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Fig. 3 Local loss and gain of Daam1a function induces opposite modulatory effects in the growth of habenular neuropil and axonal efferent connectivity to the IPN. (A) Schematics of the local electroporation procedure (a), daam1a-MO design (b) and constitutively active form of C-Daam1a (c). (B-J) Dorsal views of the habenular region (B-G) and IPN (H-J) of Tg(pou4f1-hsp70l:GFP) zebrafish at 4.5 dpf, with anterior to the top. The habenular neuropil was immunostained against acetylated α-tubulin (B-D, white), whereas the soma (E-G) and efferent projections (H-J) of dorsal habenular neurons expressing pou4f1-hsp70l:GFP were labelled in vivo (green). Vertical dotted brackets in B-D indicate the extent of neuropil development in the left Hb. Asterisks in E-G indicate the enlarged cellular domain of the Hb expressing pou4f1-hsp70l:GFP. (K-M) Dorsal (left) and lateral (right) views of the IPN at 4.5 dpf, with anterior to the top, after labelling the left (red) and right (green) Hb with DiD and DiO, respectively. Images in B-M are maximum intensity z-stack confocal projections, organised in three columns that correspond to different electroporation conditions: control-MO (left), daam1a-MO (middle) and pcDNA-C-DAAM1-HA (right). (N-P) Dorsal views of three-dimensional volumetric reconstructions of the left habenular neuropil in the three electroporation conditions. (Q) Quantification of left (grey) and right (red) habenular neuropil volumes. Asterisks indicate statistically significant decrease (daam1a-MO, n=5) or increase (pcDNA-C-DAAM1-HA, n=5) of left habenular neuropil volume with respect to controls (Co-MO, n=5; control plasmid, n=6), after Mann-Whitney test (P<0.05). Error bars represent s.d. D, dorsal; L, left; LHb, left Hb; R, right; RHb, right Hb; V, ventral. Scale bars: 20 μm.