Regulation of axonal and dendritic growth by the extracellular calcium-sensing receptor (CaSR). Thomas N. Vizard, Gerard W. O Keeffe, Humberto Gutierrez, Claudine H. Kos, Daniela Riccardi, Alun M. Davies Supplementary Figure 1. CaSR protein is expressed in the developing SCG. Western blot analysis showing CaSR protein immunoreactive bands at ~140 kd and 160 kd (representing the partially and fully glycosylated monomeric forms of the receptor, respectively) in E16, E18 and P1 SCG lysates showing barely detectable protein at E16, a high level of expression at E18 and some decrease at P1. Equal loading is demonstrated by the detection of β-iii tubulin from the same samples.
Supplementary Figure 2. CaSR protein is localised in E18 SCG neurons. Labelling of a typical E18 SCG neuron with a C-terminus anti-casr polyclonal antibody after 24 hours in culture. The neuron was double labelled with an anti-β-iii tubulin antibody. Scale bar = 20 μm.
Supplementary Figure 3. Modulation of CaSR function/expression does not affect neuronal survival. Quantification of survival of (a) E18 SCG neurons grown in a range of Ca 2+ o concentrations after 24 hr, (b) E18 SCG neurons grown in 0.7 mm [Ca 2+ ] o with and without 10 nm of the calcimimetic NPS-R 467 after 24 hr, (c) E18 SCG neurons grown in 2.3 mm [Ca 2+ ] o with and without 10 nm of the calcilytic NPS- 89636 after 24 hr, (d) E18 SCG neurons transfected with either dominant-negative CaSR (DNCaSR) or control (CTR) plasmids after 48 hr, (e) P1 SCG transfected with either wild-type CaSR (WTCaSR) or control (CTR) plasmids after 48 hr, (f) E18 SCG neurons from Casr+/+ (WT), Casr+/ _ (HET) and Casr _ / _ (KO) mice grown in 2.3 mm [Ca 2+ ] o after 24 hr. Mean ± sem of data from at least 3 separate experiments in all cases.
Supplementary Figure 4. Marginal activation of CaSR at 0.7 mm Ca 2+ o. (a) Total neurite length and (b) Sholl profiles of SCG neurons from E18 wild-type (WT) or Casr _ / _ (KO) mice cultured for 24 hrs in medium containing 0.7 mm (wild-type) or 2.3 mm (wild-type and Casr _ / _ ) [Ca 2+ ] o. Mean ± sem of data from 254 and 630 neurons per condition from 4 to 11 separate experiments.
Supplementary Figure 5. CaSR _ deficient neurons do not respond to NPS R-467. Total neurite length and Sholl profiles of SCG neurons from E18 Casr _ / _ mice cultured for 24 hrs in medium containing 0.7 mm [Ca 2+ ] o in the absence (CTR) or presence of 10 nm NPS R-467 (Calcimimetic). Mean ± sem of data from 109 and 121 neurons per condition from two separate experiments.
Supplementary Figure 6. Genetic loss of CaSR does not affect neuronal survival. Percent survival of E18 SCG neurons from Casr+/+ (WT) and Casr _ / _ (KO) mice grown for 24 hrs with a range of NGF concentrations in medium containing 2.3mM [Ca 2+ ] o and no caspase inhibitor. Mean ± sem of data from 3 separate experiments.
Supplementary Figure 7. SCG of WT and CaSR _ deficient mice are indistinguishable at P1. Immunohistochemistry revealing no difference in tyrosine hydroxylase immunofluorescence in the SCG of P1 Casr+/+ (WT) and Casr _ / _ (KO) mice (a). Scale bar = 100 μm. Mean neuronal nuclear diameter (b) and mean SCG volume (c) in P1 Casr+/+ (WT), Casr+/ _ (HET) and Casr _ / _ (KO) littermates. Mean ± sem of data from 3 mice of each genotype.
Supplementary Figure 8. CaSR protein is expressed in post-natal hippocampus. RT- PCR detection of the full-length Casr transcript (584 bp) in P4 mouse hippocampus and the full length and exon 5-deficient Casr transcript (354 bp) in kidney obtained from Casr+/ _ mice (HET), used as positive control 34 ( _ RT = no reverse transcriptase negative control) (a). CaSR immunopositive cells in P4 hippocampal cultures stained with an N-terminus anti-casr polyclonal antibody (neurons were double labelled with anti-βiii tubulin) (b). Scale bar = 50 μm.
Supplementary Figure 9. WT or CaSR _ deficient SCG neurons do not express the exon5 less splice variant of the CaSR. RT-PCR detection of the full-length CaSR transcript (584 bp) in P1 Casr+/+ (WT), Casr+/ _ (HET) but not Casr _ / _ (KO) SCG. Expression of the exon 5-deficient CaSR transcript (354 bp) was not detected in the SCG of any genotype. Both transcripts were amplified from the positive control tissue (kidney of Casr+/ _ mice 33 ). β-actin was amplified from the same samples as a positive reverse transcription control, and no-reverse transcriptase was used as negative control ( _ RT).