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First published online 5 January 2006
doi: 10.1242/dev.02211

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Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts

Institutes of Neuroscience and Molecular Biology, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA.

View larger version (33K): [in a new window]   Fig. 1. Epithelial cell contact correlates with the embryonic neuroblast division axis. (A) Lateral view of stage 10 and (B) stage 15 metaphase and telophase neuroblasts. Markers listed within each panel. To the right are summaries of neuroblast orientation relative to the epithelium. Each red mark corresponds to the angle between a straight line connecting the apicobasal neuroblast axis at telophase and a straight line through the epithelial cell center. Brackets indicate neuroblasts; asterisks indicate Mir-positive basal cortex. Apical is upwards. Scale bar: 10 µm.

View larger version (76K): [in a new window]   Fig. 2. Extrinsic cues orient the neuroblast division axis over multiple cell cycles. (A) Series of still images from a time-lapse DIC movie showing six consecutive telophase figures from a single clustered or (B) isolated neuroblast in primary cell culture. The red arrow points towards the developing GMC along the neuroblast intrinsic apicobasal polarity axis. (A,B) Diagrams on the right summarize GMC position at telophase from all six consecutive divisions, showing tight grouping of GMCs in clustered neuroblasts versus dispersal of GMCs in isolated neuroblasts. Maximum distance between two sister GMCs is uncovered by a 53° angle in clustered neuroblasts and 141° angle in isolated neuroblasts. (C) Quantitation of GMC position at telophase during successive divisions in clustered and isolated neuroblasts. Only neuroblasts dividing more than three times were scored. (D) Three serial confocal sections of a cell cluster fixed and stained for neuroblast and epithelial markers (as indicated). The Insc-positive neuroblast cortex directly contacts several DE-cadherin-positive epithelial cells. Brackets indicate the same neuroblast in the three sections, asterisks indicate the DE-cad positive epithelium only in the left panel and arrows indicate localized DE-cadherin in the middle and right panels. Scale bar: 10 µm.

View larger version (69K): [in a new window]   Fig. 3. In the presence of extrinsic cues, G2 centrosome position remains constant and correlates with a reproducible division axis. (A-E) Series of still images from a time-lapse confocal movie of a clustered neuroblast dissociated from G147-GFP embryos, during three consecutive cell cycles. Mitotic stages and cell cycles are as indicated. A red line connects both apical and basal centrosomes during mitosis through the spindle axis or the G2 centrosome and approximate cell center during interphase. The white arrows indicate the two centrosomes that remain closely apposed at G2. The asterisk indicates an epithelial cell dividing symmetrically. (F-I) Quantitation of the angle between the following two lines: (F) a line connecting the G2 centrosomes to cell center and a line through the metaphase spindle; (G) a line through the metaphase spindle and a line through the telophase spindle; (H) a line through the telophase spindle and a line connecting the G2 centrosomes to cell center in the subsequent division; and (I) a line connecting the G2 centrosomes to cell center and a line connecting the G2 centrosomes to cell center in the following division. Black bars indicate neuroblast percent. Scale bar: 10 µm.

View larger version (39K): [in a new window]   Fig. 4. In the presence of extrinsic cues, spindle orientation correlates with G2 centrosome position despite centrosome rotation at prophase. (A-F) Series of still images from two different confocal time-lapse movies from clustered neuroblasts from a single division, dissociated from G147-GFP embryos. Mitotic stages labeled above each panel. (A-C) An example of a neuroblast showing centrosome rotation prior to spindle formation and (D-F) an example of a neuroblast showing no centrosome rotation prior to spindle formation. A red line connects both apical and basal centrosomes during mitosis or the G2 centrosomes and approximate cell center during interphase. Brackets indicate neuroblasts. The white arrows indicate the two centrosomes that remain closely apposed at G2. Scale bar: 10 µm.

View larger version (59K): [in a new window]   Fig. 5. In the absence of extrinsic cues, centrosome position and the cell division axis drift over multiple cell cycles. (A-C) Series of still images from three different time-lapse confocal movies of isolated neuroblasts dissociated from G147-GFP embryos. Mitotic stages are labeled above each panel. A red line connects both apical and basal centrosomes during mitosis through the spindle axis or the G2 centrosomes and approximate cell center during interphase. The white arrow indicates the two centrosomes that remain closely apposed at G2. (D-G) Quantitation of the angle between the following two lines: (D) a line connecting the G2 centrosomes to cell center and a line through the metaphase spindle as seen in A; (E) a line through the metaphase spindle and a line through the telophase spindle as seen in A; (F) a line through the telophase spindle and a line connecting the G2 centrosomes to cell center in the subsequent division as seen in B; (G) a line connecting the G2 centrosomes to cell center and a line connecting the G2 centrosomes to cell center in the following division as seen in C. Gray bars indicate neuroblast percent. Scale bar: 10 µm.

View larger version (74K): [in a new window]   Fig. 6. In the absence of extrinsic cues, neuroblast cortical polarity does not form until metaphase. (A-D) Double-labeled clustered and (E-H) isolated neuroblasts in culture, fixed and stained for aPKC and PH3 (top row), and for -tubulin (bottom row). Mitotic stages are listed above each panel. White arrowheads indicate the center of the aPKC crescent. Scale bar: 5 µm.

View larger version (117K): [in a new window]   Fig. 7. In the absence of extrinsic cues, neuroblast cortical polarity does not form at the same position over multiple cell cycles. Series of still images from time-lapse confocal movies from a clustered (A) and an isolated (B) neuroblast dissociated from embryos ubiquitously expressing Dlg:eGFP. Mitotic stages and cell cycles are listed above each panel. White arrowheads indicate the center of the Dlg:eGFP crescent, most prominent at metaphase. White asterisks indicate the position of the newly born GMCs. Scale bar: 10 µm.

View larger version (25K): [in a new window]   Fig. 8. Model for extrinsic signaling. During the cell cycle, the neuroblast receives positional cues from the epithelium, to polarize along the apicobasal embryonic axis, and during mitosis this allows for the basal placement of all GMC daughters and their progeny after cytokinesis. At late G2, extrinsic signaling indicates the position on the neuroblast cortex to establish Par apical polarity. During prophase, apical Par crescents are forming at this pre-selected position, independently of centrosome behavior. The position of the apical crescents determines spindle orientation at prometaphase and spindle position is maintained throughout mitosis, ensuring the reproducible basal placement of all GMCs. Extrinsic cues also maintain centrosome(s) position at the epithelial/neuroblast contact site throughout the cell cycle. See Discussion for further details.