DEV02750 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Expression of c-Gcm2 in developing chicken embryonic central nervous system. c-Gcm2 in situ hybridization on whole chick embryos (A,B) and on transverse sections of embryonic spinal cord (B′-D). Embryonic developmental stages are indicated in each panel. (A) At stage 9 (E1), c-gcm2 RNA is not detected in the neural tube. Signal observed in the anterior brain is due to non-specific trapping in the ventricules. (B) From stage 10 (E1.5), c-gcm2 is expressed in a restricted rostrocaudal domain of the neural tube extending from posterior rhombencephalon to somite 4 corresponding to the presumptive hindbrain area. (B′) Transverse section through embryo in B shows c-gcm2 expression in neural progenitors. (C,D) c-gcm2 RNA remains undetectable in the developing spinal cord at later stages (E2 in C, E8 in D). Scale bars: 30 μm in B′; 20 μm in C; 80 μm in D.

• Supplemental Figure 2 -

  Fig. S2. gcm is expressed in glial and neuronal lineages during fly post-embryonic brain development. (A-D′′′) Confocal images of triple labeling on gcm-gal4,UAS-ncGFP (gcm>ncGFP) LIII CNS, four non-consecutive lateral to medial partial projections. Anterior is to the left. The dashed line defines the border between outer proliferation center (OPC) and central brain (CB). (A-D) Merge of Repo (red, A′,B′,C′,D′), GFP (green, A′′,B′′,C′′,D′′) and Elav (blue, A′′′,B′′′,C′′′,D′′′) immunolabeling. Green line in A′′′,B′′′,C′′′ indicates dorsoventral midline. In the lamina, GFP labeling is present in epithelial (eg) and marginal (mg) glia as well as in glial precursor cells (GPCs in B′′, C′′) and lamina precursor cells (LPCs in A′′) areas. Note that medulla glia (meg) does not express GFP (yellow arrow in C′). In the lamina, some GFP-positive cells coexpress Elav but not Repo (arrowhead in A,A′′′). In addition, GFP is also expressed in two distinct cell clusters in CB (white and yellow dotted areas in A,B,C,D). The medial cluster (mcbc in A′′,B′′,C′′) is shown by the white dotted area in A,B,C (corresponding to cluster detected in Fig.7A-B); the dorsolateral cluster (dcbc in D′′) is shown by the yellow dotted area in D. These cell clusters are Elav-positive but not Repo-positive. Yellow rectangle in D shows glia at the interhemispheric junction. Scale bar: 20 μm.

• Supplemental Figure 3 -

  Fig. S3. Axonal pattern of gcm-gcm2-positive neurons in fly adult brain. (A-D) Highwire (red) and GFP (green in B,D) coimmunolabeling on gcm-gal4,UAS-mCD8GFP adult brain. Right panels (B,D) show merge of GFP and Highwire immunolabeling. (A,B) Adult neurons of the dorsolateral clusters (dcbc in B) display a complex pattern of axonal projections. Ventral projections reach the suboesophageal ganglia (SOG, dashed area in A), contralateral projections form several commissures (white arrowheads in B) bridging the two brain hemispheres and extending around the ellipsoid body region (eb in B), while dorsal projections reach the region of the mushroom calyces (Ca in B) and the inner antennocerebral tract (iACTin B). Note that one neuron of each dorsolateral cluster extends one axonal projection within the lobula (lo in A) (indicated by yellow arrows in B). (C,D) Axonal projections of neurons from the medial cluster (mcbc in D) extend only within the protocerebrum neuropile (pro, dotted area in C). Scale bar: 200 μm.

• Supplemental Figure 4 -

  Fig. S4. gcm-gcm2-positive adult neurons do not express the glial marker Repo. (A-B′′′) GFP (green), Elav (blue) and Repo (Red) coimmunolabeling on gcm-gal4,UAS-mCD8GFP adult brain. A-A′′′ and B-B′′′ show high magnifications of dorsolateral (dcbc) and medial (mcbc) clusters shown in Fig.7C and 7.C′, respectively. A′′′,B′′′ show merge of GFP (A,B), Elav (A′,B′) and Repo (A′′,B′′) immunolabeling, respectively. Note that dcbc and mcbc GFP-positive cells express Elav but not Repo. Scale bar: 200 μm.

• Supplemental Figure 5 -

  Fig. S5. Glial and neuronal differentiation requires Gcm pathway in the visual system. (A-D) Immunolabeling on gcm-gal4,tub-gal80^ts;UAS-gcm^N7-4DN,UAS-gcm^N7-4DN (control in A,C) and gcm-gal4,tub-gal80^ts;UAS-gcm^DN,UAS-gcm^DN (gcm-gcm2 LOF in B,D) LIII CNS. A,B and C,D show Repo (mediolateral view) and Dachshund (Dac) (lateral view) labeling, respectively. (A,B) The number of lamina (including epithelial and marginal glia) and medulla neuropile glia (indicated by lg and mng in A, respectively) is severely reduced in gcm-gcm2 LOF (B) compared to control (A) CNS. Yellow arrowheads in A indicate migrating mng present in control CNS (note that they are absent in B). Due to projection of several confocal optical sections, some superficial glial cells not belonging to optic lobes are present in A,B (indicated by asterisks and white arrowheads). (C,D) Note that the number of lamina neurons (LN in C) is also strongly affected in gcm-gcm2 LOF (D) compared to control (C) CNS. Areas delimited by dashed lines in C,D show neurons that do not belong to lamina. Scale bar: 40 μm.