Figure 1. LRP4 is a synaptic protein expressed in excitatory neurons.

(A) Domain structure of Drosophila LRP4. Numbers indicate amino acids. EXT, extracellular side. INT, intracellular side. (B) Representative confocal image stack of a control Drosophila brain stained with antibodies against endogenous LRP4 (green) and Bruchpilot (inset, magenta) demonstrating expression throughout the brain. (C) Representative confocal image stack of an lrp4^dalek null brain stained with antibodies against LRP4 (green) and Brp (inset, magenta) demonstrating antibody specificity. (D) Representative confocal image of a Drosophila brain expressing UAS-Syt-HA via lrp4-GAL4 and stained with antibodies to HA (D, green) and N-Cadherin (inset, magenta). The expression pattern resembles that of endogenous LRP4, supporting the specificity of lrp4-GAL4. (E) Representative single slice within a single antennal lobe glomerulus of a brain processed for expansion microscopy (proExM) expressing LRP4-HA and Brp-Short-mStraw in all ORNs via pebbled-GAL4 and stained with antibodies to HA (E, E”, green) and mStraw (E’-E”, magenta). LRP4 localizes to synaptic neuropil regions. (F) High magnification image of the region bounded by dashed lines in (E) and stained as above. Arrows indicate LRP4-HA localization adjacent to / not directly overlapping with Bruchpilot-Short. Arrowheads indicate overlapping LRP4-HA and Brp-Short localization. (G–K) Representative high magnification confocal stack images of neuronal cell bodies surrounding the antennal lobe in animals expressing UAS-mCD8-GFP via lrp4-GAL4 and stained for antibodies against GFP (G-K, green) and other cell-type markers (G’-K’, magenta). Merge channels (G’’–K’’) show colocalization of lrp4 with the neuronal marker ELAV (G’’) but not the glial cell marker Repo (H’’). Neurons positive for lrp4 show colocalization with choline acetyltransferase (ChAT, I’’), and the vesicular glutamate transporter (vGlut, J’’), but little to no colocalization with the inhibitory neurotransmitter GABA (K’’), suggesting that lrp4-positive cells are largely excitatory neurons. The percentage of GFP-positive cells that are ALSO positive for the cell-type specific marker are as follows: Elav = 99.50 ± 0.19% overlap; Repo = 0.38 ± 0.18% overlap; ChAT = 59.13 ± 2.48% overlap; vGlut = 22.38 ± 1.28% overlap; GABA = 0.25 ± 0.16% overlap. For all cases, n = 8 animals, ≥ 200 cells per animal. Values = mean ± s.e.m. Scale bars = 50 µm (B–D), 150 μm (B-D, insets), 25 μm (E–F), 10 μm (G–K).

DOI: http://dx.doi.org/10.7554/eLife.27347.003

Figure 1—figure supplement 1. Sequence alignment of Drosophila, mouse, and human LRP4 homologues.

Multiple sequence alignment of Drosophila LRP4 (CG8909; accession AAF48538.1), Mus musculus LRP4 (accession NP_766256.3), and Homo sapiens LRP4 (accession NP_002325.2). Red shading = identical residues. Yellow shading = similar residues. The transmembrane domains are underlined. Considerable identity is seen throughout the extracellular side of the protein, and stretches of identity and similarity are also observed in the intracellular side. Asterisks denote a putative internalization signal, NPxY (Hussain, 2001), which is conserved on the intracellular side of all three species.

DOI: http://dx.doi.org/10.7554/eLife.27347.004

Figure 1—figure supplement 2. LRP4 reagents and patterns of LRP4 expression.

(A) Genomic region of lrp4. Top bar represents physical position on the X chromosome (in base pairs), and the blue arrow represents the lrp4 genomic region flanked by other genes (yellow). Primer sets are indicated by forward and reverse arrows (see B). The exon structure is displayed with 5’ and 3’ UTRs shaded in gray and coding exons numbered and shaded in beige. The region deleted by the lrp4^dalek mutation is indicated in pink. RNAi targets are shown below in orange. The position of the GAL4 in the GMR90B08-GAL4 line is shown below and region of the protein against which antibodies were raised are noted below. (B) PCR analysis of genomic DNA from control and lrp4^dalek adults. The presence of bands corresponding to Exon 2 and Exon 7–8 in control and heterozygous flies and their absence in lrp4^dalek demonstrate loss of the coding region. The presence of a 315 bp band in heterozygous and homozygous lrp4^dalek flies (Flank) but not in control is a result of non-homologous end joining of the 5’ and 3’ UTRs following deletion of the gene. (C) Representative confocal maximum intensity projections of the antennal lobe region of an lrp4-GAL4 animal expressing HA-tagged LRP4 and stained with antibodies to HA (C, C’’, green) and N-Cadherin (C’-C’’, magenta). LRP4-HA localizes to regions of synaptic neuropil, similar to endogenous staining (Figure 1). (D–E) Representative confocal maximum intensity projections of antennal lobes in animals expressing UAS-FRT-Stop-FRT-mCD8-GFP using lrp4-GAL4 but where FLP expression (removing the stop codon) is restricted to either ORNs using eyFLP (D) or PNs using GH146-FLP (E) and stained with antibodies to GFP (green) and N-Cadherin (magenta). Intersectional analysis reveals lrp4 expression in both ORNs as well as PNs. Scale bars = 10 µm (C), 5 μm (D–E).

DOI: http://dx.doi.org/10.7554/eLife.27347.005

Figure 1—figure supplement 3. Validation of expansion microscopy in Drosophila.

(A) A representative Drosophila brain stained with antibodies against endogenous Brp (green) and processed for proExM. Organization of the fly brain is maintained as are identifiable landmarks. (inset) Unexpanded brain stained with antibodies against Brp, shown at the same scale as the expanded brain. Individual Brp puncta are resolvable in the expanded brain but not in the unexpanded brain. (B) A representative antennal lobe in a Drosophila brain expressing Brp-Short-mStraw (magenta) in all ORNs using the pebbled-GAL4 driver and stained with antibodies against mStraw (magenta). Following proExM processing, glomerular structure and fine synaptic detail are still present. (inset) An equivalent single section from a different brain of the same genotype. In all cases, note the 4–5 fold isotropic expansion of tissue allowing for enhanced resolution while still using confocal microscopy. Scale bars as indicated.

DOI: http://dx.doi.org/10.7554/eLife.27347.006