A Forward Genetic Screen in Mice Identifies Sema3A^K108N, which Binds to Neuropilin-1 but Cannot Signal

Abstract

We have performed a three-generation, forward genetic screen to identify recessive mutations that affect the patterning of the peripheral nervous system. Using this assay, we identified Sema3A K108N, a novel loss-of-function allele of Sema3A. Class 3 semaphorins, which include Sema3A, are structurally conserved secreted proteins that play critical roles in the development and function of the nervous system. Sema3A K108N mutant mice phenocopy Sema3A-null mice, and Sema3A K108N protein fails to repel or collapse DRG axons in vitro. K108 is conserved among semaphorins, yet the loss-of-function effects associated with K108N are not the result of impaired expression, secretion, or binding of Sema3A to its high-affinity receptor Neuropilin-1 (Npn-1). Using in silico modeling and mutagenesis of other semaphorin family members, we predict that Sema3A K108N interacts poorly with the Npn-1/PlexA holoreceptor and, thus, interferes with its ability to signal at the growth cone. Therefore, through the use of a forward-genetic screen we have identified a novel allele of Sema3A that provides structural insight into the mechanism of Sema3A/Npn-1/PlexinA signaling.

Class 3 semaphorins: physiological vascular normalizing agents for anti‐cancer therapy

“…PlexinD1 however can also combine with either Nrp1 or Nrp2 to create Sema3 receptor complexes with a much higher affinity than the corresponding plexinA-containing holoreceptors [82]. Results from recent mutational studies [83,84] led to the proposal of a model in which Sema3A binds to both Nrp1 and plexins to trigger neuronal growth cone collapse (Fig. 1).…”

“…The a1 domain of Nrp1 also has a role in Sema3A signalling probably through involvement in Sema3A binding and/or receptor oligomerization. Recent studies [83,84] indicate that Sema3A must bind both Nrp1 and the signalling receptor subunit plexin. In ECs, Sema3A can signal either via plexinA4 or plexinD1.…”

Nervous vascular parallels: axon guidance and beyond

“…However, plexinD1 can also combine with either Nrp1 or Nrp2 to give rise to SEMA3 holoreceptor complexes endowed with a higher affinity than the corresponding plexinA-containing holoreceptors . Furthermore, recent mutational studies (Merte et al, 2010;Nogi et al, 2010) suggest that, similarly to SEMA3E, the collapsing activity of Nrp1-bound SEMA3A also relies on its ability to interact, albeit at lower affinity, with plexins.…”

Dlg5 Regulates Dendritic Spine Formation and Synaptogenesis by Controlling Subcellular N-Cadherin Localization

“…Mutagenesis and screening. N-ethyl-N-nitrosourea mutagenesis was performed as described previously (Merte et al, 2010a;Merte et al, 2010b). Briefly, C57BL/6 (B6) male mice were injected with three doses of 100 mg/kg body weight N-ethyl-N-nitrosourea to induce random mutations throughout the genome.…”