The connectivity of the adult nervous system underscores the complexity of processes necessary to establish this pattern. The number of cell-intrinsic and extrinsic factors required for neural development is large. Current tools of molecular biology and genomics allow us to survey the entire transcriptional profile of single cells allowing for a catalog of all the genes expressed in a particular cell type throughout the course of development to be determined. This list can be culled into interesting candidates, and animals genetically mutant at each of these independent loci can be created. Yet, the task of surveying each cell type and creating thousands of animals is daunting, particularly given that only a small subset of the genome is likely to be involved in any one specific process. As an alternative to candidate-based approaches for surveying developmental effectors, a phenotype-based, forward genetic approach can be used to survey the contribution of both spontaneous and induced mutations toward a developmental event.
We have designed and implemented a forward genetic screen to identify novel alleles critical for the developing PNS. Using a wholemount assay to label PNS neuron axons, we have identified several allelic groups with a wide array of phenotypes each affecting subsets of neurons. With each G1 mouse line, we anticipated the capacity to screen approximately 32 novel inactivating mutations. Thus, for 164 lines generated and screened using our assay we sampled the roles of ~5248 gene-inactivating mutations within the developing mouse embryo. These mutations are random and can include multiple sampling events within the same locus. Nonetheless, we have recovered interesting phenotypes in 5 out of 164 of our G1 lines. We mapped each of these phenotypes to small chromosomal regions and observed clear segregation according to Mendelian ratios. Our analysis of mice exhibiting these aberrant phenotypes uncovered mutations in previously uncharacterized genes as well as interesting mutations in genes encoding known axon guidance molecules. Using this screen, we identified Sema3AK108N
, a novel loss-of-function allele of Sema3A
. Mice harboring the Sema3AK108N
allele are homozygous viable but share guidance deficits within the PNS that are comparable to those observed in homozygous embryos in Sema3A
null alleles (Behar et al., 1996
; Taniguchi et al., 1997
). Previous characterized alleles of Sema3A
vary with respect to viability, and these effects are likely the result of variation in mouse genetic background (Taniguchi et al., 1997
mutants, like both Sema3A
null mutants, have exuberant overgrowth of the sensory neurons within the developing DRG and the trigeminal ganglia.
Sema3AK108N encodes a mutant form of Sema3A that is stable and secreted. Given the robust loss-of-function phenotypes seen of Sema3AK108N mutants, it was surprising to find that Sema3AK108N retains its ability to bind with high affinity to its receptor Npn-1 in vitro. Indeed, in silico modeling suggests that K108 is distal to the site of Sema3A-Npn-1 interaction. Instead, we suggest that K108 is an essential component of a Sema3A-Plexin interaction site. Further experiments are needed to compare and contrast the semaphorin-plexin interaction surfaces in both Npn-independent and dependent systems. It is possible that two distinct surfaces on the semaphorin exist, representing high- and low-affinity sites, that mediate Npn-independent and dependent binding to plexins, respectively. Alternatively, it is possible that the high-affinity semaphorin-plexin interaction site in Npn-independent systems is simply an extended version of a secondary low-affinity site found in Npn-dependent semaphorins. This second possibility seems likely given that K108 is conserved not only among all members of the class 3 secreted semaphorins but among all known plexin-binding semaphorins. Interestingly, K108 is not found in Sema7a, which signals guidance responses via interactions with integrins.
In addition to providing insights into the possible site of sema-plexin interaction within the holoreceptor complex, Sema3AK108N may provide an interesting mouse model in which to study Sema3A plexin-independent developmental events. Previous studies have proposed a Sema3A-Npn-1-L1 receptor complex critical for the development of DRG projection and neurons of the developing cortex. It is possible that comparisons between Sema3AK108N and Sema3A null alleles might further elucidate the role of L1 in semaphorin signaling.
In conclusion, we have identified a novel allele of Sema3A, Sema3AK108N, which abolishes Sema3A signaling in vivo. Our in vitro analysis suggests that this amino acid substitution does not globally affect the expression or stability of the protein. Moreover, Sema3AK108N appears to be able to bind its receptor, Npn-1, with high affinity. We hypothesize that K108N alters the interblade region between blades 1 and 2 of the Sema domain and affects the ability of the mutant Sema3A to interact with PlexinA in the Npn-1/PlexA holoreceptor complex. Thus, this novel Sema3A allele establishes a new mouse model system for understanding the roles of Sema3–Npn-1-Plexin signaling during mouse development and in the adult.