-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina

DEV027912 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Chx10-Cre recombination pattern in the retina. (A,B) Retina sections of Chx10-Cre; Z/EG P7 mice illustrate Chx10-CreGFP mediated recombination pattern, in which recombined cells express GFP (green) due to excision of the stop translation sequence in the Z/EG transgene. Unrecombined cells express lacZ and are immunolabeled for β-galactosidase (red). In Chx10-Cre; Z/EG retinas, the majority of retinal cells are descendents of progenitors that have undergone Cre-mediated recombination. However, occasional columns of cells spanning all layers and roughly half the cells present in the GCL are unrecombined (red; closed arrowhead). Chx10-CreGFP is also expressed postnatally in bipolar cells (A,D,E; green, open arrow). (B) Co-immunolabeling of Chx10-Cre; Z/EG retinas with GFP (green) and Brn3a (magenta) reveal that although many RGCs are spared, some RGCs are recombined (double arrow). (C-E) Immunostaining of GFP in retina sections of Pcdh-γ ^fcon3/fcon3, Chx-10-Cre;Pcdh-γ ^fcon3/fcon3 and Chx-10-Cre;Pcdh-γ ^fdel/fdel P18 mice report the extent of Pcdh-γ excision. In Pcdh-γ ^fcon3/fcon3 retinas, Pcdh-γ-GFP fusion proteins are present throughout the retina (C). In Chx-10-Cre;Pcdh-γ ^fcon3/fcon3 and Chx-10-Cre;Pcdh-γ ^fdel/fdel retinas, Pcdh-γ-GFP fusion proteins are dramatically reduced, with the exception of thin stripes of GFP sparsely and variably distributed throughout the mutant retinas (detected in the outersegment and ONL; closed arrowhead). GFP in the INL reflects postnatal GFP expression of Chx10-CreGFP transgene (D,E, open arrow). Scale bar: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Synaptic puncta density is similar in Pcdh-γ; Bax double mutant and control retinas. (A,B) Quantification of PNA-labeled cone pedicles and bassoon-immunolabeled puncta in the OPL of control and Chx10-Cre; Pcdh-γ^fcon3/fcon3; Bax^−/− retina sections of P28 mice. Bars show mean±s.e.m. of 15-18 microscope fields from three double mutant animals (red bars) and three control siblings (black bars; two Chx10-Cre; Pcdh-γ ^+/fcon3; Bax^−/− and one Pcdh-γ ^+/fcon3; Bax^−/− animals). Numbers of PNA-labeled pedicles (A) and Bassoon-positive puncta per unit area (B, left), and total Bassoon-positive puncta area (B, right) did not differ in double mutants compared to controls (by Student's t-test). (C) Cumulative histogram of Bassoon labeled puncta sizes in the OPL of double mutants compared with controls shows that they did not differ in their distribution (over 900 puncta analyzed for each; Kolmogorov-Smirnov test). (D) Quantification of Bassoon-immunolabeled puncta in the IPL of control and Chx10-Cre; Pcdh-γ^fcon3/fcon3; Bax^−/− retina sections of P28 mice. Bars show mean±s.e.m. of 13-15 microscope fields at similar regions of OFF and ON lamina of the IPL of three double mutant animals and control siblings (as above). The number (left) and total puncta area (right) did not differ between Pcdh-γ; Bax double mutants and controls (by ANOVA). (E) Cumulative histogram of bassoon puncta sizes in the IPL shows similar distributions in double mutants and controls (over 3700 puncta analyzed for each; Kolmogorov-Smirnov test).

• Supplemental Figure S3 -

  Fig. S3. Spatio-temporal receptive fields for a sample of ganglion cells in mutant and control retinas. Each panel displays the spike-triggered average stimulus of one neuron, h(x,t) (as defined in Eqn 1). Within such a receptive field, the right-most 'blob' indicates the response polarity of the receptive field center: red for ON-cells, blue for OFF cells. In some cases (asterisks) one can see regions above and below the center with the opposite polarity. These reflect the antagonistic surround of the receptive field. For further interpretation of such receptive fields, see also Kim et al. (Kim et al., 2008).