.i Hypothesis
      • VI.9.i.b.1.ii Methodology
    • VI.9.i.b.2 Zebrafish gastrulation
      • VI.9.i.b.2.i Overview
      • VI.9.i.b.2.ii Methodology
    • VI.9.i.c Models of Gastrulation
• [ Complete VI.9 Outline ]

VI.10 RELATIONSHIP TO CYTOSKELETON (PROJECT 2) AND BIOLOGICAL NETWORKS (PROJECT 1)
VI.11 TIMELINE

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VI.9.i.b.2 Zebrafish gastrulation:
VI.9.i.b.2.i Overview:

We will adapt methods to gastrulating zebrafish embryos, and we characterize normal and mutant embryos with defects in gastrulation cell movement, producing a detailed description of cell migration defects during specific phases of gastrulation.

We characterized three groups of gastrulation mutants by examining phenotypes of numerous mutant zebrafish (Myers et al., 2002). The first group of genes is required for anterior, dorsal structures to form. The second group of genes controls the formation of ventral-posterior structures and is involved in tail bud formation. The third group of genes is required for convergence and extension cell movements that elongate the body axis. Detailed analysis of gastrulation cell movements in mutant zebrafish embryos in conjunction with modeling should identify novel phenotypes and clarify tail bud formation and other gastrulation mechanisms. Systematic comparison of mutants may reveal new cell movements and points of regulation.

VI.9.i.b.2.ii Methodology:

We will examine cell movements in normal and gastrulation mutant zebrafish embryos using high-resolution confocal time lapse microscopy with either the two photon or spinning disk microscopes at the ICBM. These instruments allow rapid acquisition of image volumes with very little photo-damage. The mutant zebrafish strains are available from stock centers ( http://zfin.org/zf_info/stckctr/guide.html; http://www.eb.tuebingen.mpg.de/dept3/stockcenter/zebraf_stockcenter.html). We will produce genetic defects using Morpholino oligonucleotides. Gastrulation is completed during the first day of development in zebrafish while morpholinos last for four or five days.

We have developed methods to label single cells, e.g. embryos can be simply injected with FITC-dextran at the one cell stage. We transplant Individual blastomeres from embryos prior to the midblastula transition into a donor embryo at the same stage. We will then track these cells using epifluorescence microscopy. Tracking behavior of various cell populations that correspond to early fate mapping studies in zebrafish (Kimmel et al., 1990) will allow us to correlate specific cell movements with cell fate decisions. Comparison of these maps with mutant zebrafish will produce a detailed picture of early development cellular behavior and fate determination.

VI.9.i.c Models of Gastrulation:

Even the vast animal genome does not contain enough information to provide a detailed plan or blueprint for the development of an adult animal from a fertilized egg cell. Development must take advantage of natural physical processes that do not require detailed genetic control. A central problem of biophysics is to identify and understand these processes.

We aim to develop models to describe the complex processes of gastrulation. Modeling alternative hypothesis for the interactions between cell signaling, cell division and cell movement will be a tremendous help in guiding further experiments.

Various mechanical models (Drasdo and Forgacs, 2000; Zajac et al., 2000; Davidson et al., 1995, 1999; Weliky et al., 1991; Weliky and Oster, 1990; Odell et al., 1981) address specific types of gastrulation motion and simulations based on these models seem to show they suffice to explain the specific motions of isolated groups of cells, at least in planar arrays. In gastrulation, however, many types of motion occur in a highly coordinated way in several thousand cells arranged in a three-dimensional geometry. No models simulate this complete process.

Despite formidable obstacles, such a model may now be within reach. A principal motive for modeling early to mid-gastrulation cell movements of the embryo is to address the question of "how do cells know where to go?" Some sort of genetically controlled signaling, originating in the vegetal region of the early embryo, affects early differentiation of cells (Wolpert, 1998). However, specific cell movements are less likely to be under genetic control. Indeed, if they were, the pattern of genetic signaling, would have to be as complex as the resulting pattern of cell motion. While possible, it would only exchange the problem of understanding a complex set of cell motions for that of understanding an equally complex set of genetically controlled signals. A more attractive option, from both an explanatory and an evolutionary viewpoint, would be that a simple set of genetic signals produces an initial instability, triggering motions that are just the natural mechanical response to this instability. The challenge is to create a physically plausible model that we can simulate with present computing capacity.

We will use the CPM for our modeling. Both experiment and previous simulations suggest that cell polarity, cell deformation, cell-cell adhesive interactions, chemotaxis and cell motility and their interrelations are important. A physically plausible dynamic model using these cell properties should be possible through numerical simulation.