Chapter 10

Molecular Basis of Lamina-Specific Synaptic

Connections in the Retina: Sidekick

Immunoglobulin Superfamily Molecules

Y. Kate Hong and Masahito Yamagata

Abstract During the development of the nervous system, neurons must assem-

ble a vast network of synaptic connections to form functional neuronal circuits.

Each neuron sends axons to reach the general target region and then must

choose the appropriate target from a multitude of neurons to make proper

connections and to form synapses. How are such specific neuronal connections

established? Sidekick proteins (Sdks) are synaptic adhesion molecules of the

immunoglobulin (Ig) superfamily that have been suggested to mediate targeting

specificity in the synaptic layers in the retina. These cell adhesion molecules,

along with their close homologs, Down’s syndrome cell adhesion molecules

(DSCAMs), provide a molecular code for lamina-specific synaptic connections

that is governed by homophilic molecular interactions.

Keywords Sidekick (Sdk) � Down’s syndrome cell adhesion molecule

(DSCAM) � Synaptic specificity � Laminar specificity � Retina � Inner

plexiform layer (IPL)

10.1 Introduction

In the central nervous system (CNS), billions of neurons establish, maintain,

andmodify connections with their appropriate partners throughout the lifetime

of the organism. Over the past decade, a variety of adhesion molecules con-

centrated at synapses have been identified. Such molecules have been shown to

mediate formation, stability, and plasticity of synapses (Sudhof 2001, Scheiffele

2003, Yamagata et al. 2003, Takeichi and Abe 2005). For example, neuroligins

and neurexins participate in synaptic differentiation and maturation. Muta-

tions in these proteins are thought to underlie neurodevelopmental disorders

Y.K. Hong (*)Department of Molecular and Cellular Biology, and the Center for Brain Science,Harvard University, Cambridge, MA 02138, USAe-mail: yhong@fas.harvard.edu

M. Hortsch, H. Umemori (eds.), The Sticky Synapse,DOI 10.1007/978-0-387-92708-4_10, � Springer ScienceþBusiness Media, LLC 2009

223

such as autism spectral disorders (see Chapter 17). Classic cadherins playcrucial roles in the structural plasticity of synapses, thus in learning and mem-ory (Chapter 7). Other adhesion molecules such as neural cell adhesion mole-cule (N-CAM) are expressed bymany neurons widely in the CNS and have beensuggested to play a variety of roles, including plasticity and formation ofsynapses, cell migration, and axon guidance (see Chapter 13).

The most fascinating but perhaps least understood feature of synaptic con-nectivity is the specificity with which synapses are formed. A prime example isthe developmental process by which a retinal ganglion cell (RGC) in the eyeprojects to the optic tectum (superior colliculus in mammals), a major targetarea in the brain. Once the axon has reached its general target region within thetectum, it must find a specific cell with which to form synapses. Furthermore, itmust decide with which segment of dendrite or soma of the particular cell toform these synapses.

How do neuronal processes reach their targets with such accuracy? Toaddress this mystery, Roger Sperry (1913–1994) formulated the chemoaffinityhypothesis half a century ago, in which he speculated that wiring specificity isestablished using a ‘‘lock and key’’-like mechanism provided by unique cyto-chemical labels that denote their specific position and neuronal subtype. Thiscould be achieved by specific receptor–ligand interactions or by combinatorialinteractions of several recognition molecules that provide a code, which directsproper neuronal targeting. Several such molecules have since been identified.Perhaps the clearest example is the case of ephrins and Eph receptor tyrosinekinases. These molecules play a pivotal role in axon guidance via gradientexpression that leads to formation of topographic maps (see Chapter 16).However, the mechanisms underlying the subsequent processes of cellular andsubcellular selectivity of synapse formation are less well understood. Only a fewmolecules have thus far been implicated as determinants of synaptic selectivity.

As we will discuss in the first half of this chapter (Section 10.2), Sidekicks(Sdks) and, more recently, their close relatives, Down’s syndrome cell adhesionmolecules (DSCAMs), have been shown to play a direct role in targetingspecificity in the retina. In Section 10.3, we will review the current knowledgeconcerning the molecular structure and biological properties of Sdks.

10.2 The Role of Sdks in Laminar Specificity

10.2.1 Laminar Specificity Is a Major Determinant of SynapticSpecificity in the CNS

A major determinant of specific synapse formation in the CNS appears to belaminar specificity whereby different populations of afferent axons confinetheir terminal arbors and synapses to distinct series of laminae. Laminarorganization is widely seen throughout the vertebrate CNS, including the

224 Y.K. Hong and M. Yamagata

neocortex, olfactory bulb, hippocampus, optic tectum, lateral geniculate body,cerebellum, and spinal cord. Within each of these target regions, ingrowingafferent axons selectively synapse in just one or a few of the laminae within thetarget area (Sanes and Yamagata 1999).

10.2.2 Laminar Organization of the Retina

In many ways, the vertebrate retina is an ideal system in which to study laminarspecificity. The retina, like many other regions of the CNS, is a multilayeredstructure, whose distinct cellular and synaptic laminae are of fundamentalimportance for its ability to properly process and transmit information. Letus first examine the anatomy of the retina (Fig. 10.1). The outer nuclear layer(ONL) contains the photoreceptors that are the principle light-detecting cells ofthe retina. The inner nuclear layer (INL) contains the cell bodies of horizontal,bipolar, and amacrine cells, and the ganglion cell layer (GCL) contains theretinal ganglion cells (RGCs), which are the sole output neurons of the retinathat project directly to the brain. The outer and inner plexiform layers (OPLand IPL, respectively) contain synaptic contacts between the cellular layers.Laminar arrangement of synaptic connectivity is particularly evident withinthe IPL, where retinal ganglion cells (RGCs) receive inputs from both amacrineand bipolar cells. This lamina-specific connectivity is essential for visualinformation processing.

Fig. 10.1 The vertebrate retina is organized into multiple sublaminae. The major synaptic layerof the retina, IPL, is typically divided spatially into five sublayers (S1–5). The stratificationlevels of the RGC dendrites limit the types of cells with which they can form synapses.Modified from a drawing of a retina by Ramon y Cajal. Abbreviations: photoreceptor, PR;horizontal cell, HC; bipolar cell BP; amacrine cell, AC; retinal ganglion cell, RGC; outernuclear layer (contains PR), ONL; outer plexiform layer, OPL; inner nuclear layer (containsHC, BP, and AC somata), INL; inner plexiform layer, IPL; ganglion cell layer, GCL

10 Lamina-Specific Synaptic Connections in the Retina 225

Over a century ago, Ramon y Cajal (1851–1934) provided the first clues,suggesting that the lamina-specific arborizations of retinal neurons define theirconnectivity. He spatially divided the IPL into five different substrata andclassified retinal cells according to the branching patterns of their processeswithin different depths of the IPL (Fig. 10.1). It is clear that it is physicallyimpossible for a ganglion cell, whose dendrites are confined to the inner mostlayer of the IPL, to receive direct inputs from a bipolar cell that only hasprojections in the outer most layer. Characterization of RGCs by their dendriticarborization patterns has led to the identification of 11–15 RGC subtypes inmammals (Masland 2001, Sun et al. 2002, Dacey et al. 2003, Kong et al. 2005,Coombs et al. 2006). Importantly, each subtype is thought to be distinguishableby characteristic morphology, central projections, electrophysiological proper-ties, and neurochemical phenotypes (Karten et al. 1990, Wassle and Boycott1991, Masland 2001, Rockhill et al. 2002).

This correlation between structure and function of RGC subtypes is bestdemonstrated by ON and OFF RGCs. These functionally distinct RGC sub-types have discrete response properties to light stimuli. Moreover, cells thatmake up the ON versus OFF neuronal circuitry connect within different sub-laminae of the IPL (Famiglietti andKolb 1976). This is a clear example in whichdendritic stratifications in different IPL sublaminae directly correlate withdistinct functions of specific RGC subtypes. While Ramon y Cajal spatiallydivided the IPL into 5 layers, more recent electrophysiological studies haveshown that the IPL can be further divided into at least 10 functionally distinctparallel layers (Roska andWerblin 2001). Taken together, these studies indicatethat laminar specificity in the retina, by which RGCs of distinct morphologicalclasses are defined, clearly confers a functional classification.

Some observations suggest that these RGC subtypes are initially indistin-guishable during early development (Bodnarenko et al. 1995). Despite this, thespatial segregation and order of ON and OFF layers is always the same inindividual animals, as well as in different species. Furthermore, the accumulatingliterature suggests that characteristics of RGC subtypes appear to be cell-autonomously determined, independent of their connection partners (Yamagataand Sanes 1995,Mummet al. 2005). This raises the possibility that the connectionspecificity of each RGC subtype is genetically encoded, and that the differencesin gene expression among distinct subtypes are responsible for the distinctconnectivity.

10.2.3 Sdks Mediate Laminar Specificity

To search for molecular differences between RGC subtypes, Yamagata andcolleagues performed a differential hybridization screen of single-cell cDNAlibraries on various neurochemically distinct RGCs from the chick retina(Yamagata et al. 2002, 2006). This screen led to the identification of Sdk1,

226 Y.K. Hong and M. Yamagata

and subsequently to Sdk2 by homology. Strikingly, in situ hybridization ana-

lysis using specific RNA probes showed that the Sdk1 and the Sdk2 genes are

expressed in non-overlapping subsets of neurons in the chick retina. Both Sdks

are expressed in subsets of cells in the GCL, as well as the INL, where pre-

synaptic neurons to the RGCs reside (Figs. 10.1 and 10.2A). Antibodies specific

to the extracellular domains of Sdk1 or 2 revealed that these proteins were not

detected in the cellular layers of the retina. Rather, Sdks are highly concentrated

at the synaptic cleft, and each Sdk is localized in one or two distinct synaptic

sublaminae within the IPL. Sdk1 is predominantly found in layer S4, while

Sdk2 is in layer S2 (Fig. 10.2A). The highly restricted localization pattern of

Sdks, as well as their mutually exclusive expression pattern, suggests the possi-

bility that Sdks mediate laminar targeting of specific neurons by homophilic

adhesion (see Section 10.3).The direct proof that Sdksmediate lamina-specific targeting was provided by

a gain-of-function assay. If Sdk is sufficient to mediate laminar specificity, the

expression of Sdk protein in cells that normally do not express Sdk should drive

the cell processes to laminate in Sdk-positive laminae (Fig. 10.2B). Indeed, this

was shown to be the case for both Sdk1 and Sdk2 (Yamagata et al. 2002). In a

follow-up study, they also tested whether Sdks are necessary for laminar

Fig. 10.2 Ig superfamily molecules promote laminar specificity in the retina. (A) Expression ofSdk1, Sdk2, DSCAM, and DSCAML is restricted to specific laminae within the IPL. RGCsare represented here in the GCL; amacrine or bipolar cells are represented here on top . Cellsin the INL that express Sdk1 project to the same layer as RGCs expressing Sdk1, and likewisefor Sdk2, DSCAM, and DSCAML. Illustration of gain-of-function (B) and loss-of-function(C) experiments suggesting that Sdks and DSCAMs are necessary and sufficient to mediatelaminar specificity in the retina. In the example shown in B, ectopic expression of Sdk1 in anRGC that originally would have projected to S3 (represented here as dashed lines in black)now projects to Sdk1-positive sublamina S4 (solid lines in gray/red). Alternatively, as illu-strated in C, loss-of-function of Sdk1 results in mistargeting of processes to alternate layers inthe IPL. Processes of a cell that originally expressed Sdk1 (red/gray dashed lines) are no longerrestricted to layer S4, but wander into other layers (solid black lines)

10 Lamina-Specific Synaptic Connections in the Retina 227

specificity (Yamagata and Sanes 2008b). The expression of Sdk-specific inter-fering RNA (iRNA) resulted in a disruption of Sdk laminae, while other Sdk-negative layers remained undisrupted (Fig. 10.2C). Together, these studiessuggested that Sdks are both necessary and sufficient to mediate laminarspecificity in the retina.

An intriguing implication of these results is that other IPL layers may stilldevelop normally, even when Sdk-expressing layers are not properly formed.Because these experiments were limited to the knockdown of gene expression inonly small subregions of the retina, it remains to be determined whether a globalloss of Sdk expression would also affect the formation of other layers. It is alsounclear whether mistargeted neurons form ectopic synapses in other layers orwhether the misguided neurons are unable to form any functional synapses.

10.2.4 DSCAMs, Close Relatives of Sdks, Mediate LaminarSpecificity

If laminar specificity is in fact mediated by a genetic code as these studies imply,there must exist additional other molecules that specify other laminae. Forexample, in the chick retina, Sdk1 and Sdk2 are localized in only two of atleast five sublaminae of the IPL. This raises the possibility that similar mole-cules, other than Sdks, mediate laminar specificity. DSCAMs, which are highlyhomologous to Sdks in protein structure (Fig. 10.3A and Chapter 9), also havesimilar molecular properties to those described here for Sdks (Yamagata andSanes 2008b). Whereas multiple DSCAM genes, as well as numerous spliceisoforms, are present in insects (see Chapter 9, Schmucker et al. 2000), only twoDSCAMgenes are present in vertebrate genomes: DSCAMandDSCAM-like 1(DSCAML), each with only a single splice isoform (Agarwala et al. 2000, 2001).The two vertebrate DSCAMs, much like Sdks, mediate selective homophilicadhesion and are expressed in mutually exclusive subsets of cells in the retina(Fig. 10.2A), and loss- or gain-of-function results in diversion of neuronalprocesses into other laminae (Fig. 10.2B and C, Yamagata and Sanes 2008b).An exciting possibility is that additional, as-yet unidentified adhesive moleculesmay play similar roles in determining specific synaptic target regions (in ner-vous system structures that have a layered or laminar structure).

In themouse retina, DSCAM is thought tomediate repulsion between neuralprocesses of a subtype of amacrine cell and to maintain isoneuronal self-avoidance of neurite arborization, as well as to prevent neurite fasciculation(Fuerst et al. 2008). Similar mechanisms have been described inDrosophila (seeChapter 9), where the intracellular domain of DSCAM is required for dendriticself-avoidance (Soba et al. 2007). It is intriguing to speculate that intracellularinteracting molecules, such as PDZ proteins, might determine the cellularresponse to homophilic adhesion. The mechanism by which Sdks mediatelaminar specificity remains elusive. Further characterization of the downstream

228 Y.K. Hong and M. Yamagata

signals of DSCAMs and Sdks is needed to shed further light on the mechanismsunderlying laminar specificity. Furthermore, since there are other IPL layersthat express neither Sdks nor DSCAMs, it is intriguing to speculate that addi-tional Ig superfamily molecules might provide a molecular code for laminartarget recognition in the retinal IPL.

10.3 Molecular and Cellular Properties of Sdks

10.3.1 Structure and Expression of Sdks

Sdks are members of the Ig superfamily containing a single-pass transmem-brane segment with a large extracellular N-terminal domain consisting of 6 Igtype C2 motifs and 13 fibronectin type III (FN-III) domains (Fig. 10.3A). Sdkwas first identified in aDrosophilamutant screen for defects in eye development.

Fig. 10.3 Structural domains of Sdks and DSCAMs, members of the immunoglobulin

superfamily molecules. (A) Sdk1 and Sdk2, each consists of 6 Ig domains, 13 Fn type IIIdomains, and a highly conserved C-terminal PDZ-binding domain (amino acid sequenceGFSSFV); DSCAM and DSCAML each consist of 10 Ig domains, 6 Fn type III domains, anda C-terminal PDZ-binding motif (KSYTLV). At the amino acid sequence level, Sdk1 and Sdk2are 74% similar to one another, as are DSCAM and DSCAML; Sdks and DSCAMs are�40%similar. (B) Sdks and DSCAMs mediate homophilic adhesion, but do not bind one anotherheterophilically. In the example shown, Sdk1 only binds Sdk1, but not Sdk2. The same holds trueforDSCAMandDSCAML. Sdks are thought to be expressed at the synaptic cleft in both the pre-and postsynaptic cells and mediate homophilic adhesion

10 Lamina-Specific Synaptic Connections in the Retina 229

Sdk-null mutant animals have a rough-eye phenotype, and these studies indi-cate that Sdks play a role in controlling photoreceptor differentiation in the flyeye (Nguyen et al. 1997). While only one gene is present in Caenorhabditiselegans and Drosophila, there are two different Sdk genes in vertebrates, Sdk1and Sdk2 (Yamagata et al. 2002, Kaufman 2004, Abramowicz et al. 2005),which have an identical protein domain structure. Sdk homologs are highlyconserved across species, have similar size gene products and an identicalprotein domain organization (Fig. 10.3). In mammals, three splice isoforms ofSdk1 have been identified, whose gene products differ only in their N-terminalregion. Only one isoform has been found for Sdk2 (Kaufman et al. 2004).

In addition to their remarkable expression pattern in the retina, vertebrateSdks are also expressed in non-neuronal organs, where they appear to playadditional roles. In mice, Sdk1 expression is present in many organs includingkidney, heart, intestines, and stomach. Sdk1 may also be involved in thepathogenesis of glomerular disease in human immunodeficiency virus (HIV)-associated nephropathy. Sdk1, but not Sdk2, was found to be upregulated inHIV-infected podocytes (Kaufman 2004, Kaufman et al. 2007). It is thoughtthat cell aggregation observed in HIV-associated nephropathy may be a con-sequence of increased Sdk1 expression in HIV-infected cells (Kaufman et al.2007). It remains to be determined what specific roles Sdks play beyond theirrole in laminar specificity.

10.3.2 Sdk Ectodomains Mediate Homophilic Adhesion

Thus far the only known ligand of Sdk is Sdk itself. Sdks have been shown tomediate calcium-independent homophilic adhesion in vitro. The ectodomaindetermines the binding specificity of Sdk. Beads coated with the extracellulardomains of Sdk1 or Sdk2 aggregate only homophilically (Yamagata et al.2002). Moreover, in a cell aggregation assay of Sdk-transfected HEK cells,Sdk1-expressing HEK cells specifically aggregate with other Sdk1-expressingcells, but not with cells expressing Sdk2 (Hayashi et al. 2005, Yamagata andSanes 2008b). Likewise, Sdk2 was shown to mediate aggregation mediated byhomophilic interactions with Sdk2, but not Sdk1 (Fig. 10.3B). Similar proper-ties have been described for the two vertebrate orthologs of DSCAM (Agarwalaet al. 2000, 2001). Remarkably, each of the four molecules interacts with highspecificity, despite the strong similarities between the molecular structures ofSdks and Dcams (Fig. 10.3A).

To determine the critical domains that are required for the homophilicbinding activity of Sdks, Hayashi and colleagues generated several forms ofSdk1 and Sdk2 in which various Ig domains were deleted (2005) and tested themin the cell aggregation assay described above. Several experiments suggest thatthe first two Ig domains of Sdks are important for mediating the homophilicbinding activity. Interestingly, the gene product of a smaller splice isoform of

230 Y.K. Hong and M. Yamagata

Sdk1 fails to mediate cell aggregation. The truncated Sdk1 is identical with thefull-length Sdk1 protein, except that it lacks the first two Ig domains.Moreover,when the first two Ig domains of Sdk1 were swapped with that of Sdk2, thehybrid molecule then tended to aggregate with Sdk2, but not with Sdk1. Thesame results were seen for the reverse case. That is, the first two Ig domainsseemed to be responsible for conferring specificity of Sdk binding. As wasrecently shown for DSCAMs (Ly et al. 2008), it remains to be determinedwhether other heterophilic binding partners also exist for Sdks.

10.3.3 Intracellular Signaling of Sdks

As described above, Sdk-mediated adhesion is important for initial targetrecognition. Furthermore, adhesion triggers downstream signaling events,which may subsequently initiate synapse formation. Several results indicatedthat Sdk intracellular domains have such a signaling capacity. The intracellulardomains of Sdks are about 200 amino acids in length, and the two vertebrateand the invertebrate Sdk cytoplasmic domains differ in their protein sequences(Yamagata et al. 2002). However, a small portion of the C-terminal, whichcontains the hexapeptide GFSSFV, is strikingly conserved across all speciesexamined, as well as between Sdk1 and Sdk2. This suggests that potentialdownstream signals of Sdks mediated by this sequence are conserved through-out species. More importantly, this hexapeptide contains the canonical PDZ-binding motif, S/T-X-V. Various PDZ domain-containing proteins are presentin the postsynaptic densities of neurons. Signaling downstream of PDZ proteinsis dependent on the PDZ protein itself, which often has differential bindingspecificities to different amino acid sequences including the critical C-terminaltripeptde. In a yeast two-hybrid screen, several putative PDZ proteins havebeen reported to interact with the Sdk2 C-terminal domain, including PSD 95,MAGI-1 and 3, Shank2, and Chapsyn110 (Meyer et al. 2004, Yamagata andSanes 2008a). PDZ proteins generally function as scaffolding molecules thatbind directly to various cell surface proteins, including ion channels and otheradhesion molecules. The main role of such scaffolding proteins is thought to beto organize the signaling complexes at the postsynapse and to mediate signaltransduction (Kim and Sheng 2004).

While the downstream signaling mechanism is expected to be similar forboth Sdk1 and Sdk2, the expression of each Sdk appears to be mutuallyexclusive in RGCs. This is consistent with their putative role in conferringrecognition specificity while utilizing the same or similar signaling pathways.Furthermore, an analogous PDZ-binding motif is also present in DSCAM andDSCAML. This may indicate that the intracellular signaling by these closelyrelated adhesion molecules is similar to that of Sdk proteins. Further studies arenecessary to uncover the signaling pathways downstream of Sdks, which mayexplain the mechanism involved in their function in target-specific synapseformation.

10 Lamina-Specific Synaptic Connections in the Retina 231

10.4 Conclusions

The number of synaptic cell adhesion molecules implicated in mediating synap-

tic specificity continues to grow. SYG-1 and SYG-2 in C. elegans (see Chapter

11), flamingo inDrosophila (Lee et al. 2003) as well as Sdks and DSCAMs have

been shown to mediate synaptic specificity. Among these, only Sdks and

DSCAMs have thus far been shown to play this role in vertebrates. As discussed

above, three lines of evidence strongly suggest that Sdks and DSCAMs are

recognition molecules that mediate laminar specificity in the retina. First, all

four proteins are expressed by non-overlapping, complementary subsets of

RGCs. Second, in the retina, each molecule is expressed in separate, distinct

sublaminae of the IPL. Third, a loss- or gain-of-function experiment induces the

mistargeting of neuronal processes to alternate layers. However, Sdks and

DSCAMs mark only a few sublaminae among over 10 functionally distinct

laminae of the retinal IPL (Roska and Werblin 2001). It is exciting to speculate

that each lamina might be established by a different recognition molecule or a

combination of recognition molecules. The studies reviewed in this chapter

collectively suggest that Ig superfamily molecules are promising candidates to

carry out these functions. Sdks andDSCAMs are likely to be only the beginning

of a large number of as-yet unidentified molecules that play crucial roles in

synaptic specificity.

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