Fig. 1

Membrane-tagged GFP expression in 6 dpf th:Gal4VP16, UAS:GFP-CAAX larvae identifies peripheral catecholaminergic projections. (A–C) Whole mount immunofluorescence detection of GFP and TH in Tg(th:Gal4-VP16)^m1233, Tg(UAS:eGFP-CAAX)^m1230, Tg(UAS:SypGFP)^m1238 transgenic larvae. Images show maximum intensity projections (MIP) from confocal optical sections of larval head and trunk. TH immunoreactivity validates catecholaminergic cells with GFP expression. The immunostain reveals DA cell clusters in the ventral diencephalon/posterior tuberculum (PT) and hypothalamus, in the pretectum (PR), preoptic region (PO) and olfactory bulb (OB), in a subset of amacrine cells (AC) in the retina, as well as noradrenergic hindbrain neurons and sympathetic ganglia (SG). Arrowheads point at structures as indicated by adjacent labels. Central DA projections are labeled throughout the brain and in the spinal cord. Note that GFP expression levels in the transgenic line do not correlate with levels of TH expression, and GFP expression is often found only in a mosaic fashion in a subset of TH positive cells. In the Tg(th:Gal4-VP16)^m1233 line, at 6 dpf ectopic GFP expression occurs in a commissure at the border between mes- and rhombencephalon (#1) and in pectoral fin muscles (#2). GFP expression stemming from the heart marker of the Tg(UAS:Syp-GFP, clmc2:GFP) transgene is also indicated (#3). No TH-labeling is observed in the ectopically GFP-labeled structures. Cell nuclei were labeled with TOTO-3-Iodide to reveal anatomical landmarks (A,B). (A). Dorsal view MIP (scan from dorsal side, step size 1 μm, total depth of 410 μm, montage of three tiles from rostral part of the whole-mount) with emphasis on peripheral projections going to the lateral line neuromasts (marked with asterisks). PLLG marks posterior lateral line ganglion. (B) Ventral view MIP (scan from ventral side, step size 1 μm, total depth of 390 μm), montage of three scanned tiles from rostral part of a whole-mount larva. The ventral scan reveals GFP expression and TH-labeling in peripheral DA projections to the ventrally located lateral line neuromasts (asterisks) and otic epithelium (OE). In addition, the carotid body (CB) CA cells, the SG with their projections, and putative noradrenergic innervation of the atrial region of the heart (X1) are labeled. (C) Dorsal view of MIP from optical slices of medial subregion of the whole-mount (montage of two tiles, slices 180–240 of total 507 μm, step size: 1 μm) revealing labeled cell bodies in the depth of the brain. TH immuno-reactive cells are visible in the OB, PR and PO, among those individual cells also express GFP. A subset of TH-immunoreactive AC and their projections express GFP. In the ventral diencephalon, the DA cell clusters in the PT and dorsal hypothalamus are labeled. Double-labeled cell bodies can be observed in the anterior part of the PT in rostral (PTar) and caudal (PTac) sub-clusters, further in the cluster of the posterior PT (PTp) and in the posterior tuberal nucleus (PTN). In the dorsal medial hypothalamus small liquor contacting cells (Hdm) are labeled with GFP as well as in the caudal hypothalamus (Hc). Double labeling can also be observed in the SG. (D) Schematic representation of DA cell clusters in the PT and hypothalamus. The cells belonging to the different cell clusters can be identified and distinguished by their location, size, and shape. All scale bars: 100 μm.