A. Staging Grasshopper Embryos from 29 to 35% of Development
Our staging protocol is essentially from Bentley et al. (1979) with some changes that reflect the difference in species used (Schistocerca americana vs Schistocerca gregaria). The shape of the limb bud changes substantially from 29 to 35% of embryonic development. At 29%, the distal tip of the limb bud is only slightly pointed and the overall shape of the limb bud is rounded and unsegmented. At this stage, Ti1 cell bodies are delaminating from the distal epithelium (Fig. 4). At 30% (the appropriate culturing stage for examination of the Ti1 pathway), the distal tip becomes slightly squared (Fig. 4). In addition, the Ti1 neurons have completely delaminated but have not initiated axonogenesis. By 33% of development, the Ti1 growth cones have reached the trochanter limb segment. At this stage the limb is more tubular and the squared distal tip is more definite, with a slight invagination evident at the distal tip of the epithelium (Fig. 4, arrow). By 35%, the Ti1 pathway is established and the distal tip invagination is more evident. Segmentation is more pronounced, particularly at the tibia-femur boundary and the femur-trochanter boundary (Fig. 4).
Pods of grasshopper eggs are removed from the sand in which they were laid and are cleaned gently with room temperature water to remove excess sand or dirt. The cleaner the eggs, the lower the chance of fungal and bacterial contamination. Pods can be stored on moist towels (Kimwipes; Kimberly-Clark) in Tupperware containers in a 30° C incubator. Eggs should be sprayed with water daily to avoid desiccation. At 30° C, embryos develop at approximately 5% per day. If desired, eggs can be stored at room temperature to slow their development.
Grasshopper embryos can be cultured for up to several days without affecting nervous system development. Adult female grasshoppers lay anywhere from 30 to 80 eggs at a time, and each clutch of eggs is referred to as a pod. Because each embryo from the same pod is roughly the same age and differs by only 1% (Bentley et al., 1979), large numbers of embryos can be experimented on reproducibly. Furthermore, as each embryo has six limbs, each embryo provides six Ti1 pathways for analysis. Typical culture periods are 24 h, which allows for completion of the Ti1 pathway. At 30% of embryonic development, dorsal closure has not occurred, allowing culture media and reagents to penetrate the embryo readily. In order to label, microinject, and conduct time-lapse imaging of Ti1 growth
cones, grasshopper limbs are filleted open and the mesoderm is removed. Using Nomarski optics the Til neuronal cell bodies are readily identifiable and accessible for labeling and injecting (see Section V).
C. Blocking the Function of Known Molecules (Antibodies and Peptides)
To specifically inactivate the function of a protein, antibody blocking or peptide blocking can be employed. Site-specific antibodies (either polyclonal or monoclonal) have been used successfully to inactivate transmembrane, secreted, and basal lamina molecules. Access of blocking antibodies can be monitored accurately by simply adding the appropriate secondary antibody for visualization. Antibodies can be cleared enzymatically in order to produce Fab fragments for use in culture. An alternative method to antibody blocking is peptide blocking. If functional motifs have been identified on a molecule, for example, receptor interaction sites, then peptides can be generated that mimic this site to compete for receptor interaction. Both of these techniques have been used successfully in the past to study Til axon guidance (Kolodkin et al., 1992; Wong et al., 1997, 1999; Isbister et al., 1999; Bonner and O'Connor, 2001). Often, the best strategy is to combine these techniques with overexpression or ectopic expression studies.
The current method for ectopic overexpression in the developing limb bud requires the use of transfected cell lines. Ectopic expression by this method has been successful with insect cell lines (S2 and Sf21 cells) and a mammalian cell line (COS cells) (Wong et al., 1999). To introduce transfected cells into the limb, one of two methods can be used. If Ti1 neurons are to be visualized with real time microscopy, the limb fillet preparation can be used. In this case, after the limbs have been filleted, transfected cells are placed on top of the limb fillet. When using an intact embryo, cells can be introduced into the lumen of the limb bud by entering the medial aspect of the limb bud with a micropipette containing the transfected cells. Cells are then expelled gently into the limb lumen with a mouth pipette. The location of transfected cells can be detected by GFP expression, immunocytochemical labeling of the recombinant protein, or membrane labeling of the transplanted cells with the lipophillic carbocyanine dyes with Dil or DiO (Molecular Probes).
One of the most powerful advantages of the grasshopper limb bud system is the ability to directly observe the growing neurons using time-lapse imaging as they extend their axons and respond to guidance cues in situ (O'Connor et al., 1990; Sabry et al., 1992, 1995; O'Connor and Bentley, 1993; Isbister and O'Connor, 1999). Using this system the behaviors of growth cones at precise locations in the Ti1 pathway have been analyzed to determine how neurons make steering decisions
(O'Connor et al., 1990), and the role of differential adhesion in growth cone steering has been tested (Isbister et al., 1999). In addition, microtubules (Sabry et al., 1991, 1995) and actin dynamics (O'Connor and Bentley, 1993) have been imaged to examine the cytoskeletal dynamics that are important for neuronal growth and growth cone steering. This work has contributed, in part, to the basis of a variety of cytoskeletal models of growth cone guidance (O'Connor and Bentley, 1993; Tana-ka and Sabry, 1995; Isbister and O'Connor, 2000). Presently we are developing this system to allow for the ectopic expression of recombinant proteins in Ti1 neurons.
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