Supplementary Figure 1

The first sprout contains neuronal polarity information

a)Frequency distribution plot of hippocampal neurons in culture in which the axon emerges from the first sprout (positioned at 0’ angle), or from the sequentially formed lamellipodia. n= 90 cells.

b)Top: low magnification image corresponding to the ventricular area of embryonic-day 16 rat hippocampus. The first protrusion and longest neurites of neighbor cells (asterisks) have the same orientation (arrowheads), facing the basal surface. Bottom: enlargement of two asterisk-marked cells in upper image. BodipyCer labeling (arrowheads, fluorescence images) reveals the first bud (left) and axon (right) of these cells. Scale bar: 10m

Supplementary Figure 2

Organelle polarization marks the site of neuronal polarity

a)Distribution plot of organelle-polarization in live hippocampal neurons in culture. 0 in the X axis represents polarization from the area of initial cluster. 180 represents opposite pole growth. n=69.

b)Low magnification of coronal sections of E18 rat germinal layer stained for the Golgi antigen GM130 (left). The enlargement of the boxed area , shows that the short bud of this recently generated cell (arrowhead) presents Golgi-labeled structures at its base (right, arrow). This pattern was observed in 70% of neurons (n=30 cells per section, 5 sections analyzed). Scale bar: 10µm.

c)Distribution plot of organelle-cleavage furrow (indicated by citron-kinase staining) spatial correlation in recent post-mitotic hippocampal neurons in culture. 0 position in X represents 180° apposition. n=54

Supplementary Figure 3

Drosophila neurons polarization in vitro and in situ correlates with plane of mitotic division and the localization of the centrosomes.

a)Time-lapse video recordings of Drosphila third instar larvae neuroblasts undergoing division in vitro28. Individual neuroblasts with one daughter GMC (Nb and 1 respectively, upper left image) was constantly followed. This neuroblast gave rise to more GMC (2, 3, upper row) which in turn originated two neurons each (n1/n1’, n2/n2’, n3/n3’ in second and third rows). Axons arise perpendicular to the plane of GMC divisions, discernible by comparing their position (large image, arrowheads) with the orientation of the plane of cleavage (dotted line in the smaller images). Scale bar= 5 m

b)Representative example showing that the longest neurite of third instar larvae neurons in culture (arrows, upper image) enriches in the axonal antigen BP 102 (arrows, lower image). Scale bar: 5m

c)Confocal microscopy image of GFP-centrosomes in Drosophila melanogaster third instar larvae midbrain12. At low magnification (left) centrosomes (green dots) are visible at the base of the projecting axons (boxes 1 and 2), as revealed by HRP staining. The enlargement of the boxed areas, (right images) further highlight this correlation. Scale bar: 5m

Supplementary Figure 4

Centrosomal-mediated polarized microtubule and membrane activities precede morphological polarization in vitro and in situ

a)Embryonic hippocampal neurons treated with the microtubule-depolymerizing drug Nocodazole at the round-cell stage followed by drug-withdrawal. The first lamellipodium (arrowhead) appears from the centrosomal pole (arrow). Scale bar: 10m. Right: Distribution plot of 95 cells in which the position of the first lamellipodium after Nocodazole removal was compared to that of the centrosomes. The sphere angle 20/-20 reflects co-localization

b)BodipyCer-labelled exocytic vesicles (arrowheads, left) in hippocampal neurons in culture are preferentially delivered to the newly formed lamellipodium (asterisk, right). The Golgi apparatus (arrow) is present in the same pole. Scale bar: 10m

c)BodipyCer-labelled exocytic vesicles in whole embryonic hippocampus (arrowheads) are polarized towards the growing neurites, evidenced by phase contrast microscopy (right). The Golgi apparatus is at the base of the neurites (arrows). Scale bars=10 m

Supplementary Figure 5

Pharmacological disruption of microtubule polymerization and membrane trafficking prevents morphological polarization.

a)Microtubule de-polymerization, induced by treating round, undifferentiated, neurons with Nocodazole for 4 hours results in lack of polarization (upper right). Tubulin staining (upper left) confirms the absence of microtubules. Centrosomes remain polarized (lower panels). Scale bar= 10 m.

b)Treatment of round hippocampal neurons with the fungal metabolite Brefeldin A, that perturbs Golgi-endosomal membrane trafficking, for 6 hours results in lack of neuronal polarization. GM130 labeling (left) confirms the disruption of the Golgi apparatus. Scale bar=10 m. This result was observed in 70% of the cells thus treated. Asterisk reflects statistical significance.