Regulated temporal-spatial astrocyte precursor cell proliferation involves BRAF signalling in mammalian spinal cord

DEV077214 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Figure S1 -

    Fig. S1. Aldh1L1-GFP can be used for flow cytometric sorting of acutely dissociated spinal cord astrocyte populations. (A) 17.6% of P6 spinal cord cells are Aldh1L1-positive. (B) No co-labelling of GFP-positive cells with the pan-haematopoetic and microglial marker CD45. (C) Substantial overlap of the Aldh1L1-positive population with GFAP expression is observed (upper right corner of the plot indicates co-expression of both markers), with varying degrees of staining. Little to no GFAP positivity is seen in the GFP-negative population.

  • Supplemental Figure S2 -

    Fig. S2. Developmental epoch of astrocyte proliferation versus oligodendrocyte proliferation profile. (A) Timetable of thymidine analogue injection at five different periods is shown. The animals were then analysed at P15-18 to reveal the percentage of thymidine analogue-positive Aldh1L1-GFP cells versus thymidine analogue-positive OLIG2+ cells. (B,C) Representative pictures of BrdU+ (B) or EdU+ (C) Aldh1L1-GFP cells.

  • Supplemental Figure S3 -

    Fig. S3. The activity of PI3-K signalling pathway does not correlate with proliferative astrocyte precursors at embryonic stage. Spinal cord sections from E16 embryos are shown. Two activity readouts of PI3-K pathway, p-AKT and p-S6, were stained and largely absent in Aldh1L1-GFP+ cells.

  • Supplemental Figure S4 -

    Fig. S4. Conditional activation of BRAFV600E results in overproduction of Aldh1L1-GFP+ cells at late embryonic/early neonatal stages. (A) Activation of Braflox/lox by Blbp-lacZ-cre results in specific downregulation of BRAF in spinal cord but not in the dorsal root ganglion. (B) Activation of BRAFV600E does not increase apoptotic rate assessed by activated Caspase-3 staining. (C) Astrocyte culture from postnatal day 1 pups of hGFAP-cre; BrafCA shows increased proliferative rate compared with control pups. (D) In hGFAP-cre; BrafCA spinal cords, the activity of pERK1/2 is diminished at later postnatal stage (P4). Error bars represent s.d.

  • Supplemental Figure S5 -

    Fig. S5. EGFR transcript expression in Aldh1l1-GFP-positive cells. qPCR showing relative EGFR expression in flow-cytometrically sorted Aldh1l1-GFP positive populations at E13.5, E14.5, E17.5 and P6 (data represent three biological replicates per time point). The result was normalized to E13 expression levels and β-actin was used as internal control. Error bars represent s.d.