Fig 2: Expressing only one of two DCC isoforms differentially disrupts midline entry and exit(A) DiI tracing of spinal commissural axons from WT controls and different mutants in an open-book orientation. At E11.5, WT axons had projected across the FP and made a sharp turn upon exit. Most DccLSE axons stalled within the FP, and the axons were present in bundles (arrowheads). Only a small number of axons had exited the FP and were present as single axons (arrows). Many DccSSE axons stalled before entering the FP at E11.5 (arrowheads). In DccLSE; DccSSE transheterozygotes, the axons entered and exited the midline normally. A heterozygous Robo3 KO allele facilitated midline exit when it was introduced into DccLSE mutants. Robo3+/- by itself does not cause significant guidance defects.7,18,43 Scale bar, 100 µm.(B and C) Quantification of midline entry (B) and exit (C), respectively, by DiI tracing at E11.5. The signals from four boxed areas were measured with ImageJ. The ratio between areas 2 and 1 represents midline entry, and the ratio between areas 4 and 3 represents midline exit. Data were normalized to WT controls and are presented as mean ± SD. One-way ANOVA with a Tukey post hoc test; p values and animal numbers as indicated.

Fig 3: Generation of CRISPR-Cas9-engineered mouse models for Dcc isoforms(A) Design of guide RNAs (gRNAs) for Dcc isoforms. Targeted nucleotides of the gRNAs are underlined, and the alternative splice sites are in red.(B) Genomic mutations in two independent founder lines for each Dcc isoform. Deleted nucleotides are in lighter gray and inserted nucleotides in lower case.(C) Only one isoform is produced in Dcc isoform mutants, measured by semi-quantitative RT-PCR. Both isoforms are expressed in wild-type (WT) controls at E11.5.(D) Anti-DCC staining of transverse spinal cord sections from WT and Dcc isoform mutant embryos at E11.5. DCC is present in both pre- and post-crossing axonal segments (yellow arrows). Brackets indicate the floorplate (FP) area. Dcc is also expressed in ipsilateral-projecting interneurons and motor neurons.37 Scale bar, 100 µm.

Fig 4: ROBO1 localization is unaffected in Dcc isoform mutants and ROBO1 binds both DCC isoforms(A) Anti-ROBO1 and anti-ROBO2 staining of transverse spinal cord sections at E11.5. In both WT and Dcc isoform mutants, ROBO1 and ROBO2 are present at low levels in pre-crossing axonal domains but become highly upregulated post crossing. Robo1/2 are also expressed in ipsilateral-projecting interneurons and motor neurons.18 Scale bar, 100 µm.(B) CoIP between DCC isoforms and ROBO1. Hemagglutinin (HA)-tagged DCC (L, long; S, short) and V5-tagged ROBO1 were coexpressed in COS-1 cells in the absence or presence of Netrin1 and SLIT2N stimulation (250 ng/mL each for 30 min).(C and D) Quantification of DCC and ROBO1 interaction from immunoblotting (IB) with anti-HA (C) and anti-V5 (D). Data were collected from three independent experiments and are presented as mean ± SD. Two-way ANOVA was performed with a Tukey post hoc test (not significant for all data points). Both DCC isoforms interacted with ROBO1 and at comparable levels, with or without ligand stimulation.

Fig 5: Expressing only one of two DCC isoforms differentially affects commissural axon outgrowth and guidance(A and C) Axon outgrowth from dorsal spinal cord (DSC) explants in response to bath-applied Netrin1 in 3D collagen matrix (A) and to Netrin1 coated on 2D surface (C), respectively (Netrin1 concentration and culture period as indicated). Under both conditions, axon outgrowth was increased in DccLSE mutants but was reduced in DccSSE mutants.(B and D) Quantification of axon outgrowth in (A) and (C), respectively; 3–5 explants were examined for each embryo. Data were normalized to WT controls and are presented as mean ± SD. One-way ANOVA with a Dunnett post hoc test; p values and animal numbers as indicated.(E) Axon projection around the midline in cultured whole embryos. The spinal cord was cut open at the dorsal midline and flattened in an open-book configuration to allow visualization of the FP area. A Gfp reporter was electroporated unilaterally into the DSC. Within 36 h of culturing (E10 to the equivalent of E11.5), WT axons were able to enter and exit the FP and make a sharp turn on the contralateral side (dashed lines indicate the FP boundaries). DccLSE axons entered the FP normally, but the majority stalled within or at the contralateral boundary of the FP (two examples are highlighted with arrowheads). By contrast, most DccSSE axons had not reached or entered the FP by the end of culturing (examples highlighted by arrowheads). Scale bars, 100 µm.