Figure 9.

Layering of the medulla neuropil in the adult and mid-stage pupa. (A-L′) Frontal sections of medulla neuropile, labeled by anti-DNcadherin (magenta in A-L; white in A′, B″, C′-L′), which reveals distinct strata with different signal intensity. Double labeling with cell type-specific markers, traditionally used to define layers m1-m10, permits one to assign the DNcadherin pattern to these layers. The layer-specific markers used are: anti-Chaoptin (24B10; labeling of photoreceptors R7/8; green in A, C, I; blue in E, K); c202-Gal4 (labeling of lamina L1 neurons; blue in C); bsh-Gal4 (labels medulla Mi6 neurons; green in D, J); ln-Gal4 (labels lamina L3/4 neurons; green in E, K); wg-Gal4 (labels precursors of medulla tangential neurons; green in F, L). The medulla layers (m1-10) defined by the specifically neuronal populations are indicated next to the marker (lower left of panels (A-F) and (G, K). Note that anti-Chaoptin labels adult layers m3 and m6; at P8 and earlier (see Fig.9) it labels m6 and transient layer distal of m1 (“tm1”). The top two rows of panels (A-F′) show adult stage; bottom rows (G-L′) pupa at 48h after puparium formation (P48). For a given pair of panels (e.g., C, C′), the upper one shows anti-DNcadherin label (magenta) in conjunction with marker; the lower one is anti-DNcadherin only (in white). Panels of left column (A, A′, G, G′) present overview of medulla neuropil at low magnification; hatched lines outline domain of neuropil shown at high magnification in the four columns to the right. Panels (B-B″) and (H) show labeling of neuropil glia expressing the marker NP3233-Gal4 (green). White arrowhead in (C, C′, F, F′, I, I′, L, L′) indicates serpentine layer (boundary region between m7 and m8. White arrowhead in (H) marks the DNcadherin^low stratum (nascent layer m3) prior to the formation of significant glial processes. Note differences in anti-DNcadherin pattern between late pupal and adult stage illustrated in panels (C′) and (I′), respectively. Layer m3, in the adult, is represented by a thin band with moderate DNcadherin signal (“3b”) flanked by bands of low signal (“m3a”, “m3c”). In the 48h pupa, m3 is narrower and typically represented by a single band with low or moderate DNcadherin level. Most conspicuously, the DNcadherin^low band representing layers m5/6 in the adult (C, C′) is absent in the pupa (I, I′); here, terminals of m5/6 specific neurons (L1, R8), that coincide with a DNcadherin^low stratum in the adult, are found in the lower part of a DNcadherin^high band (I). This band represents the “protolayer” for m4-m6. For other abbreviations, see legend of Fig.2. Bar: 50 μm (A, G)