Semaphorins are a large and diverse family of widely expressed secreted and membrane-associated proteins, which are conserved both structurally and functionally across divergent animal phyla. This diversity in expression, structure, and function is highlighted in the manner in which a number of the semaphorins were originally characterized. The first semaphorin to be discovered, the grasshopper transmembrane protein semaphorin-1a (Sema-1a; originally named Fasciclin IV), was identified in a screen for molecules with distinctive temporal and spatial distributions in the developing grasshopper nervous system 1. In parallel experiments, a neuronal growth cone collapsing factor associated with chicken brain membranes was biochemically purified and found to be a secreted semaphorin (Sema3A; originally named Collapsin) 2. Separate experimentation and molecular characterization revealed that an antigen first observed in the 1970s as present in high frequency on human red blood cells, the John Milton Hagen (JMH) human blood group antigen, was a glycosylphosphatidylinositol (GPI)-linked semaphorin (Sema7A; also known as CDw108) 34. And work in the human immune system showed that an antigen first characterized in 1992 for its presence on the surface of T lymphocytes was a transmembrane semaphorin (Sema4D; originally named CD100) 5.

Sequences encoding a number of different semaphorins have since been identified in nematode worms, insects, crustaceans, vertebrates, and viruses, but to date they have not been described in protozoans, plants, or the most primitive metazoans. Although initially given various and often conflicting names, these sequences have now been consolidated into one family called the semaphorins; the name is derived from the word 'semaphore', meaning to convey information by a signaling system 67. The semaphorin gene family currently includes 20 members in mice and humans and five in Drosophila, and they can be divided into eight classes, 1-7 and V (Figures 1, 2) 7. Vertebrates have members in classes 3-7, whereas classes 1 and 2 are known only in invertebrates and class V only in viruses.

Semaphorin genes are dispersed throughout the genome, typically including several exons per gene, and are known to be alternatively spliced. There is considerable sequence diversity within the family: with a few exceptions, individual members are not more than about 50% identical to each other at the amino-acid level (see Additional data file 1).

The eight main classes of semaphorins 7 differ in sequence and overall structural characteristics, but all members of the family contain a conserved extracellular domain of about 500 amino acids termed the semaphorin (sema) domain (Figure 2). This domain shows considerably higher conservation among the different semaphorins and across phyla than do the full-length proteins (see Additional data file 2). In addition to several blocks of conserved amino acids, the sema domain is characterized by highly conserved cysteine residues that have been found to form intrasubunit disulfide bonds 8. Crystal structures have revealed that the sema domain of both the mouse secreted semaphorin Sema3A and the human transmembrane semaphorin Sema4D fold in a variation of the β propeller topology, a common topology that occurs in proteins with diverse functions (reviewed in 8). Interestingly, these sema domains fold in a manner that is most similar to the β propeller topology of integrins and low-density lipoprotein (LDL) receptors.

The sema domain is also a critical component through which semaphorins mediate their effects 91011. In particular, an approximately 70-amino-acid region within the sema domain is important for the effects of Sema3A on repulsive axon guidance and the collapse of the growing tip or growth cones of axons, which stops their extension 9. Structurally, this portion of the sema domain of Sema3A and Sema4D appears to correspond to blade three of the seven-bladed β propeller topology 8. Interestingly, a small stretch of amino acids homologous to tarantula hanatoxin, a K⁺ and Ca²⁺ ion-channel blocker, is also important for the growth-cone-collapsing effects of Sema3A 12.

Immediately to the carboxy-terminal side of the sema domain, semaphorins contain a plexin-semaphorin-integrin (PSI) domain (Figure 2). This small stretch of cysteine-rich residues has also been referred to as a MET-related sequence (MRS) or a cysteine-rich domain (CRD). With the exception of some viral semaphorins, all examples of proteins containing a sema domain have a PSI domain 8. Crystal-structure analysis indicates that this domain is highly conserved, but its three-dimensional position relative to the sema domain can vary among semaphorins 8. Semaphorins also have consensus N-linked glycosylation sites and may be alternatively spliced (as in Drosophila Sema-1a 13, and mammalian Sema3F 14 and Sema6A 15), although little is known about the significance of these modifications.

In contrast to these defining characteristics, individual semaphorins have a number of distinguishing features. Semaphorins vary in their membrane anchorage, and include secreted, transmembrane, and GPI-linked family members (Figure 2). They may also contain additional sequence motifs, including a single C2-class immunoglobulin-like (Ig) domain, a stretch of highly basic amino acids, and/or seven canonical type 1 thrombospondin repeats (TSRs; Figure 2). These additional domains are responsible for at least some of their functional effects; for example, the Ig domain and basic tail of chicken Sema3A potentiate the effect of its sema domain in growth-cone collapse 9, and the thrombospondin repeats of mammalian Sema5A are important in regulating the effect of Sema5A on axon guidance 1116.

As a group, semaphorins are expressed in most tissues and this expression varies considerably with age. The expression patterns of the individual semaphorins are best characterized in the nervous system, particularly during development, where most, or perhaps all, semaphorins are widely expressed in the nervous system by neuronal and non-neuronal cells (reviewed in 17; see Table 1 for details of the expression and functions of all members of the family and associated references). Semaphorins are also widely expressed in many organ systems and their derivatives, including the cardiovascular, endocrine, gastrointestinal, hepatic, immune, musculoskeletal, renal, reproductive, and respiratory systems.

No particular pattern of expression appears to define each of the different classes of semaphorins, but many are dynamically expressed in particular areas during development, and this expression often decreases with maturity. In the nervous system, for example, semaphorin expression is often associated with growing axons as they form axonal tracts, but this expression often decreases following the formation of the tracts. Interestingly, changes in the adult expression levels of semaphorins have been described following injury in neuronal and non-neuronal tissues, during tumorigenesis, and in association with other pathological conditions.

The diverse expression patterns of the different semaphorins suggest that they are important in a variety of functions during development and into adulthood. Indeed, genetic analyses in both invertebrates and vertebrates indicate that semaphorins are often required for viability and reveal, in combination with additional functional assays, distinct roles in various physiological and pathological processes in most or perhaps all tissues. These studies reveal that semaphorins on cellular processes such as adhesion, aggregation, fusion, migration, patterning, process formation, proliferation, viability, and cytoskeletal organization.

Semaphorins are best known for their roles in nervous system development, and a number of approaches in vivo and in vitro indicate that semaphorins can enable axons to find and connect with one another and their other targets (reviewed in 18). An important way in which semaphorins guide these growing axons is by repelling them or preventing them from entering certain regions. For example, characterization of their normal expression patterns, the defects observed in particular semaphorin mutants, and assays in vivo and in vitro have revealed that at least some semaphorins form molecular boundaries to prevent axons and cells from entering inappropriate areas. Semaphorins also have roles in physiological and pathological processes in the adult. In the nervous system, altered semaphorin function has been linked to epilepsy, retinal degeneration, Alzheimer's disease, motor neuron degeneration, schizophrenia, and Parkinson's disease 19202122.

Semaphorins may also limit the ability of axons to regrow after injury and prevent abnormal sprouting of axons involved in pain or autonomic function 23242526. In the immune system, semaphorins are critical for various phases of the immune response (Table 2; reviewed in 27). Semaphorins are also involved in cancer progression, by affecting chemotaxis, viability, tumorigenesis, metastasis, and angiogenesis (reviewed in 28). More recently, semaphorins have also been implicated in vascular health and heart disease (reviewed in 29).

Despite considerable progress in our characterization of members of the semaphorin family, much remains to be learned about their functions and molecular mechanisms of action. Several semaphorins have yet to be functionally characterized, and many have undergone only a cursory examination. A number of questions remain, including the purpose of having so many related semaphorins and the underlying logic to their complex expression patterns and physiological roles. The degree of interaction among semaphorins is also poorly understood. Do they regulate each other's signaling cascades? Do they physically associate? What special attributes and abilities do the secreted, transmembrane, and GPI-linked forms of semaphorins functionally provide?

Understanding the signaling cascades that underlie the different functional effects of semaphorins will provide insights into these important proteins. Are there differences in the signaling cascades activated by the different semaphorins? How much do their signaling cascades vary in order to mediate their different cellular effects? How do semaphorins exert their dramatic effects on the cytoskeleton?

A more detailed understanding of the role of semaphorins in the normal functioning adult is important. In the nervous system, the role of semaphorins in forming neural connections is well established, but the role of semaphorins in neural connectivity as it pertains to thought, emotion, memory, and behavior is unknown. The role of semaphorins in human disease and pathology is also poorly understood. Mutations in semaphorins are associated with patients with cancer 28, retinal degeneration 51, decreased bone mineral density 52, rheumatoid arthritis 53, and CHARGE syndrome (a disorder characterized by cranial nerve dysfunction, cardiac anomalies, and growth retardation) 54. Further characterization of the semaphorins and a better understanding of their signaling mechanisms will undoubtedly uncover additional roles for semaphorins and semaphorin signaling in human disease.

Given the role of semaphorins in a wide range of tissues and functions including neurobiology, vasculobiology, cancer biology, and immunobiology, further characterizing the semaphorins and their signaling cascades will reveal fundamental mechanisms of how these systems work and strategies for preventing and treating pathologies associated with them.

The following additional data files are available: tables of the protein sequence identities between different semaphorins over the whole sequence (Additional data file 1) and the sema domain (Additional data file 2).