glial cells missing and gcm2 Cell-autonomously Regulate Both Glial and Neuronal Development in the Visual System of Drosophila Carole Chotard, Wendy Leung and Iris Salecker Supplemental Data Supplemental Results Epithelial and Marginal Glial Cells Originate from Multipotent Progenitor Pools To determine the relationship between gcm gene expression and the developmental sequence leading to glial fate commitment, we conducted lineage analysis experiments of epithelial and marginal glial cells using MARCM. The MARCM stock w hs-flp tubp-gal80 FRT19A; UASnlacZ UAS-cd8GFP; tubp-gal4/tm6b (kindly provided by A. Gould; Bello et al., 2003) was crossed to a wild type FRT19A line. Mitotic recombination was induced by heat shocking larvae in a 37 C water bath for 70 minutes at 48 hours after egg laying. Animals were analyzed during the late third instar larval stage. To score the composition of clones, glial cells and neurons were visualized using antibodies against the differentiation markers Repo and Elav, respectively. From 98 clones (corresponding to 92 optic lobes), images were taken at different focal planes throughout the optic lobe and some of these were processed to obtain a 3D view. The majority of clones (n=95) within GPC areas contained glial cells and neurons. Epithelial and marginal glial cells emanated from the distal half of clones within GPC areas, whereas the proximal half included a subpopulation of Elav-positive medulla neurons (Figures S1A-S1D). These proximal medulla neurons extended prominent axon bundles into ventral or dorsal parts of the medulla neuropil, which are not innervated by R-cell axons (Figures S1B and S1D; Fischbach and Dittrich, 1989). Epithelial and marginal glial cells are thus derived from multipotent progenitor pools. In three samples, we obtained small clones that consisted exclusively of glial cells (Figures S1G and S1H). Clones could be traced to the areas immediately adjacent to the margins of GPC areas, where epithelial and marginal glial cells accumulate before entering the R-cell projection field. Staining with an antibody specific for phospho-histone H3 labeled individual cells in mitosis adjacent to Repo-positive glial cells in 1
this area (Figure S1F). This indicates that common progenitors give rise to clusters of committed, mitotically active glial precursor cells at the lamina margins. Intriguingly, some differentiated epithelial and marginal glial cells are mitotically active after completing their migration into the R-cell projection field (Figure S1E). High magnification images further revealed that individually labeled glial cells at the lamina border extend leading processes towards neighboring glial cells to form a migratory chain in direction of the R-cell projection field (Figures S1G and S1H). In summary, our clonal analysis revealed the following developmental sequence underlying the generation of epithelial and marginal cells in the optic lobe: GPC areas include multipotent progenitors because epithelial and marginal glial cells share the same lineage with a subtype of medulla neurons that specifically innervate the proximal medulla neuropil. These common progenitors give rise to committed glial precursor cells at the border of the R-cell projection field, which in turn produce differentiated epithelial and marginal glial cells. Gcm and Gcm2 expression begins within these committed glial precursor cells at the lamina margins. Supplemental References Bello, B.C., Hirth, F., and Gould, AP. (2003). A pulse of the Drosophila Hox protein Abdominal-A schedules the end of neural proliferation via neuroblast apoptosis. Neuron 37, 209-219. Fischbach, K.F., and Dittrich, A.P.M. (1989). The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure. Cell Tissue Res. 258, 441-475. 2
Supplemental Figures Figure S1. Figure S1. Epithelial and Marginal Glial Cells Are Derived From Multipotent Progenitor Pools. (A) The schematic diagram summarizes the lineage of epithelial (eg) and marginal (mg) glial cells. These glial cells originate from the distal part of dorsal and ventral glial precursor cell (GPC) areas, whereas proximal medulla neurons (prox mn) and some lateral medulla neurons (lat mn) innervating the medulla neuropil (asterisk) are derived from the lower part of GPC areas. The black arrow indicates the location of committed glial precursors, which accumulate at the border of the R-cell projection field. R1-R6 axons terminate in the lamina, whereas R7 and R8 3
axons project into the medulla. OPC, outer proliferation center; LPC, lamina precursor cells; ln, lamina neurons; mn, medulla neurons; meg, medulla glia; mng, medulla neuropil glia. (B-D, G and H) Lineage analysis was performed using MARCM. Wild type clones express GFP (green). Glial cells were visualized using anti-repo (blue C-H) and R-cell axons using mab24b10 (red D, G and H) or anti-hrp (green E and F). The 3D reconstruction (B) and staining of another clone (C), which displays proximal Elav-positive cells (red, arrowhead) and distal Elav-negative cells (arrow), underscore the multipotent nature of GPC areas. (D) Successive focal planes of the clone shown in B illustrate that a continuous bridge of cells emanating from the distal half of the clone within the GPC area is directly connected to the rows of epithelial and marginal glial cells. Glial cells accumulate at the lamina margin before entering the R-cell projection field (arrows). The lower half of the clone gives rise to proximal medulla neurons innervating the medulla neuropil (arrowheads), as well as scattered lateral medulla neurons. (E and insets) Some Repo-positive differentiated glial cells (blue) retain their ability to divide and are positively labeled with phospho-histone H3 (red) after migrating to their positions along the lamina plexus (arrowheads). (F and insets) Repo-positive glial cells accumulate and intermingle with mitotically active precursor cells at the border of the GPC area (arrowheads). (G and H) Glial cells extend leading processes as they migrate towards the R-cell projection field (PF). All samples were orientated in a frontal view (see Figure S3), as this enabled us to unambiguously identify different glial cell types based on their position relative to R-cell axons. Figure S2. Figure S2. gcm and gcm2 are not Required for Asense expression in the Outer Proliferation Center. Clones in the target area were generated using the ELF system and visualized by the absence of GFP expression (green). In wild type (A), neuroblasts in the Outer Proliferation Center (OPC) and lamina precursor cells (LPC) express Asense (red). In Df(2L)200 mosaic animals (B), expression of this marker is not affected. 4
Figure S3. Figure S3. Schematic Drawings Illustrating Frontal, Horizontal and Lateral Orientation of Optic Lobes. GPC, glial precursor cell areas; LF, lamina furrow; LPC, lamina precursor cells; OPC, outer proliferation center. 5