Mutation in asl affects the fidelity of polarity orientation in larval NBs. (A and B) Still images from time-lapse recordings of larval NBs expressing tubulin-GFP. Division axes and daughter cells are highlighted by color-coded lines and circles, respectively, for successive divisions (yellow, first; and green, second). The orientation of each division was judged by the position of the midbody (arrowheads). (A) In control NBs, the orientation of successive divisions varies little, and the interphase aster (t₂, asterisk) coincides with the location of the apical spindle pole of the previous division (t₀ and t₁) and allows predicting the position of the apical spindle pole in the next division (t₄). (B) asl mutant NB dividing twice (Video 9). Interphase asters are absent (t₂), and variations in the orientation of the division axis were observed: in contrast to controls, daughter cells resulting from two consecutive divisions were born, in this case, separated by several daughter cell diameters (t₁ and t₄). (C) Plot of daughter cell bud site variations in asl mutant NBs (green) compared with control NBs (yellow). Time is shown in hours:minutes:seconds. Bar,10 µm.

Probing the mechanism that controls the fidelity of polarity orientation in larval NBs. (A–D) Still images from time-lapse recordings of larval NBs. Fluorescent reporters are indicated to the left of each row. Division axes, daughter cells, and apical crescents are highlighted by color-coded lines, circles, and asterisks, respectively, for successive divisions (yellow, first; green, second; and red, third). (A) An NB mutant for pins. This cell, which lacks interphase asters, divides three times, producing daughters that are scattered over an arc of ∼180° (t₀ and t₂; Video 10). (B) An NB mutant for polo. Two inactive centrosomes (arrowheads) are visible in interphase (t₁). (C) An NB expressing Khc-73 RNAi. The arrow in t₁ points to the stable microtubule-nucleating apical centrosome. (D) An NB expressing Par-1–GFP. The arrow in t₁ points to the apical aster during interphase. (E–H) plots of apical crescents or daughter cell budding site variations in NBs mutant for pins (E) or polo (F) or expressing Khc-73 (G) or Par-1–GFP (H). Green indicates mutant NBs and yellow indicates control (worniu-Gal4 UASCherryTubulin) NBs. Time is shown in hours:minutes:seconds. Bars,10 µm.

The role of the microtubule network in the orientation of asymmetric cell division in Drosophila NBs. (A) Under normal conditions, the apical position of the main MTOC is maintained through the cell cycle and passed on from mother to daughter NB (1 and 2), polarity orientation is retained in consecutive mitoses, and the small differentiating cells (GMCs) are delivered in a cluster (3). (B and C) Under microtubule depolymerization conditions, the interphase aster is disassembled, and the cortical attachment of the apical centrosome is lost. If microtubule dynamics are restored during interphase, the memory of cortical polarity orientation is often unaffected (B, 1, asterisk). However, if microtubule-depolymerizing conditions are kept until the cell enters mitosis, the orientation of cortical polarity is randomized (B, 2), and once microtubule dynamics are restored, asymmetric cell division takes place along the new, ectopic axis of cortical polarity (C, 1). This situation results in the ectopic delivery of a GMC, the relocation of the interphase aster of the NB (C, 2), and the resetting of the axes of cortical polarity and asymmetric cell division orientation to the new ectopic orientation (C, 3). (D) In different mutant conditions that affect the assembly or the stability of the interphase asters, the memory of polarity orientation is partially lost (1 and 2) and daughter cells are not clustered (3).

The loss of centrioles severely compromises the fidelity of polarity orientation in larval NBs. (A–D) Still images from time-lapse recordings of MARCM clones labeled by the expression of nuclear GFP (NLS-GFP [nlsGFP]). Genotypes and fluorescent reporters are indicated to the left of each row. Colored asterisks, lines, and circles mark the position of apical crescents, the division axes, and small daughter cells, respectively, in successive cell cycles (yellow, first; green, second; red, third; and white, fourth). Dashed circles indicate the position where GMCs will be delivered. (A) NB in a control wild-type (wt) clone dividing three times. Polarity orientation, as judged by apical centrosome position (arrows) and small daughter cell budding sites, changes very little from one cycle to the next (t₀–t₃). The resulting GMCs are clustered (t₃; Video 7). (B–D) dsas-4 mutant NB clones. (B) The position of apical crescents and the place of daughter cell delivery, which appear to be aligned, vary by nearly 90° between two successive cell cycles (t₀ and t₂; Video 8). (C) Projections made to reflect small daughter cell budding sites in successive rounds of division, which are scattered around the NB cortex in four successive divisions (t₀–t₃). (D) Apical crescent positions scatter similarly over three consecutive cell cycles (t₀–t₃). (E) Plot of apical crescent position variations between two consecutive cell cycles of control dsas-4/+ NBs (yellow) and homozygous dsas-4 mutant NBs (green) from mosaic brains. (F) Plot of daughter cell budding site variations between two consecutive cell cycles of control dsas-4/+ NBs (yellow) and homozygous dsas-4 mutant NBs (green). Time is shown in hours:minutes:seconds. Bar,10 µm.

Microtubule depolymerization severely compromises the fidelity of cortical crescent position in larval NBs. (A–G) Still images from time-lapse recordings of larval NBs before (t₀) and after (t₁ and t₂) treatment with colcemid. NBs are shown oriented with their apical side up in all t₀ panels. Fluorescent reporter constructs are indicated to the left of each row. (A–F) Yellow and green asterisks mark pre- and posttreatment crescent position, respectively. Arrows mark apical centrosomes; white arrowheads refer to the basal centrosome in t₀ and motile centrosomes in t₁ and t₂. (A) Before colcemid treatment, the apical centrosome nucleates an aster, and the basal shows migratory behavior (t₀). When microtubules are depolymerized, the apical centrosome detaches from the cortex, and both centrosomes localize randomly (t₁ and t₂; Video 1). (B–D) In the control division, metaphase crescents and position of centrosomes reflect the polarity axes (t₀; yellow lines). Behavior of Dlg (B), Pins (C) and Baz (D; Video 2) crescents upon colcemid treatment is shown. Loss of microtubules renders both centrosomes motile (t₁). The place of crescent formation becomes unpredictable, suggesting a change in polarity axis orientation that bears no apparent relation with centrosome position at the time of crescent assembly (t₂; green lines). (E and F) Yellow and green lines represent pre- and posttreatment cortical polarity orientation, respectively. (E) Basal crescent positioning responds similarly to colcemid treatment (Video 2). (F) Alignment of apical with basal crescents is unperturbed in colcemid-arrested cells despite occurring at an ectopic position (t₀ and t₂; yellow and green arrowheads, highlighting the limits of the basal crescent, before and after microtubule depolymerization, respectively). Depolymerizing microtubules can prolong interphase, but crescent formation occurred always closely preceding or at the time of NEB, as in controls (not depicted). (G) A crescent assembled before colcemid treatment (t₀, yellow asterisk, arrow [apical centrosome], and arrowhead [basal centrosome]) does not change position when microtubules are depolymerized (visible with the loss of centrosome anchoring; t₁ and t₂, arrowheads). (H and I) Plot of the angle of crescent position in two successive cycles in control cells (yellow) and before and after colcemid treatment (green). Time is shown in hours:minutes:seconds. Bar, 10 µm.

Ectopic division orientations induced by transient microtubule depolymerization are permanently kept once microtubule dynamics are restored. (A and B) Still images from time-lapse recordings of larval NBs under normal microtubule dynamics (t₀), after microtubule depolymerization by colcemid (t₁), and after colcemid inactivation (t₂–t₄). Fluorescent reporter constructs are indicated to the left of each row. Arrows mark apical centrosomes; arrowheads refer to motile interphase centrosomes and to the basal centrosome in cells in metaphase. Colored circles highlight GMCs, and colored lines highlight the division axis. Dashed circles indicate the position where GMCs will be delivered. Each are colored according to birth order (yellow, first; green, second; and red, third). (A) An NB dividing three times (colcemid added, 13:55:46; effect detectable, 14:11:46; UV pulse delivered, 15:05:17; and total time exposed, 70 min). The orientation of the apical crescent and the spindle reflect the polarity axis (t₀, yellow line). Under microtubule depolymerization conditions, the apical crescent forms ectopically (t₁, green asterisk). After the drug is inactivated, the newly assembled spindle reorients to align with the ectopic crescent (t₂, green asterisk/line). The same ectopic orientation is kept in the next cell cycle so that the third GMC is delivered on top of the second, away from the first (t₄, red asterisk/line). In this NB, the original apical centrosome ended up in the GMC, and the other remained in the NB (t₁ and t₂). (B) An NB dividing three times (colcemid added, 15:34:41; UV pulse delivered, 16:45:23; and total time exposed to colcemid, 71 min; Video 6). (t₀) The dashed line indicates the control polarity axis, as judged by the place of the most recent GMC. The cell rounds up upon colcemid treatment and upon release from colcemid arrest, divides asymmetrically with an ectopic orientation (t₁ and t₂, green line). This orientation is kept in the next mitosis, which delivers the third GMC on top of the second, diametrically opposed to the first GMC (t₃ and t₄). (C) Quantification of GMC budding sites plotting the variation (yellow sector) between the first and second cell division orientation (green and red, respectively) with respect to the control division (dashed line, 0°). Time is shown in hours:minutes:seconds. Bar, 10 µm.

Transient loss of microtubules establishes ectopic cortical polarity that drives cell division once microtubule dynamics are restored. (A–D) Still images from time-lapse recordings of larval NBs dividing twice. Fluorescent reporter constructs are indicated to the left of each row. White arrows mark apical centrosomes; white arrowheads refer to motile interphase centrosomes and to the basal centrosome in cells in metaphase. GMCs are highlighted by circles and are colored and numbered according to birth order (yellow, first; and green, second). (A–C) Yellow and green lines refer to pre- and posttreatment division orientation, respectively, in the transient microtubule depolymerization experiments followed by colcemid inactivation during (A and B) or before (C) mitosis. (A) The first GMC is delivered basally opposing the apical aster (t₀). Microtubule depolymerization mispositions the centrosomes (t₁). After microtubule polymerization is restored (t₂), the cell enters metaphase (t₃) and divides asymmetrically, delivering the second GMC ectopically (t₄). Then one centrosome organizes an aster at the ectopic apical pole, whereas the other is down-regulated and motile (colcemid added, 19:06:12; effect detectable, 19:20:22; UV pulse delivered, 20:13:17; and total time exposed, 67 min; Video 3). (B) Transient colcemid treatment produces an ectopic basal cortex (t₀–t₃; yellow and green arrowheads outline pre- and posttreatment crescents, respectively). Upon colcemid inactivation, the spindle, reflected by the centrosomes at the spindle poles, aligns with this crescent (t₂) that is segregated to the ectopically delivered GMC (t₃ and t₄; colcemid added, 13:35:43; effect detectable, 14:00:43; UV pulse delivered, 14:51:54; and total time exposed, 76 min). (C) The control division delivers the GMC basally (t₀). 25 min later, the apical centrosome has detached, yet microtubules remain detectable (t₁ and t₂, red arrow). When the UV pulse is delivered shortly thereafter, before entry into mitosis, microtubules regrow only over the ectopically localized apical centrosome (t₃), and once assembled, the spindle rotates, and the following division occurs at the pretreatment orientation (t₄; colcemid added,16:00:46; effect detectable, 16:09:12; UV pulse delivered, 16:32:25; and total exposure time, 32 min; Video 4). (D) Apical crescents form before (t₀, yellow asterisk) and after the addition of Taxol (t₄, green asterisk) at roughly the same sector of the cortex (Video 5). (E) Plot of GMC budding site variations induced by transient microtubule depolymerization (microtubule dynamics restored after entry into mitosis, green; two consecutive control divisions, yellow). (F) Plot of variations of the place of apical crescent assembly after the addition of Taxol (green) compared with two successive control divisions (yellow). Time is shown in hours:minutes:seconds. Bar, 10 µm.