MOLECULAR MECHANISM OF
NEURONAL CONNECTIVITY
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Chi-Hon
Lee, M.D. Ph.D., Head, Unit on Unit
of Neuronal Connectivity Saiyda Khan, Biological Laboratory Technician |
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Using the Drosophila visual system as a model, our laboratory is studying the molecular mechanisms by which neurons established specific connections during development. The fly retina contains three classes of photoreceptor neurons (R1-6, R7, and R8), each responding to a specific spectrum of light, and connecting to a specific layer in the brain (Fig. 24). We focus on the layer-specific targeting of R7 neurons, which are essential for visual function and, moreover, amenable to genetic manipulation. By using visu-al-driven behavior assays, we previously identi-fied N-cadherin (Ncad) and the receptor tyrosine phosphatase LAR because of their requirement for R7 target selection (Lee et al., 2001). Mosaic analysis showed that Ncad and LAR are required "cell-autonomously" in R7 neurons for selecting the proper target layer. To understand how Ncad regulates R7 target selection, we developed a genetic method to analyze single Ncad mutant R7 axons during development. Our data suggest that Ncad provides adhesive interactions between R7 growth cones and their synaptic partners. Furthermore, we have uncovered the striking molecular diversity of Ncad, which is generated by alternative splicing. We are testing the hypothesis that combinatory use of Ncad isoforms forms a synaptic code that matches pre- with post-synaptic partners. FIGURE 24 Connectivity of Drosophila retina Molecular Diversity of N-Cadherin As removing Ncad results in only about 75 percent R7 axon mistargeting (Lee et al., 2001), we suspected the existence of an additional molecule(s) that is functionally redundant with Ncad. Genomic sequence analysis revealed a cadherin gene (designated Ncad2) located next to the Ncad gene (Fig. 25). The Ncad2 gene is smaller than the Ncad gene and appears to result from partial duplication of the Ncad gene. RNA transcript analysis indicated that Ncad2 is expressed in the developing eye disk. In addition, mature Ncad2 transcripts consist of two alternative spliced forms, one of which contains exon 9 and encodes a type I receptor that is similar to Ncad while the other form does not contain exon 9 and encodes a secreted molecule. The soluble form of cadherins has been shown to disrupt cell-adhesion. These two Ncad2 forms are present in about equal proportion in the eye disk mRNA pool; in the mRNA of the whole animal, the receptor type is predominant. Thus, this alternative splicing appears to be regulated in a tissue-specific fashion. We are currently generating Ncad2 mutants for analyzing Ncad2 function in vivo.
FIGURE 25 N-cadherin loci contain
constant regions and variable regions. We detected the striking molecular diversity of Ncad generated by alternative splicing. The genomic sequence analysis revealed that the Ncad locus contains three pairs of exons (exon7-exon7', exon13-exon13', and exon18-exon18') in modular arrangement (Fig. 25). RNA tran-script analysis indicated that these exon pairs are used in a mutually exclusive manner, i.e., each mature transcript contains only one of the two alternative exons. All six exons are used in developing eye tissue, although exons 7, 13, and 18' are used predominantly in the eye disk. Using these three "exon modules" in a combinatorial fashion, the Ncad locus is capable of encoding eight isoforms, which each share the same mo-lecular architecture but has a unique amino acid sequence. Similar molecular diversity generated by modular arrangement of alternative exons has been observed in other receptors and might be a general mechanism for neuronal diversity and connection specificity. The notion that the different Ncad isoforms might have distinct functions
was first suggested by protein sequence analysis and later verified by
cell aggregation assay. Exons 7 and 7' encode the C-terminal half of the
eighth cadherin repeat and N-terminal half of the ninth cadherin repeat,
and exons 13 and 13' encode the eleventh and twelfth cadherin repeats
in a similar manner. Exons 18 and 18' encode one and one-half of the EGF
repeat and one-half of the transmembrane region. Interestingly, we found
nonconservative amino acid changes in two regions that potentially mediate
calcium binding and homophilic interactions, suggesting that these isoforms
might have different adhesive activity. To test such a possibility, we
expressed individual isoforms in S2 cells and assayed their ability to
induce cell aggregation. Our preliminary data showed that the isoforms
encoded by exon 7 but not exon 7' are capable of mediating homophilic
interaction. In contrast, the region of Ncad encoded by exon13 and exon13'
does not significantly affect Ncad's ability to induce cell aggregation.
We are currently testing whether these isoforms can mediate heterophilic,
or graded, interaction. Cellular and Molecular Mechanism of R7 Target Selection During development, R8, R7, and laminal interneurons (LNs) sequentially
innervate the medullar neuropil. R8s differentiate first and extend their
axons into the medulla. Approximately 12 hours later, R7 axons project,
along the R8 axon shafts, into the medulla, followed shortly thereafter
by the LNs. The first stage of R7 target selection involves R7 growth
cones defasciculating from R8 axons, thereby allowing R7 growth cones
to advance even deeper into the medulla. The separation of R7 and R8 growth
cones requires LNs whose axons follow R7 and project into the region between
the R8 and R7 growth cones. Genetic ablation of LNs, using an eye-specific
allele of hedgehog mutants (hh1), blocks
the separation of R7 and R8 growth cones. Although the interaction between
LNs and R7 (or R8) appears to be critical, the molecular nature of these
afferent-afferent interactions is not currently understood. In the second stage, it is likely that the R7 growth cones enter the
target layer by recognizing envi-ronmental cues provided by their target
neurons. This process requires Ncad function. Removing Ncad in single
R7 neurons causes premature arrest of R7 growth cones at the intermediate
layer (M4-5) instead of their entering the presumptive R7 recipient layer
(M6). R7 target neurons sprout dendritic processes in this layer and express
Ncad. In vitro, Ncad is capable of mediating
homophilic interaction. We favor the view that Ncad provides adhesive
interaction between R7 growth cones and their targets. Once R7 growth cones reach the presumptive R7 recipient layer, they stop
and undergo a conformational change from a spear-like structure into an
expanded conformation. This morphological change signifies the beginning
of the third stage, the stabilization phase. In LAR
mutants, R7 axons project into the correct layer at earlier stages but
later retract to the R8 recipient layer, suggesting that LAR is required
for stabilizing the interactions between R7 and their targets (Clandinin
et al., 2001). In vertebrates, LAR has
been suggested to up regulate N-cadherin-mediated homophilic adhesion
by dephosphorylating â-catenin. We envision that LAR stabilizes
R7 target interaction by up-regulating Ncad activity in R7 growth cones.
Our analyses have dissected R7 target selection into a series of complex
cellular and molecular interactions that involve afferent-afferent interactions
as well as afferent-target interactions executed in a temporally and spatially
controlled fashion. At the molecular level, the Ncad-based adhesion system
appears to play a key role in R7 target selection. The targeting specificity,
we believe, is achieved by both differential modulation of Ncad adhesive
activity in different growth cones and combinatorial use of Ncad and Ncad2
isoforms as well as by other surface receptors. It is likely that other
determinants of connection specificity remain to be discovered. The developmental
analysis presented here provides a conceptual framework upon which the
molecular details can be built. |
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SELECTED PUBLICATIONS
COLLABORATORS Akira Chiba, Ph.D., University
of Illinois, Urbana, IL |
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