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.iii Retinotectal and Muscular Innervation
  • VI.9.iii.a Retinotectal pathfinding and optic nerve fasciculation and defasciculation
    • VI.9.iii.a.1 Hypothesis
    • VI.9.iii.a.2 Methodology
  • VI.9.iii.b The Influence of Spatial Distributions of Receptors on Growth Cones and Variable Ligand Density on Target Cells on Topographic Map Formation
  • VI.9.iii.c Innervation in the Presence of Skeletal Elements
• [ 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.iii Retinotectal and Muscular Innervation:

We propose three subprojects on innervation:

1. The role of cadherins on retinotectal pathfinding.
2. The influence of spatial distributions of receptors on growth cones and of variable ligand density on target cells on topographic map formation
3. Innervation in the presence of skeletal elements.

VI.9.iii.a Retinotectal pathfinding and optic nerve fasciculation and defasciculation:

Retinal ganglion cells project axons from the eye through an elaborate pathway, forming connections with several targets in the brain (Burrill and Easter, 1995; Burrill and Easter, 1994; Stuermer and Bastmeyer, 2000). Axonal projections respond to a variety of positive and negative cues that restrict their growth and adhesion to appropriate pathways (Sanes and Yamagata, 1999; Sperry, 1963), including cell adhesion molecules, ephrins/Eph receptors and semaphorin/collapsin.

N-cadherin regulates retinal ganglion cell axon targeting to retinorecipient lamina in the optic tectum (Inoue and Sanes, 1997), suggesting that cadherins regulate retinotectal pathfinding and target cell recognition. In Drosophila, N-cadherin mutation causes defasciculation of central nervous system tracts (Iwai et al., 1997). R-cadherin and N-cadherin are expressed in ganglion cells in zebrafish (Liu et al., 2001; Liu et al., 1999a; Liu et al., 1999b), and R-cadherin is expressed in most, if not all target brain nuclei for ganglion cell axons (Liu et al., 1999a; Liu et al., 1999b). R-cadherin expression strongly correlates with axonal fasciculation within the optic nerve and defasciculation at target nuclei within the brain (Liu et al., 1999a; Liu et al., 1999b).

Homotypic cadherin-mediated adhesion between a growth cone and the substrate facilitates axon outgrowth (Bixby, 1992; Hall et al., 1996; Lilien et al., 1999; Riehl et al., 1996), and in addition, cadherins provide homophilic adhesion forces for axons to fasciculate and migrate in concert.

VI.9.iii.a.1 Hypothesis:

We hypothesize that cadherins expressed in retinal ganglion cell axons and their substrate and target tissues provide specific chemoaffinity signals for growing axons to use as guideposts in pathfinding and optic nerve fasciculation.

VI.9.iii.a.2 Methodology:

We propose to test our hypotheses directly and to provide an experimental test of model predictions by inhibiting cadherin function using Morpholino knockdown or dominant negative cadherin expression to examine the consequences of cadherin inhibition on ganglion cell guidance and retinotectal projection.

We will examine ganglion cell axons trajectories from the eye to visual centers of the brain and retinotopic map formation in zebrafish embryos (Burrill and Easter, 1995; Burrill and Easter, 1994; Picker et al., 1999; Stuermer, 1988; Stuermer and Bastmeyer, 2000; Stuermer et al., 1990) to examine the consequences of cadherin inhibition on visual system development. To examine ganglion cell axon trajectories, we will stain embryos using acetylated tubulin antibodies or anterograde DiI or DiO labeling of retinal ganglion cells. Acetylated tubulin staining provides an extensive view of axons within developing embryos showing whether inhibiting cadherin function produces gross changes in retinal ganglion cell pathfinding and targeting to central visual centers. DiI or DiO injection into the eye reveals specific changes in retinal ganglion cell axon trajectories. Whole eye filling by injecting saturated solutions of DiI or DiO into fixed embryos allows anterograde labeling of axons after overnight incubation.

Visualization of fluorescent dyes will use confocal or two-photon at the IBMC. For cryosectioning we can photoconvert the fluorescent molecules to a dark brown precipitate by photoexcitation in the presence of DAB (diaminobenzidene). These techniques combine with immunohistochemistry or in situ hybridization to double label retinal ganglion cell axons with specific cadherin molecule expression patterns. We have used these methods successfully to study zebrafish R-cadherin expression (Liu et al., 1999a; Liu et al., 1999b).

We propose to use antisense Morpholinos to produce a graded series of inhibition of N-cadherin and R-cadherin protein expression to examine the consequences of varied levels of cadherin expression in the visual system, and this variable effect will permit us to test the validity of simulations using variable adhesive force values. We expect downregulating cadherin adhesion to induce defasciculation of the optic nerve and other fasciculated nerves. This study will test of our predictions and validate our computational models.