Regulators of spine maturation are distinct from regulators of spine precursor formation.

A) Representative Images of GFP-expressing DIV-16 neurons transfected with the indicated shRNA targeting sequence for 48 hours. B) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine density later in synaptic development (DIV-16). Spine density is expressed as the percentage of the average control spine density. n = 44 control, 16 Ophn-1 shRNA #1, 5 Ophn-1 shRNA #2, 17 β-pix shRNA #1, 7 β-pix shRNA #2, 15 Frabin shRNA #1, 8 Frabin shRNA #2 neurons (Spine density was not significantly different from control as determined by t-test, except for β-pix shRNA #1 which was determined by Mann-Whitney Rank Sum Test). C) Arhgap23 shRNAs significantly increase spine density later during synaptic development (DIV-16). n = 44 control (same as B), 22 Arhgap23 shRNA #1, and 12 Arhgap23 shRNA #2 neurons; p = 0.02 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p = 0.002 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test). D) Regulators of spine precursor formation, OLIGOPHRENIN-1 (OPHN-1), β-PIX, and FRABIN, do not alter spine length later in synaptic development (DIV-16) neurons. Cumulative distribution plot of spine length in DIV-16 primary rat hippocampal neurons co-expressing GFP and the indicated shRNA targeting sequence. Spine length is expressed as a percentage of the average control spine length. n = 3273 control, 651 Ophn-1 shRNA #1, 130 Ophn-1 shRNA #2, 729 β-pix shRNA #1, 449 β-pix shRNA #2, 556 Frabin shRNA #1, 688 Frabin shRNA #2 spines (Spine length was not significantly different from control as determined by Mann-Whitney Rank Sum test). E) Arhgap23 and Vav2 shRNAs significantly increase spine length later in neuronal development (DIV-16). n = 3273 control (same as D), 1207 Arhgap23 shRNA #1, 1182 Arhgap23 shRNA #2, 962 Vav2 shRNA #1, and 551 Vav2 shRNA #2 spines; p < 0.001 for Control vs Arhgap23 shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs Arhgap23 shRNA #2 (Mann-Whitney Rank Sum Test), p = 0.006 for Control vs Vav2 shRNA #1 (Mann-Whitney Rank Sum Test), p < 0.001 for Control vs Vav2 shRNA #2 (Mann-Whitney Rank Sum Test).