1 The IrreC/Nephrin/SYG-1 Family of Proteins

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and NEPH3). syg-1 and syg-2 are two closely related genes in the C. elegans

genome, suggesting that they might have derived by a gene duplication event

from a common ancestoral precursor gene. Two Drosophila genes, IrreC and

Duf, are homologous to syg-1 and the other two are to syg-2. In some publications, IrreC has also been referred to as Roughest, and DUF (dumbfounded) as

Kirre (Kin of IrreC). The four human genes are also part of this gene family.

Nephrin is the sole homolog to syg-2, while the other three human genes are

most homologous to syg-1. NEPH1, 2, and 3 have also been named Kirrel1, 3,

and 2, respectively (Fig. 11.1).



Fig. 11.1 Phylogenetic analysis of the IrreC/Nephrin/SYG-1 family proteins. Amino acid

sequences of full-length proteins were analyzed with sequence cluster method



11.2 SYG-1 and SYG-2 Encode Synaptic Target Choice of the

HSNL Neuron in C. elegans

The general specificity of neuronal connections is established through a series of

developmental events, including cell migration, axon and dendrite outgrowth,

and guidance, followed by target recognition and synapse assembly. Each step

gradually limits the pool of possible connecting targets, eventually leading to

the target choice. Although a large body of experimental data have provided us

with a detailed understanding of the molecular mechanisms of axon guidance,

little is known about how neurons make final decisions in selecting their

synaptic partners. Based on anatomical and physiological experiments, it is

well documented that synaptic connections in the brain are precise and stereotyped (Benson et al. 2001). Therefore, it is very likely that there are molecular

mechanisms by which neurons select their correct synaptic partners to initiate

synaptic assembly, while rejecting other contacting cells in the same target field.

Naturally, one might expect that the molecules mediating the recognition event

are directly or indirectly involved in the assembly of the pre- and the postsynaptic apparatus. The most intuitive model that has been generally accepted is

that cell adhesion molecules (CAMs) found on pre- and postsynaptic cells

mediate specific cell recognition events and that the interaction between these

CAMs also initiates synaptogenesis.

Surprisingly, little experimental evidence is available to support the existence

of membrane molecules that can effectively perform the synapse-inducing

cell–cell recognition events. For any adhesion molecules to qualify for this



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job, several criteria must be fulfilled. First, these molecules need to be

expressed by the synaptic partners at the time of synaptic target selection

and synapse formation. Second, these molecules should be present at

synapses. Third, the interaction between these adhesion molecules should

trigger a molecular assembly program, which results in the construction of

the pre- and the postsynaptic apparatus. The fourth and probably the most

stringent criteria is that in the absence of these molecules, there should be

defects in synaptic target choices.

A number of CAMs, which fulfill at least some of these requirements, are

likely candidates for acting as synaptic specificity determinants. For example, the neurexin and neuroligin families of membrane adhesion molecules

are expressed in neurons and are localized at synapses. More importantly,

when expressed in exogenous cells, a neurexin and neuroligin interaction is

sufficient to trigger formation of pre- and postsynaptic specializations (Craig

and Kang 2007). However, in knockout mice where most or all of the

neurexins and neuroligins are deleted, little synapse development phenotypes

can be detected (Missler et al. 2003, Varoqueaux et al. 2006). These results

suggest that the neurexin and neuroligin molecules are synaptic localized

adhesion molecules with their abilities to induce synapse formation (see

Chapter 17). But whether they encode specificity is still an open question.

Immuoglobulin domain family proteins, called SynCAMs, are also synaptically localized adhesion molecules that have synapse-promoting activities

(Biederer et al. 2002) (see Chapter 8). However, their precise roles in

synapse development have not been validated by a loss-of-function genetic

analysis. EphrinB and Eph receptors are another class of molecules that

qualify as prime candidates for molecules for synaptic specificity (see Chapter 16). Both ephrinB and its receptors are localized at synapses and have

pre- or postsynaptic inducing activities (Aoto and Chen 2007).

Forward genetic analysis using one set of motor neuron synapses in the

nematode C. elegans led to the identification of two immunoglobulin super

family (IgSF) proteins that fit the bill as synapse-inducing adhesion molecules. The egg-laying behavior of C. elegans is controlled by two pairs of

motor neurons, HSNL/HSNR and VC4/VC5. The HSNs form en passant

synapses onto vulval muscle cells and onto the VC neurons. Although HSN

axons contact many other cells, they do not normally form synapses with

them. Furthermore, the egg-laying synapses elaborated by HSNs are clustered in a short and stereotyped segment (about 10 mm) of the HSN axons

(at least 100 mm). As expected, the position of the synapses matches the

physical location of the postsynaptic targets: the VC neurons and the vulval

muscles (Shen et al. 2004). Because of the simplicity of the worm nervous

system and the ability of specific labeling of HSN synapses, it is possible to

ask several fundamental questions about synapse formation in vivo using

this system. For example, do the postsynaptic cells induce the development

of presynaptic specializations directly? And what are the molecules that

mediate the specificity of synapse development in vivo?



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Fig. 11.2 SYG-1 and SYG-2 determine the localization of presynaptic terminals in HSN axons.

A model illustrating the cellular action of SYG-1 and SYG-2. SYG-2 is expressed in guidepost

vulval epithelial cells. SYG-1 functions in the HSN axon and is recruited to future presynaptic

location via a direct interaction with SYG-2. The SCFsel-10 ubiquitin E3 complex is diffusely

localized throughout the HSN axon and is responsible for the degradation of the presynaptic

apparatus. SYG-1 binding to Skr-1 inhibits the assembly of the SCF complex and hence

locally protects synapses through the suppression of SCF activity



The first surprise that resulted from an analysis of this system was the

observation that the postsynaptic cells (VC neurons and vulval muscles) are

dispensable for the correct localization of the presynaptic specializations in

HSN. In animals in which the postsynaptic cells were ablated by laser-assisted

methods prior to the axon guidance event, HSNs still cluster presynaptic

vesicles at the right locations. This suggests that the synapse-inducing signal

comes from a source other than the postsynaptic cell (Shen and Bargmann

2003). Shen and Bargmann reported that a group of epithelial cells play an

essential guidepost role for HSNL synaptogenesis. These guidepost cells contact the HSNL axon and induce the clustering of synaptic vesicles at the site of

contact, shortly before the normal postsynaptic targets are innervated. In the

absence of guidepost cells, clusters of HSNL synaptic vesicles accumulate at

ectopic locations. Further analysis of the guidepost cells showed that they

physically contact the HSN axons during the initial specification of the presynapse in HSN. The exact location of the contact between HSN and the

guidepost cells defines the location of the synapses. These results suggest that

the guidepost cells are not required for HSNs to form synapses per se, but

instead, they are required to specify the location of the HSN synapses.

A forward genetic screen yielded several mutants with abnormal HSN

synapse localizations. In syg-1 and syg-2 mutants, HSN synapses are drastically

reduced at the wild-type location and robustly form at anterior ectopic locations along the HSN axon. Interestingly, this aberrant localization pattern

closely mimics the synapse localization pattern found in animals with ablated

guidepost cells. Molecular cloning of the genes affected in these mutants



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revealed that both the SYG-1 and the SYG-2 genes encode transmembrane

IgSF proteins. Furthermore, SYG-1 and SYG-2 are homologous to each other,

and they both belong to an evolutionarily conserved family of molecules (Shen

and Bargmann 2003, Shen et al. 2004).

A further genetic and developmental analysis of these two genes showed that

SYG-2 is expressed transiently by the guidepost cells during the early stages of

HSNL synaptogenesis. SYG-1 functions in the presynaptic HSNL neuron and

localizes to synapses early during synapse formation. In loss-of-function syg-1

and syg-2 mutants, the HSNL axon fails to form synaptic connections with its

normal targets (VC neurons and vulval muscles) and instead forms synapses

with adjacent cells that do not normally receive synaptic input from the HSNL

axon (Shen and Bargmann 2003, Shen et al. 2004). When SYG-2 is expressed in

the secondary vulval epithelial cells, which are located next to the guidepost

cells and do not normally express SYG-2, both SYG-1 and synaptic vesicles

localize to the segment of the HSNL axon that contacts these secondary vulval

epithelial cells (Fig. 11.2). This gain-of-function phenotype supports the idea

that interactions between SYG-1 and SYG-2 are sufficient to trigger synaptic

vesicle clustering. A biochemical analysis showed that the extracellular domains

of SYG-1 and SYG-2 are likely to directly interact with each other (Shen et al.

2004). Taken together, these results suggest that SYG-2 is the guidepost molecule. It binds to SYG-1 on the HSN axon and localizes SYG-1 to the future

synaptic region. This interaction between SYG-1 and SYG-2 eventually leads to

the localized assembly of the presynaptic machinery.

These results left several questions unanswered. How does the interaction of

SYG-1 and SYG-2 induce synapse formation? Why do synaptic vesicles accumulate at ectopic sites in the syg-1 and syg-2 mutants? How does SYG-1 ensure

formation of presynaptic sites at the appropriate location in HSNL? Insights

into these questions were obtained by studying the developmental process that

leads to the specific distribution of HSN synaptic vesicles. During development,

transient presynaptic sites form at multiple locations along the HSNL axon.

However, by adulthood, most of these presynaptic sites are eliminated and only

those that contain SYG-1 remain. As it turns out, SYG-1 helps achieve this

stereotypical presynaptic pattern by playing a protective role. Ding and colleagues recently showed that an E3 ubiquitin ligase, a Skp1-Cullin-F-box (SCF)

complex, acts in HSNL to eliminate unwanted presynaptic sites (Ding et al.

2007). Animals with loss-of-function mutations in components of this complex

have delayed or incomplete elimination of superfluous presynaptic sites. These

results argue that the SCF complex is at least in part responsible for eliminating

ectopic synapses during development.

However, it is still not clear how SYG-1 can protect synapses format areas

where SYG-1 protein is localized. The answer to this question came from

experiments examining the subcellular localization and the activity of the

SCF complex. It turns out that the SCF complexes are diffusely localized

throughout the entire HSN axons, implicating that active SCF can be found

on the whole axon. Binding studies revealed that SYG-1 binds to the Skp1



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homolog SKR-1 and prevents it from interacting with the rest of the SCF

complex. These results indicate that SYG-1 plays a protective role by locally

inhibiting the SCF complex, thus preventing the degradation of presynaptic

sites at locations marked by SYG-2 (Fig. 11.2). In the absence of SYG-1 or

SYG-2, the activity of the synapse-degrading SCF complex becomes redistributed more toward the normal synaptic region, which leads to fewer synapses in

the wild-type location and the appearance of ectopic synapses in the anterior

area.



11.3 Kirre/DUF, IrreC/Roughest, SNS, and Hirbris Mediate

Myoblast Fusion in Drosophila

The body wall musculature of the Drosophila embryo consists of 30 muscles in

each abdominal hemisegment (see Fig. 2.2). During development, each muscle

is formed by the fusion of two cell types: a founder cell and fusion-competent

myoblasts. The founder cell defines the identity of a particular muscle, and the

fusion-competent myoblasts are attracted by and fuse to the founder cell. The

location and number of fusion events are thought to determine the shape and

size of the muscles (Chen and Olson 2004).

Through forward genetic analysis, a large number of mutants were isolated

in which myoblast fusion is blocked (Richardson et al. 2008). Among the genes

affected by these mutations are four transmembrane IgSF proteins. Dumbfounded/Kirre (Duf) and Roughest/IrreC (Rst), the orthologs of SYG-1, function in the founder cell, while Sticks and Stones (SNS) and Hibris (Hbs), which

are orthologous to SYG-2, function in the fusion-competent cells. Similar to the

SYG-1 and SYG-2 heterologous interaction, Duf and Rst bind directly to SNS,

and these proteins are the primary mediators of myoblast adhesion. Loss-offunction genetic analysis showed that duf and rst act redundantly in the founder

cells and removal of both genes leads to a complete fusion defect (Strunkelnberg

et al. 2001). Interestingly, removal of SNS also causes a complete fusion defect

(Bour et al. 2000). Loss of Hbs causes a mild fusion defect, and thus it is possible

that Hbs regulates SNS during particular stages of fusion. Additionally, a

zebrafish Kirre/Duf-like molecule is also required for myoblast fusion, suggesting that this pathway is conserved in vertebrates (Srinivas et al. 2007).

It is interesting to compare HSN synapse formation and myoblast fusion.

These two seemly distinct processes share certain similarities. Both processes

involve asymmetric cell–cell recognition. In both cases, cellular junction structures form. Interestingly, both these junctional structures are transient. The

epithelial–HSN junction is eventually replaced by the mature synapses between

HSN and its postsynaptic targets. The myoblast fusion junction leads to the

perforation of the membrane and the fusion of the two cells. Another intriguing

parallel is the asymmetric expression of SYG-1 and SYG-2 and of IrreC/Rst,

Kirre/DUF and SNS. In the case of HSN synapse specification, SYG-1



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functions in the HSN neurons, while SYG-2 is specifically expressed and is

required in guidepost epithelial cells. SYG-1 predominantly binds to SYG-2 in

a heterologous fashion. In the case of myoblast fusion, while weak homophilic

interactions of IrreC/Rst and Kirre/Duf have been demonstrated in vitro,

heterophilic interactions between SNS and IrreC/Rst, as well as between SNS

and Kirre/Duf, are thought to be critical for myoblast fusion.



11.4 Kirre/DUF, IrreC/Roughest, SNS, and Hirbris Are Required

for Proper Patterning of the Drosophila Eye

The formation of the Drosophila compound eye involves the generation and

alignment of hundreds of identical eye units (ommatidia), which are organized

into an ordered array. In the last step of its development, a line of pigment cells

forms between neighboring ommatidia to insulate them from each other. During

this process, the pool of undifferentiated cells found between the ommatidial

clusters – the interommatidial precursor cells (IPCs) – undergoes morphogenetic

movements that eventually create a precise pigment cell lattice. This final patterning process includes carefully regulated cell shape changes, cell movements, and

cell death (Rusconi et al. 2000). During this process, IPCs contact other IPCs and

the primary pigment cells (18), but selectively form adherent junctions with the

primary pigment cells. Thus, one important aspect of this complex morphogenesis event is the cell–cell recognition between IPCs and primary pigment cells.

The first hint that the IrreC/Nephrin/SYG family protein might play an

important role in this process came from the analysis of mutant lines. When

ommatidia morphogenesis fails, the mutant eyes exhibit a ‘‘rough’’ appearance

compared with wild-type controls. Interestingly, both Roughest (IrreC) and

Hibris mutants exhibit a rough-eye phenotype. Expression analysis revealed

that Hibris and IrreC are expressed in complementary cell types. Hibris is made

by primary pigment cells, while IrreC is predominantly expressed by IPCs at the

time of the adhesion event. Furthermore, both Hibris and IrreC proteins are

localized to the interface between these two cell types. In vitro binding assays

confirmed the specific interaction between IrreC and Hibris (Bao and Cagan

2005). Taken together, these experiments suggest that the heterologous binding

between the IrreC/Nephrin/SYG family proteins across two different cell types

specifies another asymmetric cell–cell recognition event.



11.5 Vertebrate NEPH1 and Nephrin Are Critical Proteins

in Kidney Development

While the function of the IrreC/Nephrin/SYG proteins in synapse formation

and muscle fusion in vertebrate animals still awaits further confirmation, these

proteins are essential for the formation of the slit diaphragm, a cellular junction