Figure 6. Embryonic expression patterns of side genes.

Fluorescent in situ hybridization (magenta) of side-II, side-III, side-VI, side-VII and side-VIII genes in fillet preparations. All preparations are co-stained with anti-Fasciclin II antibody (1D4) to reveal all motor nerves (green). (A) side-II is predominantly expressed in the CNS, where it has an increasingly broad expression pattern as development progresses. (B) side-III expression pattern in a stage 14 embryo in the developing trachea (dashed line). (C) side-IV expression pattern in a stage 12 embryo in ventral muscle precursors. (D) At stage 16 expression of side-IV is localized to the ventral oblique muscles (muscles 15, 16, 17), the ventral transverse (25), the lateral oblique (5) and the segment border (8) muscles. (E) Expression of side-VI at stage 14–15 co-stained for RN2-Gal4 > UAS tau-LacZ. side-VI is broadly expressed in the CNS and in specific tissues in the periphery. XZ sections are indicated and represented underneath the main panel and magnifications of selected areas (G, H) are presented in individual panels. Orthogonal views show a cross section of a dorsal set of sensory neurons (1) and the junction of the ISN at its first branch, FB (2). The location of the ISN is marked with an arrowhead. (F) In a stage 16 embryo the ISN tip explores a group of side-VI expressing dorsal sensory neurons. (G) 3D projection of the ISN FB region where side-VI is expressed at high levels in the PT cell. The path of the ISN is overlaid with a dashed line. (H) The lateral bidendritic neuron (LBD), a synaptic target of the transverse nerve (TN), expresses high levels of side-VI. (I) side-VI is expressed in the dorsal median cell (DMC, arrowheads) in the CNS. (J) side-VI expression in ventral muscles in a stage 16 embryo (dorsal and ventral borders of muscles 12 and 13 are indicated by dotted lines). (K) side-VII expression in a S14 embryo is broad in the CNS and in the trachea (outlined). (L, M) side-VIII expression at stages 15–16 in RP1, 3, 4, and five motoneurons co-stained for lim3 >Tau myc (L), and in the pCC interneuron co-stained for RN2-Gal4 > UAS tau-myc (M). (N) Synopsis of side-III, IV, VI and VII expression in the periphery. Anterior is left and the ventral CNS is down in all panels except C, D, I, L, M and N where anterior is up. Scale bars indicate distances.

Figure 6—figure supplement 1. Analysis of side-VI expression patterns in larvae.

Side-VI-T2A-GAL4 > eGFP third instar larvae were dissected and stained with anti-GFP (green) and anti-HRP (magenta) to visualize neuronal membranes. GFP staining was observed on motor axons and NMJs. GFP channel (A) and HRP channel (A₁) of muscle 4. Arrows denote Type Ib and Type Is arbors on muscle 4. GFP channel (B) and HRP channel (B₁) of muscles 12 and 13. Arrows denote Type Is, Type Ib and Type III branches of the muscle 12 NMJ. The punctate green staining on muscles 12 and 13 may indicate that there is residual expression of side-VI in these muscles at third instar. Scale bar, 25 µm.