Rapid Wallerian degeneration in mice lacking DR6 in comparison to multiple mutants with delayed degeneration, assessed at 3 days after axotomy

(A) Representative semithin (1st and 2nd columns) and electron micrographs (3rd column) of transverse sciatic nerve sections of distal nerve stumps from wild-type (WT) and the indicated mutant mice 3 days after sciatic nerve transection. Note complete structural disintegration of transected axons with absent or floccular cytoskeleton and collapsed myelin sheaths in the preparations from WT, DR6-/-, and DR6-/-, CMV-Cre mice. In contrast, the majority of disconnected axons from heterozygous WldS mice, and from Phr1 and SARM1 knockout mice, are structurally preserved with uniform cytoskeleton and intact myelin sheaths. Scale bars: 10 µm (B) Quantification of preserved axons in transverse sciatic nerve sections of distal nerve stumps from mice with the indicated genotypes. Each symbol in the scatter dot plots represents the quantification from one animal (% of control axon numbers in micrographs from uninjured contralateral nerve preparations for each animal).

Normal rates of nerve injury-induced axon disintegration in DR6-deficicient mice at earlier stages of Wallerian degeneration

(A) Representative semithin micrographs of transverse tibial nerve sections of distal nerve stumps from mice with the indicated genotypes (top) 30h after sciatic nerve transection, and corresponding quantifications of relative axon survival (bottom). (B) Representative semithin micrographs of transverse tibial nerve sections of distal nerve stumps from mice with the indicated genotypes (top) 36h after sciatic nerve transection, and corresponding quantifications of relative axon survival (bottom).

Scale bars: 10 µm

Normal Schwann cell c-Jun injury responses during WD in mice lacking DR6

(A, C) Representative immunofluorescence confocal micrographs for c-Jun with DAPI nuclear counterstaining on transverse frozen sections from contralateral uninjured nerve (uncut) and distal sciatic nerve stumps 3 days and 30 h following axotomy in mice with the indicated genotypes.(B, D) Corresponding quantifications of percentage of c-Jun immunoreactive and DAPI+ cells on nerve sections.

Scare bars: 50µm.

Normal degeneration kinetics of injured DRG neurites in primary neuronal cultures from DR6 knockout mice

(A, D) Western blot analysis (cropped blot images) of DR6 protein expression in dorsal root ganglia (DRGs) from DR6-/- embryos and from DR6 LoxP/LoxP embryos infected with a lentivirus expressing Cre recombinase (DR6Cre), together with the respective control preparations. (B, E) Time course of neurite fragmentation, quantified as degeneration index (DI) (see Experimental Procedures) in DRG preparations from embryos with the indicated genotypes or lentiviral infection conditions. The data points shown in (B) represent the averaged neurite DI values calculated from multiple DRG preparations from each embryo at the indicated time point after injury (one data point = one embryo). The data points shown in (E) represent the averaged neurite DI values calculated from multiple micrographs acquired from each DRG preparation in a cell culture well at the indicated time point after injury (one data point = one well). (C, F) Representative phase-contrast micrographs of disconnected DRG neurites from embryos with the indicated genotypes and lentiviral infection conditions at the indicated time points after axotomy. Scale bars: 50µm.

Disrupted DR6 expression in two DR6 knockout mouse models

(A, C) Quantitative real-time PCR analysis of relative brain DR6 mRNA levels from control and mutant mice with the indicated genotypes. Each dot represents the measurement from one mouse. (B, D) Western blot analysis (cropped blot images) of brain lysates from control and mutant mice with the indicated genotypes probed with the shown antibodies. Each western blot lane represents the brain lysate data from one individual mouse.

Normal developmental myelination in sciatic nerves from mice lacking DR6 at postnatal day 1

(A, C) Representative semithin micrographs (A, C) of transverse sciatic nerve sections from control and mutant mouse pups at postnatal day 1 with the indicated genotypes. Red arrows depict examples of axons with nascent myelination. Scale bars: 10µm. (B, D) Quantification of myelinated axons in transverse sciatic nerve sections from control and mutant pups at postnatal day 1 with the indicated genotypes. Each dot represents the quantification obtained from one mouse pup.

Normal nerve histomorphometry in DR6-deficient mice

(A, C) Quantification of g ratios (left: scatter plots show g ratios of individual myelinated axons as function of axon diameter, right: corresponding cumulative g ratios per animal) in tibial (A) and sciatic nerves (C) from adult control and mutant mice with the indicated genotypes. (B, D) Axon caliber and fiber caliber (axon plus myelin sheath) distributions in tibial (B) and sciatic nerves (D) from adult control and mutant mice with the indicated genotypes. Each dot represents the measurement from one mouse.

Normal JNK activation in sciatic nerves from DR6-deficient mice

(A, C) Representative western blots (cropped blot images) of lysates from uninjured (0 min) and axotomized distal sciatic nerve stumps (30 min after axotomy) from control and mutant mice with the indicated genotypes probed with the shown antibodies. Each lane represents the result from one mouse sciatic nerve. (B, D) Densitometric quantifications with statistical analysis of western blot data for all experimental animals used. Each dot represents a measurement from sciatic nerve lysate from one mouse.

Normal ultrastructure of transdifferentiated Schwann cells and normal myelin remodeling dynamics during Wallerian degeneration in DR6-deficient mice

(A) Representative electron micrographs of transdifferentiated Schwann cells (red arrowheads) that lost axonal contact from distal sciatic nerve stumps 3 days following nerve transection in control and DR6 knockout mice with the shown genotypes. Note similar ultrastructure with marked cytoplasmic expansion, increased organelle content, and myelin debris (myelin ovoids) in Schwann cell bodies in all genotypes. Scale bar: 2µm. (B, C) Quantification of area occupied by myelin sheaths and myelin debris in transverse nerve sections from control and mutant mice with the indicated genotypes. Each dot represents the quantification obtained from one nerve.