Figure 4. Cholinergic 11A neurons adopt GABAergic identity in unc-4 mutant animals.

(A–D) The adult VNC from control (left) and unc-4 mutant (right) animals shown; images are from maximum Z projection of confocal slices. (A) All Unc-4⁺ neurons were visualized with unc-4^DBD-tub^AD driven GFP expression (green). No major difference was observed between the control and mutant. (B) unc-4^AD-ChAT^DBD split-GAL4 combination used to label cholinergic Unc-4⁺ neurons. Most Unc-4⁺ neurons were marked with this combination in both the control and mutant VNC and no major difference was seen between two. (C) unc-4^AD-VGlut^DBD driver marked a number of embryonic-born Unc-4⁺ glutamatergic motor neurons in the control VNC, which included flight related motor neurons MN1-4 (arrowheads) and MN5 (arrows). In the mutant, these motor neurons were disorganized and some showed reduction in cell size; for example, MN5 neurons. (D) unc-4^DBD-gad1^AD combination did not consistently show any GABAergic Unc-4⁺ neuron in the control CNS; however, in the unc-4 mutant background GABAergic interneurons were found in both the T1 (9 +/- 5 neurons; N = 30) and T2 (17 +/- 12 neurons; N = 30) segments. (E) A BAC transgene containing unc-4 locus suppressed the induced GABAergic phenotype and reduced the number of these neurons to 1.5 +/- 1 neurons per T1 segment and 1.3 +/- 1 neurons per T2 segment (N = 11 VNCs each). (F) A partial Z projection showing the main neuronal processes of unc-4^DBD-gad1^AD marked neurons in the VNC of an unc-4 mutant animal. They extend ipsilateral processes both anteriorly and posteriorly (arrows). (G) NB7-2 intersected reporter immortalization used to label all lineage 11 neurons in unc-4 mutant background and partial Z projection made to show only 11A neuronal processes but not that of 11B. The projections by the mutant cells match those of the 11A neurons (arrows). (H–K) unc-4^DBD-gad1^AD expression (outlined with dashed yellow lines) in the T2 segment from unc-4 mutants shown. Nkx-6, a marker for 11A neurons, is expressed in a subset of unc-4^DBD-gad1^AD marked neurons (arrows in H) while Eve, a marker for 11B neurons, is not (I). (J) ChAT (blue) and gad1(magenta) mRNAs were visualized via RNA in situ hybridization. Majority of unc-4^DBD-gad1^AD marked neurons expressed gad1 mRNA but not ChAT mRNA. (K) unc-4^DBD-gad1^AD marked neurons were immunopositive for GABA reactivity (magenta). Scale bar is 20 microns.

Figure 4—figure supplement 1. Emryonic-born Unc-4⁺ neurons become flight motor neurons in the adult VNC.

(A–C) unc-4^AD-VGlut^DBD driven GFP expression in the larval (A, C) and adult (B) VNC shown. Eve expression (magenta) marks segmentally repeated larval U/CQ motor neurons (C). Based on our tracing experiment, these motor neurons are remodeled during metamorphosis to be adult MN1-4 and haltere motor neurons in thoracic segments, and dorsal body-wall innervating motor neurons in abdominal segments (B). (A) Unc-4 is also expressed in two pairs (in T1 and T2) and one pair (in T3 and abdominal segments) neurons, which are located on the dorsal surface of the pupal VNC. Based on lineage clones we generated (not shown) and previously published work (Rogulja-Ortmann et al., 2008), these neurons are likely embryonic progeny of NB2-4. (B) In T1, these neurons become adult neck motor neurons. In T2, one of these neurons becomes MN5 motor neuron while its cousin is eliminated by apoptosis during pupal development (asterisks). Genotype: unc-4^AD/+;VGluT^DBD/+;10XUAS-IVS-myr::GFP/+.

Figure 4—figure supplement 2. ChAT (magenta) and gad1(blue) mRNAs were visualized via RNA in situ hybridization.

unc-4^DBD-tub^AD marked Unc-4⁺ hemilineages (green) express ChAT mRNA but not gad1 mRNA. Single confocal slices from the T2 segment of the dorsal (A) and ventral (B) VNC shown. Scale bar is 20 microns. unc-4^DBD/+;tub^AD/+;10XUAS-IVS-myr::GFP/+.