Fig. S1

Related to Figure 1. Morphological development and emergence of neurotransmitter identity of newborn neurons

(A) Picture of the electroporation preparation with the electrodes positioned on both sides of the larva. White arrow: the capillary containing DNA introduced in the tectal ventricle. Scale bar: 200 μm.

(B) i, DNA constructs used for labeling newborn neurons in Tg(huC:GCaMP5G). Long-term expression of the vectors was achieved using the co-electroporation of a transposase enabling the stable incorporation of the constructs flanked with Tol2 sites, into the larva’s genome. ii; Constructs used for labeling newborn neurons in Tg(gad1b:GFP;vglut2a:loxP-DsRed-loxP-GFP).

(C) Quantification of the survival of electroporated larvae and age-matched controls, at 1 and 7 dpe. Note that electroporations did not show any significant effect in the survival of the larvae. ns.: p > 0.05.

(D) Time-course of the zebrafish larva development from 0 to 8 dpf, in terms of morphology, behavior and maturation of the visual system. Performing electroporations at 4 dpf enables studying the development of newborn neurons as they incorporate into an already functionally mature brain structure. OKR: optokinetic response and OMR: opto-motor response. Red dots: the developmental stages at which we performed the experiments. Arrow-head: the developmental stage at which we performed the electroporations.

(E) Two examples of mature neurons in 5 dpf larvae (i and ii). Left, position of the soma of the labeled neuron (arrow-head), within the PVZ layer of the optic tectum. Scale bar: 50 μm. Middle, a 3D projection of the mature neuron showing the neuron’s morphology. Scale bar: 20 μm. Right, reconstruction of the neuron in the middle panel. Note that in contrast to the newborn-labeled neurons at 5 dpf (1 dpe), neurons far from the neurogenesis site already show mature morphologies. The neurons were labeled using single-cell electroporation in Tg(huC:GCaMP5) larvae.

(F) Examples of Tg(gad1b:GFP;vglut2a:loxP-DsRed-loxP-GFP) larvae electroporated with Tol2- huC:Gal4 and Tol2-10xUAS:mTagBFP2CAAX constructs. Electroporated newborn neurons are indicated with white arrow-heads. Top, a newborn neuron not yet differentiated at 1 dpe. Middle and bottom, differentiated excitatory and inhibitory neurons at 4 dpe. First column: optical plane of the optic tectum showing inhibitory (green), excitatory (red) neurons and a single newborn neuron (blue and arrow-head). Scale bars: 20 μm. Second, third and fourth columns, zoom of the dashed region in the first column, for the red, green and blue channels, respectively. Fifth column, all channels merged. Scale bars: 50 μm. L: left, R: right, A: anterior and P: posterior.

(G) Based on the morphology of the dendritic arbors at 4 dpe, newborn-labeled neurons developed into different neuronal types. Fraction of the different neuronal types. nsPVIN: non-stratified periventricular interneurons. PVPN: periventricular projection neurons. bsPVIN : bi-stratified periventricular interneurons.