Regulation of spindle orientation and neural stem cell fate in the Drosophila optic lobe

Abstract

Background: The choice of a stem cell to divide symmetrically or asymmetrically has profound consequences for development and disease. Unregulated symmetric division promotes tumor formation, whereas inappropriate asymmetric division affects organ morphogenesis. Despite its importance, little is known about how spindle positioning is regulated. In some tissues cell fate appears to dictate the type of cell division, whereas in other tissues it is thought that stochastic variation in spindle position dictates subsequent sibling cell fate.

Results: Here we investigate the relationship between neural progenitor identity and spindle positioning in the Drosophila optic lobe. We use molecular markers and live imaging to show that there are two populations of progenitors in the optic lobe: symmetrically dividing neuroepithelial cells and asymmetrically dividing neuroblasts. We use genetically marked single cell clones to show that neuroepithelial cells give rise to neuroblasts. To determine if a change in spindle orientation can trigger a neuroepithelial to neuroblast transition, we force neuroepithelial cells to divide along their apical/basal axis by misexpressing Inscuteable. We find that this does not induce neuroblasts, nor does it promote premature neuronal differentiation.

Conclusion: We show that symmetrically dividing neuroepithelial cells give rise to asymmetrically dividing neuroblasts in the optic lobe, and that regulation of spindle orientation and division symmetry is a consequence of cell type specification, rather than a mechanism for generating cell type diversity.

Figure 1

GAL4^c855a reveals the proliferation centers of the developing optic lobe. (a) A late third instar larval central nervous system (CNS): ventral nerve cord (VNC), central brain (CB) and optic lobes (OL). Subsequent images show frontal confocal sections, as shown in the inset diagram (OPC in green). Anterior and posterior refer to the neuraxis of the larval CNS. (b) A frontal section through a brain lobe at mid third instar: the OPC (green), the inner proliferation centre (IPC, yellow) and the medulla cortex (me). Discs large (Dlg; grey) outlines all cell cortices in the larval brain and highlights the morphology of the two optic lobe proliferation centres. (c) GAL4^c855a begins to drive expression of UAS-pon-gfp (green; Dlg in red) at first instar. At late first/early second instar (24 hours ALH; after hatching), the OPC and the IPC can be distinguished as two closely associated epithelia. The cells belonging to the proliferation centers (green) are clearly distinguishable by their columnar shape, in contrast to the round, isolated central brain cells. (d) At the end of second/early third instar (48 hours ALH) the epithelia of the OPC and IPC separate from each other and smaller progeny cells are located between the two epithelia. (e) As development progresses during second to mid third instar (72 hours ALH) the OPC cells at the medial edge of the epithelium loose their columnar shape (to the left of the arrowheads). (f) At late third instar (96 hours ALH) the OPC epithelium decreases in size while the number of round neuroblast-like cells increases at the medial edges (to the left of the arrowheads). All images are single confocal sections, with anterior on top and lateral to the right. Scale bar is 50 μm (a) and 20 μm (b-f).

Figure 2

Optic lobe neuroepithelial cells and neuroblasts are arranged in distinct medio-lateral zones. (a) The developing optic lobe generates a lateral epithelial region (to the right of the arrowheads). Epithelial cells express three proteins that localize to cellular junctions: Scrib (green) localizes to the basolateral septate junctions in epithelial cells; DE-Cad (red) localizes to the basolateral zonula adherens; and PatJ (blue) localizes to apical and subapical regions in epithelial cells. Medial neuroblasts (to the left of the arrowheads) are more round and lack the clear subcellular localization of these junctional proteins. (b) sc-lacZ (red) is expressed in the lateral epithelium of the optic lobe (to the right of the arrowheads). Expression is diminished in medial optic lobe neuroblasts (to the left of the arrowheads). Medial neuroblasts express the bHLH transcription factor Dpn (blue), which is not expressed by neuroepithelial cells. Dlg (green) outlines all cell cortices but is enriched at adherens junctions. (c) Asense (red) shows weak cytoplasmic expression in medial Dpn (blue) positive neuroblasts (to the left of the open arrowheads). Asense is nuclear in the progeny of neuroblasts (filled arrowheads). (d) Pros protein (red) forms a basal crescent (inset) in mitotic medial optic lobe neuroblasts (filled arrowhead). Dpn (blue) is restricted to the neuroblasts but Pros (red) is inherited by the basal progeny cells where it localizes to the nucleus. (e) Mira (red) forms a basal crescent in mitotic neuroblasts (filled arrowhead) (metaphase; n = 9 and telophase n = 9). Note that neither Pros nor Mira are present in neuroepithelial cells (to the right of the arrowheads). (f) Insc protein (red) forms an apical crescent in mitotic medial optic lobe neuroblasts (filled arrowhead and inset). These neuroblasts reveal weak cytoplasmic Dpn (blue). Dlg (green) is enriched apically, where it co-localizes with the Insc crescent at the apical cortex (inset). Some progeny cells in the medulla cortex also express insc (arrow). All images are single confocal sections from third instar brains, with anterior to the top and lateral to the right. Open arrowheads mark the boundary between the neuroepithelium (to the right) and the neuroblast zone (to the left).

Figure 3

Proliferative symmetric and differentiative asymmetric division depends on the medio-lateral location within the optic lobe. (a, b) mCD8-GFP MARCM clones (green) are shown in mid third instar brains. Dlg is in red and DNA in blue. (a) A lateral clone contains columnar shaped epithelial cells that presumably were generated by proliferative symmetric divisions (the single confocal section shows three epithelial cells). The clone located at the medial edge of the optic lobe contains neuroblasts with attached strings of progeny cells (the single confocal section shows one neuroblast and three progeny cells). (b) A clone at the medial edge of the optic lobe comprises four progenitor cells and one progeny cell (the single confocal section shows two progenitor cells and one progeny cell). (c-e) GAL4^c855a driven UAS-pon-gfp (green) reveals the division mode of optic lobe neuroepithelial cells and neuroblasts. Dlg is in red and DNA in blue. Brains at mid-third (c, d) and early third (e) instar. (c) Neuroepithelial cells undergoing mitosis round up at the apical surface of the epithelium and show basolateral Pon-GFP (metaphase: filled arrowheads). Upon cytokinesis Pon-GFP is partitioned equally to both daughter cells (telophase: open arrowhead). At the medial edge of the epithelium optic lobe neuroblasts reveal a basal crescent of Pon-GFP at metaphase (arrow; enlarged in (c')). (d) At the medial edge of the epithelium a neuroblast in anaphase segregates Pon-GFP asymmetrically to the basal daughter cell (arrow; enlarged in (d')). (e) A more dorsal confocal section reveals a neuroepithelial cell in anaphase segregating Pon-GFP symmetrically to both daughter cells (arrowhead) and a neuroblast (arrow; enlarged in (e')) in anaphase segregating Pon-GFP to the basal daughter cell. All images are single confocal sections, with anterior on top and lateral to the right.

Figure 4

Optic lobe neuroblasts derive from the neuroepithelium in a medial transition zone. (a-c) Single FLP-out clones expressing nuclear β-gal (red) in the optic lobe at late third instar (96 hours ALH). Dpn is in green, Scrib in blue. (a) An epi only clone containing Dpn negative epithelial cells (marked with β-gal in red, open arrowhead) but no Dpn positive neuroblasts (green, arrowhead). (b) An epi/NBs/progeny clone containing Dpn negative epithelial cells (open arrowhead), Dpn positive neuroblasts (arrowhead, yellow) and progeny cells (arrow). (c) A NBs/progeny clone containing Dpn positive neuroblasts (arrowheads, yellow) and progeny cells (arrow).

Figure 5

Misexpression of Insc in neuroepithelial cells can induce vertical spindle orientation. Spindle orientation at prometaphase/metaphase was analyzed in neuroepithelial cells at mid third instar (72 hours ALH). (a) In control brains the great majority of neuroepithelial cells have a horizontal spindle axis (arrowhead, enlarged in (a')) (n = 29). Note that neuroepithelial cells do not express Insc. (b)GAL4^c855a driven UAS-insc results in apical Insc in neuroepithelial cells and forces spindles into a vertical orientation (n = 42). (c, d) Spindle orientation in control optic lobes (c) and optic lobes misexpressing Insc (d). A horizontal spindle axis is 0°; a vertical spindle axis is 90°. The number of neuroepithelial cells is shown in red within six 15° angle sectors from 0° to 90°.

Figure 6

Model of neuroepithelial to neuroblast transition at the medial edge of the optic lobe. At the medial edge of the optic lobe columnar neuroepithelial cells disassemble adherens junctions and undergo a transition to neuroblasts. Neuroepithelial cells divide symmetrically with horizontal spindle orientation, which results in the expansion of the progenitor pool. Medial neuroblasts divide asymmetrically with vertical spindle orientation and bud off smaller ganglion mother cells (GCMs) towards the presumptive medulla cortex.