Antibody:	Anti-HCN4 gamma
Detection system:	Secondary antibody coupled to Cy3
Assay notes:	Two anti-Hcn4 antibodies were described in the methods Table S1. Either of the following could have been used: MGI:6450086 or MGI:6450087.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	3J , 3L	Hcn4 labels type 3a bipolar cells and co-labeled with Irx6:b-gal positive bipolar cells.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	6B , 6D	Type 3a bipolar cells were shown to be triple labeled by Cabp5, Hcn4, and Irx6:b-gal.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	6J , 6H	In the mutant, Hcn4 expression continued, but Cabp5 expression was reduced in type 3a cells.
TS28: retina inner plexiform layer	Present		6H , 6J

Structure	Level	Pattern	Image	Note
TS28: retina inner plexiform layer	Present	Regionally restricted	7A	Expression was detected in the type 3a bipolar cell axon terminals.

Structure	Level	Pattern	Image	Note
TS28: retina inner plexiform layer	Present	Regionally restricted	7B	In the mutant, expression expanded into the entire OFF region of the inner plexiform layer.

Structure	Level	Pattern	Image	Note
TS28: retina inner plexiform layer	Present	Regionally restricted	7C	In the mutant, expression expanded into the entire OFF region of the inner plexiform layer.

Structure	Level	Pattern	Image	Note
TS28: retina inner plexiform layer	Present	Regionally restricted	7D , 7F	The axon terminals of Hcn4 immunopositive type 3 bipolar cells were normally positioned in sublamina 2 (between the two outermost Calb2-labeled bands).

Structure	Level	Pattern	Image	Note
TS28: retina inner plexiform layer	Present	Regionally restricted	7I , 7G	Immunolabeled axon terminals in the mutant spanned both sublamina 1 and 2 (located above the outermost Calb2-labeled band). The expansion of label was observed across the entire retina. Hcn4-labeled axon terminals were not observed in sublaminae 3-5.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	8A , 8G	Expression was detected in type 3a bipolar cells.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	8H , 8B	Expression was detected in type 3a bipolar cells.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	9A , 9C	Expression was detected in type 3a bipolar cells, but not the Tacr3-labeled type 1 and 2 cells.
TS28: retina inner plexiform layer	Present	Regionally restricted	9C' , 9A'	Expression was detected in type 3a axon terminals. There was no overlap with Tacr3 expression.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	9F , 9D	Ectopic Tacr3 expression was observed in Hcn4-immunolabeled cell somas of putative type 3a cells. Type 1 and 2 bipolar cells were not labeled with Hcn4.
TS28: retina inner plexiform layer	Present	Regionally restricted	9D' , 9F'	Ectopic Tacr3 expression was observed in Hcn4-immunolabeled putative type 3a axon terminals.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	9G , 9I	In the double mutant, all of the Hcn4-positive bipolar cells co-immunolabeled with Tacr3.
TS28: retina inner plexiform layer	Present	Regionally restricted	9G' , 9I'	Hcn4 and Tacr3 co-immunolabeling was noted in the inner plexiform layer.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	S4_merge Irx6LacZ/LacZ;Vsx1+/AltB5 , S4_Hcn4 Irx6LacZ/LacZ;Vsx1+/AltB5	Expression was detected in type 3a bipolar cells. Expression of Hcn4 was not detected in all Irx6:bgal-positive cells.

Structure	Level	Pattern	Image	Note
TS28: retina inner nuclear layer	Present	Regionally restricted	S4_merge Irx6LacZ/LacZ;Vsx1AltB5/AltB5 , S4_Hcn4 Irx6LacZ/LacZ;Vsx1AltB5/AltB5	Expression was detected in type 3a bipolar cells. Expression of Hcn4 was not detected in all Irx6:bgal-positive cells.