Dscam1 Has Diverse Neuron Type-Specific Functions in the Developing Drosophila CNS

Abstract

Two key features endow Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) with the potential to provide a ubiquitous code for neuronal arbor self-avoidance. First, Dscam1 contains three large cassettes of alternative exons, so that stochastic alternative splicing yields 19,008 Dscam1 isoforms with different Ig ectodomains. Second, each neuron expresses a different subset of Dscam1 isoforms, and isoform-specific homophilic binding causes repulsion. This results in even spacing of self-arbors, while processes of other neurons can intermingle and share the same synaptic partners. In principle, this Dscam1 code could ensure arbor spacing of all neurons in Drosophila This model is strongly supported by studies on dendrite spacing in the peripheral nervous system and studies on axonal branch segregation during brain development. However, the situation is less clear for central neuron dendrites, the major substrate for synaptic input in the CNS. We systematically tested the role of Dscam1 for dendrite growth and spacing in eight different types of identified central neurons. Knockdown of Dscam1 causes severe dendritic clumping and length reductions in efferent glutamatergic and aminergic neurons. The primary cause for these dendritic phenotypes could be impaired self-avoidance, a growth defect, or both. In peptidergic efferent neurons, many central arbors are not formed, arguing for a growth defect. By contrast, knockdown of Dscam1 does not affect dendrite growth or spacing in any of the five different types of interneurons tested. Axon arbor patterning is not affected in any neuron type tested. We conclude that Dscam1 mediates diverse, neuron type-specific functions during central neuron arbor differentiation.

Keywords: Drosophila; Dscam1; dendrite; interneuron; motorneuron; self-avoidance.

Figure 1.

Temporal and spatial expression patterns of Dscam1. A, Representative projection images of immunocytochemistry with α-GFP (green) to detect Dscam1^GFP and with the active zone marker α-brp (magenta) to label neuropil regions in Drosophila brain at different developmental stages of postembryonic life. B, Dscam1^GFP expression through VNC development. C, Images for all stages have been acquired with identical settings, Dscam1^GFP labeling intensities measured from projection views of VNC neuropils of three preparations per stage, normalized to maximal intensities at stage P7, and plotted over development. Time periods of motoneuron dendrite growth and synaptogenesis in VNC are indicated at the bottom. Dscam1 expression levels in VNC neuropils peak at pupal stages of fast motoneuron dendrite growth and synaptogenesis. L3 is larval stage 3, P3, P4, P5, P7, P10, P11, and P15 are pupal stages 3 to 15.

Figure 2.

Dscam1-RNAi knocks down protein and reproduces known CNS phenotypes. A, Colabeling of Dscam1^GFP (green) and synaptic neuropil (brp; magenta) in the larval CNS of homozygous Dscam1^GFP animals. B, Heterozygous Dscam1^GFP/Dscam1 animals show similar expression patterns but lower Dscam1^GFP labeling intensity (reduction by 42 ± 5%). C, D, Pan-neuronal RNAi knockdown of Dscam1 under the control of elav^C155-GAL4 with two different UAS-RNAi transgenes (C; catalog #GD36233, VDRC; D; catalog #KK108835, VDRC) reduced the Dscam1^GFP label below detection threshold (reduction by 90 ± 4% compared with control). E, Western blotting for Dscam1^GFP and hsp90 (loading control) from adult Drosophila VNC homogenate. The Dscam1^GFP is detected at its expected size of ∼270 kDa in homozygous and heterozygous Dscam1^GFP animals but absent in controls. Both Dscam1 knockdowns reduce signal by ∼80%. F–I, α-Fas2 immunolabel of adult MB in control (F) and following the expression of Dscam1-kd (catalog #KK108835, VDRC) in MB under the control of C739-GAL4 in three animals with representative kd phenotypes (G–I).

Figure 3.

Dscam1 is required for larval MN dendrite but not axon differentiation. A–F, Maximum intensity projection views of CLSM image stacks of GFP-labeled RP2 motoneurons in third instar larvae in control (A), with two different Dscam1-RNAi-knockdowns (B; catalog #KK108835, VDRC; C, catalog #GD36233, VDRC), and with a Dscam1²³ (D), a Dscam1⁴⁷ (E), and a Dscam1²¹ (F) mutant allele. All mutant RP2 motoneurons were homozygous mutant in an otherwise heterozygous background with MARCM. G, Dendritic coverage area as measured from projection views is significantly (***p < 0.001, ANOVA with Tukey’s post hoc comparison) reduced in all genotypes with reduced Dscam1. H–K, Morphometric analysis reveals a significant reduction of TDL (H; ***p < 0.001, unpaired two-sided Student’s t test) and number of branches (I; **p < 0.01, unpaired two-sided Student’s t test) with reduced Dscam1, but the MDLs (J; n.s. unpaired two-sided Student’s t test) and mean dendritic radius (MDR; K; n.s. unpaired two-sided Student’s t test) remain unaltered. L, Dscam1^GFP expression (green) and active zone labeling with brp immunolabel (magenta) in the VNC during the first (L1, left), second (L2 middle), and third larval instar (L3, right) stages. Larval motoneuron total dendrite length is increased ∼10-fold between L1 and L3. M, N, Using a flippase strategy (see Materials and Methods), we created mosaic larvae with RP2 motoneurons that either express GFP and Dscam1-kd or not. α-HRP (cyan) and α-brp (magenta) labeling reveals no obvious differences in axon terminals and active zones between GFP-negative control (M) and GFP-positive Dscam1-kd RP2 cells (N).

Figure 4.

Dscam1 is not required for larval MN axon terminal differentiation. A, B, Representative motoneuron axon terminals on muscle 4 in control (A) and with Dscam1-kd (B). Quantification reveals no significant differences (n.s., unpaired two-sided Student’s t test) in active zone numbers (C), bouton size (D), evoked postsynaptic current (PSC) amplitude (E), miniature PSC amplitude (F) and frequency (G), or paired pulse ratio (H). F–H were recorded in two-electrode voltage-clamp mode. I, Crawling speed was significantly reduced (***p < 0.001, unpaired, two-sided Student’s t test) in animals with Dscam1-kd in larval motoneurons.

Figure 5.

Dscam1 is required for normal dendrites in aminergic and peptidergic neurons. A, B, Projection views of ventral unpaired median aminergic neurons in the larval VNC that are labeled with UAS-GFP expressed under the control of TDC2-GAL4. White boxes in control (A) and with Dscam1-kd (B) are selectively enlarged in Ai and Bi. Dscam1-kd eliminates higher-order dendritic branches (see white arrows). C, D, Projection views of larval peptidergic neurons with UAS-GFP expression under the control of CCAP-GAL4. White boxes in control (C) and with Dscam1-kd (D) are selectively enlarged in Ci and Di. Dscam1-kd eliminates higher-order dendritic branches and causes dendrite clumping (see white arrows). E, F, Projection views of ventral unpaired median aminergic neurons in the adult VNC that are labeled with UAS-GFP expressed under the control of TDC2-GAL4. White boxes in control (E) and with Dscam1-kd (F) are selectively enlarged in Ei and Fi. Dscam1-kd causes dendrite clumping (see white arrows). G, H, Projection views of adult peptidergic neurons with UAS-GFP expression under the control of CCAP-GAL4. Dscam1-kd reduced dendritic branches in VNC abdominal segments and nearly eliminates CCAP neuron dendrites from the thoracic VNC neuromeres. I, Dscam1-kd in adult CCAP neurons goes along with wing defects. Both CCAP neuron morphologic defects and wing defects show variable severity, but there is no correlation between the severity of neuronal and wing phenotypes.

Figure 6.

Dscam1 is not required for VNC interneuron arbor differentiation. A, Expression of tdTomato under the control of per-GAL4 in animals with GFP-tagged endogenous Dscam1 reveals that all axonal and dendritic arbors of PMSIs (red) are located Dscam1^GFP (green)-positive VNC neuropil areas. B, Dscam1-kd in PMSIs has no obvious effects on the axonal or dendritic structure of these interneurons. C, Mosaic labeling of basin-1 interneurons (green) in larval VNC counterstained with the synaptic marker brp (magenta) in control and with Dscam1-kd in basin-1 neurons. D, Selective enlargement reveals no differences between control and Dscam1-kd basin-1 interneurons.

Figure 7.

Dscam1 is not required for brain interneuron arbor differentiation. A, Representative projection view of GFP expression in serotonergic CSD interneurons (green) in the adult Drosophila brain counterstained with the synaptic marker brp (magenta, middle). CSD interneuron structure shows no obvious differences between control (top left, top middle) and Dscam1-kd (bottom left, bottom middle). Selective enlargements of CSD neuron arbors in antennal lobe (right) reveals no obvious differences between control (top right) and Dscam1-kd (bottom right). B, Overview of LC4 neuron structure in the adult brain counterstained with brp (left). Dscam1-kd (right) has no obvious effect on LC4 neuron arbor lengths or spacing compared with control (middle). C, Left, Representative projection views of GF interneuron dendrite structure in control (left), with Dscam1-kd (middle), and following Dscam1 overexpression. All three dendritic subtrees (numbered) contain higher-order dendrites, which are normally spaced relative to each other. Right, Representative quantitative reconstructions of control and Dscam1-kd GF interneurons with similar total dendrite length and mean dendritic branch length.