Figure 5. Endoglycan expression reduces adhesion of commissural neurons in vitro.

(A-C) Commissural neurons dissected from HH25/26 chicken embryos were cultured on a layer of control HEK cells or HEK cells stably expressing human Endoglycan. Neurons were allowed to attach for 16 hr. Staining for Axonin-1 revealed a pronounced decrease in the number of commissural neurons on HEK cells expressing Endoglycan compared to control HEK cells (A and D). For each replicate, the number of neurons attached to HEK cells expressing Endoglycan was normalized to the number of cells attached to control HEK cells (D). The number of commissural neurons attached to control HEK cells was more than twice the number on Endoglycan-expressing HEK cells (only 0.47 ± 0.34 compared to 1 ± 0.35). Treatment with Neuraminidase (+N) or O-glycosidase (+O) abolished the difference. See Table 2 for values. N(replicates)=4. ns (not significant), ***p<0.001, ****p<0.0001, one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent standard deviations. O, O-glycosidase; N, Neuraminidase; vehic, vehicle; ctrl, control. Scale bar: 50 µm. Source data and statistics are available in Figure 5—source data 1 spreadsheet.

Figure 5—figure supplement 1. Endoglycan expression reduces adhesion of motor neurons in vitro.

Dissociated motoneurons dissected from HH26 chicken embryos were cultured on control HEK cells (A) or HEK cells stably expressing human Endoglycan (C). Neurons were allowed to attach for 40 hr. Staining for Neurofilament revealed a pronounced decrease in the number of motoneurons on HEK cells expressing Endoglycan (D) compared to control HEK cells (B). On control HEK cells (A), we found 52.8 ± 2.4 motoneurons per view field. On a layer of HEK cells expressing Endoglycan, only 18.3 ± 1.4 motoneurons were counted. Similar results were obtained in five independent experiments. Bar: 100 μm. ****p<0.0001, unpaired t test, two-tailed. Source data and statistics are available in Figure 5—figure supplement 1—source data 1 spreadsheet.

Figure 5—figure supplement 2. Post-translational modification of Endoglycan is required for its anti-adhesive effects on motoneurons.

Motoneurons (stained with anti-neurofilament antibodies in B,D,F,H) were cultured on control HEK cells (A–B) or Endoglycan-expressing HEK cells (C–H). Before adding the motoneurons, HEK cells were either left untreated (A–D), or treated with neuraminidase (E,F) or O-glycosidase (G,H) for 2 hr. Attached motoneurons were counted after 40 hr. Removal of sialic acid by neuraminidase (E,F) or O-glycosidase (G,H) abolished the anti-adhesive effect of Endoglycan (C,D). Quantification of the number of attached motoneurons under the different conditions is shown in (I). ns p>0.05, *p<0.05, **p<0.01, ****p<0.0001. One-way ANOVA with Tukey’s multiple comparisons test. Source data and statistics are available in Figure 5—figure supplement 2—source data 1 spreadsheet.