Genetic analysis of DSCAM's role as a Netrin-1 receptor in vertebrates

Abstract

Down syndrome cell adhesion molecule (DSCAM) has mainly been characterized for its function as an adhesion molecule in axon growth and in self-recognition between dendrites of the same neuron. Recently, it has been shown that DSCAM can bind to Netrin-1 and that downregulation of DSCAM expression by siRNAs in chick and rodent spinal cords leads to impaired growth and turning response of commissural axons to Netrin-1. To investigate the effect of complete genetic ablation of DSCAM on Netrin-1-induced axon guidance, we analyzed spinal commissural neurons in DSCAM-null mice and found that they extend axons that reach and cross the floor plate and express apparently normal levels of the Netrin receptors DCC (deleted in colorectal carcinoma) and Neogenin. In vitro, commissural neurons in dorsal spinal cord explants of DSCAM-null embryos show normal outgrowth in response to Netrin-1. We therefore conclude that DSCAM is not required for Netrin-induced commissural axon outgrowth and guidance in mice.

Figure 1.

Normal commissural axon trajectory in DSCAM mutant spinal cord. A–H, TAG-1 immunostaining revealed that commissural neurons of DSCAM^−/− embryos (E–H) reached and crossed the floor plate as in control littermates (A–D). n = 4 embryos per genotype. I–P, While DSCAM mRNA (I, M) was not detected in DSCAM^−/− e11.5 embryos (M), DCC (O) and Neogenin (N) expression was maintained at levels comparable to wild-type (J, K) and position and numbers of motor neurons was not affected as revealed by Isl1 immunostaining (L, P). n = 4 embryos per genotype. Arrowheads, commissural neurons; asterisks, motor neurons. Scale bar (in P), 65 μm in all panels. Q, Western blot analysis of e11.5 DSCAM^−/− spinal cords reveal that DCC and Neogenin expression was maintained at levels comparable to wild-type (n = 3 per genotype).

Figure 2.

Netrin-induced axon outgrowth is unaffected by DSCAM mutation. A–F, Dorsal spinal cord explants of e11.5 DSCAM mutant (C, F), DSCAM^+/− (B, E) and control littermates (A, D) were cultured in the presence or absence of 200 ng/ml Netrin-1. G, Quantification of axon outgrowth. N > 6 explants per condition; n = 4 wild-type, 7 DSCAM^+/− and 4 DSCAM^−/− embryos. Scale bar (in F), 20 μm in all panels.

Figure 3.

Normal commissural axon floor plate crossing in DSCAM-null mutants. A–B, DiI injections into e12.5 dorsal spinal cords (on the left side of the shown “open book”) of control littermates (A, A′) and DSCAM mutants (B, B′) show most commissural axons crossed the floor plate (dashed line) and turned anteriorly. C, Quantification of DiI labeled axonal projections. N = 36 injections in DSCAM^−/− and 37 in wild-type embryos; n = 4 embryos per genotype. Scale bar (in B′), 50 μm in all panels.

Figure 4.

DCC/DSCAM compound mutants have commissural axon trajectory similar to DCC mutants. A–D, TAG-1 immunostaining at e11.5 brachial spinal cord embryos. Commissural neurons of DCC^+/−; DSCAM^−/− embryos (B) reached and crossed the floor plate as in control littermates (A). Like DCC mutants (C), a small percentage of TAG-1 commissural neurons of e11.5 DCC/DSCAM compound mutant embryos (D) reached and crossed the floor plate (arrows) while the majority were not able to reach the floor plate (arrowheads). N = 3 DCC^−/−; DSCAM^−/−, 3 DCC^−/−, 3 DCC^+/−; DSCAM^−/−, and 3 control embryos. Scale bar, 50 μm in all panels.