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MOLECULAR MECHANISM OF NEURONAL CONNECTIVITY
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Chi-Hon
Lee, MD, PhD, Head, Unit on Neuronal
Connectivity Shuying
Gao, PhD, Postdoctoral Fellow Chun-Yuan
Ting, PhD, Postdoctoral Fellow Shinichi
Yonekura, PhD, Postdoctoral Fellow Phoung Chung, BA, Biological Laboratory Technician |
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We
study the molecular mechanisms that specify neuronal connections during
development. Most parts of animal brains are divided into layers, each innervated
by one or a few distinct types of afferents. This type of connection
specificity, called layer-specific targeting, facilitates information
processing. We use the Drosophila visual system as a model to study
how different retinal afferents choose their target layers during
development. Our first objective is to determine the cellular events involved
in the target selection process and have discovered that the layer selection
of the R7 type of photoreceptor neurons occurs in two distinct stages, each
involving a distinct set of molecular and cellular interactions. Our second
objective is to identify the molecular components involved in the selection
process and to determine their mechanisms of action. Using a genetic
technique that allows the generation of single mutant neurons in an otherwise
wild-type animal, we revealed that N-cadherin functions in the first target
selection stage. We further determined that the N-cadherin (Ncad) gene
undergoes alternative splicing to generate 12 protein isoforms. Our third
objective is to determine the function of the molecular diversity of
N-cadherin. We are currently testing the hypothesis that different cadherins
form a synaptic code to direct connection specificity. Occurrence
of R7 and R8 neuronal layer selection in two distinct stages Ting, Lee In
flies, the visual information received from the retina converges to the
external part of the medullary neuropil (M1 to M6 layers). R7 and R8
photoreceptors directly connect to the M6 and M3 layers, respectively,
whereas laminal interneurons (LNs) relay R1 to R6 to multiple layers (M1 to
M5) in the medulla. To gain insight into the developmental mechanisms
governing the formation of layer-specific connectivity in the medulla, we
examined the innervation of medulla by R7, R8, and LN afferents at various
developmental stages. We found that medulla innervation by retinal afferents
occurs in two distinct stages: the early and midpupal stages. During the
first stage, R7, R8, and LN axons independently target to their temporary
layers. At the second stage, the R7 and R8 growth cones regain motility to
reach their destined layers. The
first stage of layer selection occurs immediately after retinal afferents
reach the medullary target area. Within each basic visual unit, the R8, R7,
and laminal axons project sequentially into the medulla. R8 axons first
project into the superficial layer (the R8 temporary layer). R7 axons follow
the R8 axons and terminate at a layer below the R8 temporary layer. Finally,
LN axons project past the R8 layer and terminate between the R7 and R8 growth
cones. Thus, the layer-specific targeting of R8, LNs, and R7 (positioned from
proximal to distal layers) at the early pupal stage does not arise simply from
the order of their innervation (R8 first, then R7, and finally LNs) but most
likely reflects specific properties of different afferents. The
second stage of R7 and R8 target selection starts approximately 50 hours
after pupal formation. During this stage, the R7 growth cones project
approximately 2 micrometers farther into the deeper medulla while the R8
growth cones project another 6 micrometers or so past the L2 growth cones to
reach a layer below. By 70 hours after pupil formation, the R7 and R8 growth
cones reach their final layers to assume their adult configurations. In
contrast to the initial target selection, which occurs immediately after the
axonal projection, all R7 and R8 axons enter the second target selection
stage at approximately the same time, regardless of the time of their arrival
at the medulla. We speculate that a global signal is responsible for
triggering the initiation of the second stage. As the inhibition of nitric
oxide (NO) synthase has been shown to disrupt layer-specific targeting of R
cell, we are currently investigating whether NO plays a role in regulating
the second stage of R7 layer selection. To
address whether afferent-afferent interaction is required for proper target
selection, we examined target selection in mutant animals lacking LNs or R7s.
First, we observed that, in hh1 mutants that lack LNs, the R7 and R8
axons still project to their temporary layers, although they fail to
separate. We further confirmed this observation by blocking LN
differentiation with a dominant negative form of EGF receptor. Thus, LN
growth cones intercalate between the R7 and R8 growth cones to separate them,
but the LN axons are not required for R7 and R8 target selection. Second, we
observed that the R8 and LN axons target correctly in sevenless
mutants, which lack R7s. Third, the removal of Ncad in single R8s disrupts R8
targeting without affecting the targeting of the neighboring R7s. Based on
these results, we conclude that afferent-afferent interactions play a minor
role in layer-specific targeting. Instead, we favor the view that R8, R7, and
LN growth cones read an existing cue in the medulla to select their temporary
target layers. Clandinin TR, Lee C-H, Herman T, Lee RC, Yang AY, Ovasapyan S,
Zipursky SL. Drosophila LAR regulates R1-R6 and R7 target specificity
in the visual system. Neuron 2001;32:237-248. Lee RC, Clandinin TR, Lee C-H, Requirement
for N-cadherin at the first stage of the R7 target selection Yonekura, Ting, Lee By
examining Ncad phenotypes at the adult stage, we previously
demonstrated that N-cadherin is required for R7 layer–specific
targeting. To determine the developmental stage during which Ncad functions
and to identify the developmental defects in Ncad mutant R7s, we
modified the single-cell mosaic method previously used for adult phenotypic
analysis to allow us to analyze single wild-type or mutant R7 axons during
development. We observed that, during the early pupal stage, about a quarter
of the Ncad mutant R7 growth cones failed to reach the R7 temporary
layer. In addition, more than half the Ncad mutant R7 growth cones
exhibit various morphological defects; they fail to expand fully in the
medulla, with some expanding prematurely before reaching the appropriate
layer. We next examined the Ncad mutant phenotype at 35 percent pupal
stage, during which the wild-type R7 and R8 growth cones form two separate
layers. We observed that approximately half the Ncad mutant R7 axons
terminated incorrectly at the R8 layer or the layer between the R7 and R8
layers. We conclude that, at the midpupal stage, at least a quarter of the Ncad
mutant R7 growth cones reach the R7 temporary layer at the early pupal stage
but retract from the correct layer at the midpupal stage. We propose that
N-cadherin mediates the adhesive interaction between R7 growth cones and
their temporary target layer. Lee C-H, Herman T, Clandinin TR, Lee R, Zipursky SL. N-cadherin
regulates target specificity in the Drosophila visual system.
Neuron 2001;30:437-450. Dynamic
regulation of Ncad alternative splicing during development Ting, Lee; in
collaboration with Chess, Chiba, Hsu, Neves Genomic
sequence analysis revealed an unusual modular organization of the Ncad
gene that contains three exon modules, corresponding to exons 7, 13, and 18;
each module is composed of a pair of highly similar but distinct exons
designated exons 7a/7b, exons 13a/13b, and exons 18a. We identified similar
genomic organization in the Ncad-orthologous gene in the honeybee (Apis
mellifera), the malaria mosquito (Anopheles gambiae), and another
member of the Drosophila family, D. pseudoobscura, which
all diverged from D. melanogaster approximately 380, 250, and 25
million years ago, respectively. In addition, we uncovered an additional
exon, designated as exon 7a´. Exon 7a´ encodes three amino acid residues and
was found in the exon 7a-containing Ncad transcripts, but not in those
containing exon 7b. By combinatorial use of these alternative exons, the Ncad
locus is capable of generating 12 isoforms (encoded by exon 7a, exons 7a+7a´,
or exon 7b; exon 13a or 13b; exon 18a or 18b). All 12 Ncad isoforms share the
same modular structure of a distinct class of classical cadherin receptors
composed of a large extracellular domain, a single-pass transmembrane region,
and a cytoplasmic tail that can interact with catenins. The extracellular
domains of this cadherin class contain 16 cadherin repeats (CA), four
EGF-like calcium-binding cysteine-rich repeats (EGF-CA), and two lamina-A
globular (LmA-G) domains. Drosophila Ncad homologs were found in
vertebrates (chicken, zebrafish, and fugu) and worms. However, our
preliminary analysis fails to reveal the Ncad alternative splicing in
vertebrates. To
test whether Ncad alternative splicing is regulated during
development, we developed a Tagman®-based
real-time PCR assay to examine the Ncad expression profiles in
different tissues and developmental stages. The assay provides high accuracy
and specificity for detecting each alternative exon in the range of 10 to
3,000 fg cDNA. Using this assay system, we found that all six alternative
exons are expressed at some level throughout development. However, we
observed several significant differences in the Ncad expression
profiles at different developmental stages, indicating that Ncad
alternative splicing is developmentally regulated, especially during the
embryonic stages. At the early embryonic stage, exons 7a and 18b were used
predominantly (88.3 and 94.8 percent, respectively) while exons 13a and 13b
were expressed at approximately equal levels. In sharp contrast, exons 7b,
13a, and 18a are used predominantly (89.7, 94.7, and 74.1 percent,
respectively) at the late embryonic stage. Moreover, from the larval, to
pupal, and to adult stages, the relative levels of 7b, 13a, and 18a gradually
increase and, in the adult stage, constitute over 90 percent of the total Ncad
transcripts (90.0, 93.6, and 94.7 percent, respectively). These data indicate
that the isoform 7b-13a-18a is prevalent in the adult stages while the other
isoforms encoded by exons 7a, 13b, and 18b were expressed primarily during
development. We
further examined the Ncad expression profiles in the eye discs at
different developmental stages. We found that all six alternative exons are
expressed in the retina throughout development. However, the expression of
exons 18a/18b is significantly regulated: from the third instar larval, to
the pupal, and to the adult stages, the exon 18a level gradually increases
from 15 to 92 percent. In contrast, the relative levels of exons13a/13b and
exons 7a/7b show only modest variations throughout development. The
7b-13a-18b and 7b-13a-18a isoforms are the predominant form in the larval eye
discs and adult retina, respectively, while these two forms are similarly
expressed during pupal stages. We next examined whether different subtypes of
photoreceptor neurons express different isoforms. We adapted a cell-sorting
method to isolate R3/4 and R7 neurons from the third instar larval eye discs
and subjected the extracted RNA to quantitative PCR analysis. We found that
R3/4 and R7 neurons exhibit virtually identical expression profiles on exons
7a/b and 18a/b and a similar preference for exon 13a over 13b, indicating
little difference between the expression profiles of the two distinct R-cell
types. In summary, the alternative splicing of exons 18a/18b is dynamically
regulated in the developing eye discs. However, the R3/4 and R7 types of
photoreceptor neurons have similar expression profiles. Mediation
of homophilic interactions with graded affinities by Ncad isoforms Yonekura, Ting, Chung,
Lee We
have taken two approaches to determine the function of the Ncad isoforms.
First, we used transgene-rescue and overexpression assays to determine
whether different Ncad isoforms have distinct functions in R7 neurons.
Second, we used a cell-aggregation assay to test whether Ncad isoforms can
mediate homo- and heterophilic interactions in vitro. In the
transgene-rescue experiments, we combined the GMR-FLP/MARCM system with
UAS-Ncad isoform transgenes to express a single type of Ncad isoform in Ncad
mutant R7s. The experiments are designed to avoid non–cell-autonomous
effects associated with Ncad expression because the transgene-mediated
Ncad isoform expression is restricted to the R7 neurons that are homozygous Ncad
mutants. We found that expressing a single Ncad isoform, including
7a-13a-18a, 7b-13a-18a, 7b-13b-18a, and 7b-13a-18b, is sufficient to rescue
the Ncad phenotypes in R7 axons. We next determined whether, in the
wild-type background, mis- or overexpressing a single Ncad isoform in R7s
alters the cell’s target specificity. We found that expressing any of
the isoforms in R7 axons causes modest mistargeting and growth cone
morphological defects. However, both defects were reduced in the older R7
axons and were not observed in the later stage. This finding indicates that
overexpressing single Ncad isoforms in R7 neurons is insufficient to change
R7 target specificity permanently. We concluded that Ncad molecular diversity
does not play an important role in R7 targeting. Using
an S2 cell-aggregation assay, we found that the Ncad 7b-13a-18a and the
alternative exon-substituted isoforms, including 7a-13a-18a, 7b-13b-18a, and
7b-13a-18b, are capable of inducing cell aggregates in the presence of
calcium, indicating that they mediate homophilic interaction. We observed
that the Ncad isoform 7b-13a-18b is capable of inducing very large aggregates
while Ncad 7b-13a-18a induces mostly small aggregates, suggesting that the
isoforms have different homophilic binding affinities. After careful
quantification, we conclude that Ncad isoforms mediate homophilic
interactions and that the region encoded by exon 18 dictates the binding
affinity. In addition, we found that all tested Ncad isoforms induced
mixed-cell aggregates, indicating that they can mediate heterophilic
interactions. However, the Ncad-expressing S2 cells did not intermix with the
S2 cells expressing E-cadherin, another Drosophila classical cadherin;
instead, they formed separate cell aggregates. In summary, Ncad isoforms
mediate type-specific but not isoform-specific heterophilic interactions. The lack of isoform specificity
revealed by the transgene rescue, overexpression experiments, and
heterophilic interaction assays strongly argues against the hypothesis that
the Ncad isoforms constitute an adhesion code to direct targeting
specificity. Instead, we suggest that the Ncad isoforms encoded by exon 18b
are expressed predominantly during the axon outgrowth stages to provide
strong axon-axon interactions while the exon 18a-encoding Ncad isoforms
provide weak interactions between growth cones and their targets. COLLABORATORS Andrew Chess, PhD, Whitehead Institute, Akira Chiba, PhD, Shu-ning Hsu, BA, Guilherme Neves, PhD, Whitehead Institute,
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