The similar projection pattern of two identified ems expressing secondary lineages with the adPN and lPN lineages in the third instar larval brain suggested that ems might be expressed
in GH146 negative cells of the PN lineages during development. This prompted us to apply the dual-expression-control MARCM system (Lai and Lee, 2006) to the third instar larval brain when ems expression is observed in the E2 and E3 lineages. The dual-expression- control MARCM system allows the simultaneous labelling of homozygous GAL80-minus cells with two reporter systems that are differentially expressed within the Nb clone. Therefore, we have used the ubiquitous LexA::GAD driver labelling all the progeny of the GAL80-minus Nb, in combination with the GH146 driver to specifically label a subset of identified cells in the PN lineages (Lai and Lee, 2006). In case ems was expressed in the adPN and/or lPN lineage at third instar larval stage, we would expect to find single Nb clones labelled with the ubiquitous LexA::GAD driver that express ems in one subset and GH146 in a different subset of the clonally related cells.
In order to test this hypothesis, we have induced dual-expression-control MARCM clones in the early embryo for labelling of the complete lineage and analyzed the PN clones at 96 hours after larval hatching (ALH) in the late third instar brain (Fig. 3-2 A, B). Based on the morphology of the GH146 clones, we could identify the adPN and lPN lineages, respectively, and examine all GAL80-minus cells within the Nb clone using the ubiquitous lexA::GAD reporter system. As described in the adult, many additional cells that are negative for GH146 could be detected due to lexA::GAD expression (Lai and Lee, 2006). Furthermore, we have repeatedly found ems expression in one big cell, supposedly the Nb, and a number of adjacent smaller cells in both the adPN and lPN lineages. In both PN clones ems expression was mainly restricted to cells located close to the outer surface of the cortex (Fig. 3-2 A1, B1), whereas the GH146 signal was more frequent in cells deeper in the cortex and close to the antennal lobe (Fig. 3-2 A2, A3, B2, B3). Again, no overlap between the anti-ems and the GH146 signal in the same cell could be observed. Thus, applying the dual-expression-control MARCM system we have found molecular genetic evidence that ems is expressed in both the adPN and lPN lineages in the late third instar brain.
Figure 3-2 ems is expressed in adPN and lPN lineages during larval stages. Dual-expression control
MARCM with ubiquitous tubP-lexA::GAD driving rCD2::GFP (green) and the subtype specific GAL4-GH146 driving mCD8 in PN (red; see Materials and Methods for details). Co-localization of the two membrane-bound reporter constructs in yellow. Blue represents Anti-Ems. Clone induction by heat shock at 3-6 hours after egg laying. Clones were analyzed in late third instar larvae (A and B) at 96 hours after larval hatching (96 h ALH) and at second to third instar transition (C and D) at 48 hours ALH (48 h ALH). GAL80-minus adPN and lPN clones were identified via the GH146 signal and the whole lineage was labelled by the ubiquitous driver. (A – D) partial views from a ventral region including antennal lobes of single hemispheres. Anterior to the top, medial to the right. Image series (1 – 3) in (A – D) show single optical sections of same confocal stack with section (1) close to the outer cortical surface, (2) at deeper cortical level, (3) through antennal lobe at neuropile surface. ems expression is found in neuroblast (arrowhead) and adjacent cells but not in GH146-positive cells. (E) Model of transient ems expression during larval development (see text for details). Scale bars: 10μm.
Given the limited number of ems-positive cells and the lack of overlap with the GH146- positive cells within the PN lineages, we next wanted to test if ems expression in PN lineages is restricted to late third instar stage or if ems is also expressed earlier in larval development. Therefore, we have again induced dual-expression-control MARCM clones in the early embryo but now brains were analyzed at 48 h ALH. As expected, clones of both PN Nbs appeared smaller with respect to the late third instar stage. However, as in the late third instar brain, lexA::GAD expression revealed the presence of GH146-negative cells within the Nb clones. In both PN lineages, ems expression was again found in one Nb-like large cell and in adjacent smaller cells that were located close to the outer surface of the cortex (Fig. 3-2 C1, D1). No co-expression of ems and the GH146 signal in the same cells was found in any of the brains examined (Fig. 3-2 C2, C3, D2, D3). The same expression pattern of the ems gene within the adPN and lPN lineages was observed at 72 hours ALH and at 0 puparium formation (data not shown).
The restricted expression of ems to the putative Nb and its adjacent progeny is consistent with the observation that ems is transiently expressed in seven out of eight secondary lineages, during larval and early pupal development. Furthermore, ems expression has been shown to completely disappear from all transiently expressing brain lineages, including the E2 and E3 lineages, after 24 hours APF (Lichtneckert et al., 2007). Taken together, this suggests that ems is transiently expressed during the early differentiation steps of all larvally born PNs. As the PNs mature ems expression is down-regulated and stays off in the adult brain (Fig. 3-2 E).