In wild-type adPN and lPN lineages, ems is only transiently expressed in postmitotic cells and expression disappears before the GH146-signal becomes detectable in the differentiating neurons. In both lineages, no ems expression is found later than 24 hours APF (Lichtneckert et al., 2007). We therefore, wanted to test if down-regulation of ems expression in the
differentiating PNs is necessary for correct innervation of the antennal lobe glomeruli and the lateral horn. For this, we have misexpressed ems using a UAS-ems construct in otherwise wild-type GH146 MARCM clones that were induced in the early first instar larva.
To our surprise, no lPN clone could be detected in the 53 adult brains examined. This suggests that GH146-positive cells misexpressing ems in this lPN lineage did not survive to the adult stage. In contrast to the lPN lineage, 9 adPN neuroblast clones could be recovered from 53 adult brains and cell counts revealed that the average clonal cell number corresponded to wild-type levels (m = 33; s.d. = 0.4; see above). We next focused on the dendritic innervation of antennal lobe glomeruli by the adNb clones. We found that in adNb clones which misexpressed ems (expression confirmed by immunostaining; data not shown) 7 glomeruli were mistargeted which were never innervated by wild-type adNb clones induced in the early larva (Fig. 3-7 A – D). 2 glomeruli, DC1 and VM4, were mistargeted with a medium frequency (44%), whereas innervation of other 5 glomeruli was observed in more than 78 % (78-100%) of the cases. Interestingly, innervation of the VA2 glomerulus was found in all ems misexpressing adPN neuroblast clones induced in the early first instar larva. Although VA2 class PNs belong to the adPN specific set of GH146-positive glomeruli (see Fig. 3-4 B), they are only produced during embryonic development and no VA2 innervation is found in wild-type adNb clones induced during larval stages.
In addition to the 7 glomeruli which had ectopic innervation by ems misexpressing adNb clones we have found loss of innervation in one adPN specific glomerulus, the DL1 glomerulus. In all ems misexpressing adNb clones examined innervation of the DL1 glomerulus was completely absent (Fig. 3-7 C, D). In order to test if the lack of DL1 innervation is due to the absence of DL1 class neurons or the misprojection to other dendritic targets, we have analyzed DL1 class single-cell clones misexpressing ems that were induced in the early first instar larvae. Wild-type adPN single-cell clones induced by early larval heat shock (0 – 36 h) invariably innervate the DL1 glomerulus (see above; Jefferis et al., 2001). All 7 examples of ems misexpressing DL1 single-cell clones showed abnormal dendritic innervation. Whereas innervation of the DL1 glomerulus was never observed, two categories of misprojections were repeatedly found. In 5 of 7 single-cell clones dendritic innervation was mistargeted to the DA2 and DM6 glomeruli (Fig. 3-7 E, F). All of these clones showed additional dendritic branching in the dorsal part of the antennal lobe which could not be attributed to single glomeruli. In 2 of the 7 single-cell clones a diffused pattern of dendritic
branches was observed without dense innervation of single glomeruli (Fig. 3-7 G). Thus, misexpression of ems in GH146-positive adPNs causes dendritic mistargeting and the lack of DL1 innervation.
Figure 3-7 Misexpression of ems in mature adPN causes dendritic and axonal targeting defects. Green
represents GFP labelled GH146-GAL4 UAS-Ems misexpression MARCM clones. All clones were induced in the early first instar larva. (A – C) Antennal lobe dendritic innervation pattern of ems misexpressing adNb clones shown in anterior (A), intermediate (B) or posterior (C) single confocal sections (For wild-type adNb clone innervation pattern see Fig. 3-4 E). The ems misexpression clone ectopically innervated the DA2, VA2, VA6 (A), DC1 (B), DL2, VL2 andVM4 (C) glomeruli (outlined with white dots) and lacked innervation in the DL1 glomerulus (outlined with red dots). (D) Frequency of innervation of 7 ectopic glomeruli (white background) and the adPN specific glomerulus DL1 (red background) in totally 9 ems misexpressing adNb clones. (E – G) Antennal lobe dendritic innervation pattern of ems misexpressing DL1 single-cell clones shown in anterior (E) or posterior (F) single confocal sections and as z-projection through the whole anennal lobe (G). The ems misexpressing DL1 class single-cell clone never innervated the DL1 glomerulus but either ectopically innervated the DA2 and DM6 glomeruli (E) or extended unspecific arborizations through a major part of the antennal lobe (G). (H and I) Two examples of lateral horn axon terminal arborization of ems misexpressing DL1 single-cell clones. A dorsal branch first extends dorsally (arrowheads) but then turns and expands towards the tip of the lateral branch (arrows). (J) Z-projection of ems misexpressing adNb clone shows a bundle of dorsal branches
(arrowhead) and dense innervation (arrowhead) on a hypothetical line connecting the tip of the dorsal branch with the lateral tip.
We next wanted to test if misexpression of ems could affect the axonal projection pattern of DL1 single-cell clones. All 7 single-cell clones extended an axonal projection from the antennal lobe to the mushroom body calyx and the lateral horn, suggesting that overall PN morphology was not affected. In order to examine the axonal morphology at higher resolution we have again focused on the terminal arborization in the lateral horn. In all 7 DL1 class single-cell clones misexpressing ems the two major branches characteristic for DL1 axon terminals could be observed. However, in all examples, the dorsal branch first projected dorsally, as in wild-type, but then made a 90° turn and continued to extend towards the tip of the lateral branch. This lateral turn and overgrowth of the dorsal branch was never observed in wild-type DL1 cells (compare Fig. 3-6 B with Fig 3-7 H, I).
Subsequently, we expanded the analysis from DL1 class single-cell clones to ems misexpressing adNb clones. For this, we examined their general axonal pattern as z- projections encompassing the entire lateral horn and compared it to the wild-type control. In both cases, the dorsally projecting branch is clearly distinguishable (arrowheads in Figs. 3-6 C and 3-7 J). However, as compared to the wild-type, the ems misepxressing adNb clones contained additional axonal branches in the dorsal part of the lateral horn (arrows in Figs. 3-6 C and 3-7 J). The position of these additional branches in the ems misexpressing adNb clones was coincident with the site where the overshooting dorsal branch in ems misexpressing DL1 single-cell clones was observed (arrows in Figs. 3-7 H – J). This suggests, that the axonal phenotype observed in the DL1 class neurons could, at least in part, account for the phenotype observed in the whole adNb clone.
Taken together, misexpression of ems in PNs beyond the time of endogenous ems expression leads to dendritic and axonal targeting defects of adPNs and the absence of GH146-positive lPN in the adult brain.