Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons

Abstract

Background: Developing neurons form dendritic trees with cell type-specific patterns of growth, branching and targeting. Dendrites of Drosophila peripheral sensory neurons have emerged as a premier genetic model, though the molecular mechanisms that underlie and regulate their morphogenesis remain incompletely understood. Still less is known about this process in central neurons and the extent to which central and peripheral dendrites share common organisational principles and molecular features. To address these issues, we have carried out two comparable gain-of-function screens for genes that influence dendrite morphologies in peripheral dendritic arborisation (da) neurons and central RP2 motor neurons.

Results: We found 35 unique loci that influenced da neuron dendrites, including five previously shown as required for da dendrite patterning. Several phenotypes were class-specific and many resembled those of known mutants, suggesting that genes identified in this study may converge with and extend known molecular pathways for dendrite development in da neurons. The second screen used a novel technique for cell-autonomous gene misexpression in RP2 motor neurons. We found 51 unique loci affecting RP2 dendrite morphology, 84% expressed in the central nervous system. The phenotypic classes from both screens demonstrate that gene misexpression can affect specific aspects of dendritic development, such as growth, branching and targeting. We demonstrate that these processes are genetically separable. Targeting phenotypes were specific to the RP2 screen, and we propose that dendrites in the central nervous system are targeted to territories defined by Cartesian co-ordinates along the antero-posterior and the medio-lateral axes of the central neuropile. Comparisons between the screens suggest that the dendrites of peripheral da and central RP2 neurons are shaped by regulatory programs that only partially overlap. We focused on one common candidate pathway controlled by the ecdysone receptor, and found that it promotes branching and growth of developing da neuron dendrites, but a role in RP2 dendrite development during embryonic and early larval stages was not apparent.

Conclusion: We identified commonalities (for example, growth and branching) and distinctions (for example, targeting and ecdysone response) in the molecular and organizational framework that underlies dendrite development of peripheral and central neurons.

Figure 1

Embryonic da dendrite screen – examples of phenotypes.(a) Cartoon showing the relative positions of cell bodies of dorsal cluster multidendritic (md) neurons in late stage 17 embryos. The dotted box indicates the region of dendritic field chosen for enlargement in each of (b',c',d',e',f'). (b,b') In control animals, the dendritic trees of the six da neurons in the dorsal cluster can be visualized with GAL4^109(2)80 driving membrane-targeted GFP (UAS-mCD8::GFP). (c,c') Misexpression of GSd034: dilation of primary and reduced outgrowth of higher order dendritic branches. (d,d') Misexpression of GSd231: reduced dendritic field size with residual branching. (e,e') Slightly younger control animal, though still late stage 17, for comparison with (f,f'). (f,f') Misexpression of GSd422: production of filopodial spine-like protrusions. All images are maximal Z-projections of stacked confocal images. Anterior is left and ventral is down in all panels. Scale bar in (b) = 20 μm and applies to (c-f) also.

Figure 2

Larval da dendrite screen – examples of phenotypes. (a) In a control third instar larva (GAL4^109(2)80, UAS-mCD8::GFP/+), one can visualize the eight multidendritic neuron cell bodies of the dorsal cluster and the fields occupied by da neuron dendrites. Dotted boxes indicate fields examined at higher power in (c,e,g) to illustrate regions occupied primarily by the dendrites of class IV (c) (ddaC), class I (e) (ddaE), and class III (g) (ddaA) da neurons. (b) Misexpression of GSd302: severe reduction of higher order branches in class III and class IV da neurons, though the growth of primary branches of these and other da neurons appears intact. (c) Region in control highlighting (in red) the higher order branches of the class IV ddaC neuron. (d) Misexpression of GSd239 reduced the length of higher order branches of ddaC, with no obvious reduction in branch number. (e) Control ddaE (arrowhead at cell body), a class I da neuron that ordinarily has a simple pattern of lower order dendrite branches (highlighted in red). (f) Misexpression of GSd458 caused numerous small branches to emerge from ddaE (arrowhead at cell body). (h) Compared to controls (as in (g)), misexpression of GSd236 severely reduced numbers of spine-like protrusions in the class III neuron ddaA (cell body marked with arrowhead, dendrites highlighted in red). (i-k) Misexpression GSd402 and GSd450 caused severe reduction of dendrite outgrowth and branching (i,k), often with fewer GFP-labelled da neurons and signs of neuronal degeneration (higher power in (j,l)). All images are maximal Z-projections of stacked confocal images. Anterior is left and ventral is down. Scale bar in (a) = 100 μm and applies to (b) also. Scale bar in (c) = 30 μm and applies to (d-h) also. Scale bar in (i) = 100 μm and applies to (k) also.

Figure 3

GS misexpression with class-specific Gal4 drivers in larval da neurons.(a) Control class I da neuron ddaE visualized with GAL4²²¹ driving UAS-mCD8::GFP. (b) Misexpression of GSd458 caused increased numbers of small dendritic branches, though the primary branches were unaffected. (c) C161-GAL4 drives expression in classes I-III, but not class IV, allowing better visualization of spine-like protrusions on the class III neuron ddaA. Arrowheads in (c,d) mark the cell body of ddaA. (c') Tracing of ddaA cell body and dendrites in (c). (d) Misexpression of GSd236: primary dendrites of ddaA are devoid of spine-like protrusions. (d') Tracing of ddaA cell body and dendrites in (d). (e) ppk1.9-GAL4 is a class IV da neuron driver, revealing the complex dendritic tree of ddaC. (f) Misexpression of Gsd454: reductions in the number and growth of higher order branches of ddaC. (g) Quantification of branch ends per neuron for the genotypes shown in (a-f), showing class specificity of branching defects. In class I ddaE neurons (left), GSd458 increases branching dramatically (asterisk denotes t-test, P < 1e^-5), while GSd454 has no effect. In class IV ddaC neurons (right), both GSd458 and GSd454 reduce branching relative to controls (wild type (WT); asterisks denote t-tests, both P < 1e^-8). In both cases, the total length of the dendritic arbor was dramatically reduced (control (WT) = 17,389 ± 422 μm versus GSd454 = 8,544 ± 657 μm (t-test P < 1e^-10) or versus GSd458 = 2,650 ± 296 μm (t-test, P < 1e^-16). Since higher order branches were reduced but the growth of primary dendrites was mostly unaffected, there was no effect on dendritic field area (for example, control = 304,899 ± 7,115 μm² versus GSd454 = 301,475 ± 9,141 μm²; t-test P > 0.8). In class III ddaA neurons (middle), GSd236 dramatically reduced the number of short spine-like protrusions (t-test, P < 0.003), but had no effect on the total length of primary dendrites (control = 1,736 ± 137 μm versus GSd236 = 2,132 ± 157 μm; t-test P > 0.1). All images are maximal Z-projections of stacked confocal images. Anterior is left and ventral is down. Scale bars: (a-d) = 50 μm; (e,f) = 100 μm.

Figure 4

RP2 dendrite screen – phenotypic categories. Rows show examples representing the main phenotypic categories recovered from the central (RP2) neuron dendrite misexpression screen. Left and centre columns: confocal images (maximal Z-projections) of RP2 neurons at 25–31 hours AEL, visualised with UAS-mCD8::GFP. (a) Control RP2 neuron with brackets indicating the dendritic tree. (b) Control RP2 neuron in the context of a set of axon tracts visualised by anti-FasciclinII staining (magenta), with arrowheads pointing from top to bottom to the lateral, intermediate and medial FasciclinII tracts and the midline indicated by a dotted line. Dendrites between the lateral and central intermediate Fasciclin II fascicle are defined as 'lateral'; dendrites located between the central intermediate fascicle and the midline as 'medial'; the same applies to (o,p). (c) Same neuron as in (b) but with sectors of its dendritic tree pseudo-coloured to highlight branches targeted to anterior lateral (magenta), anterior medial (yellow) and posterior lateral (cyan) regions. Anterior is left and the ventral midline is down. (d,e,g,h,i,j,l,m,o,p) Experimental cells: misexpression lines are indicated in the bottom right-hand corner of each panel. Right column: (f,k,n,q) quantifications of the dendritic phenotypes shown in the left and central columns. As illustrated in (f), both dendritic tree length and number of branching events are reduced in the 'Growth' and 'Branching' categories. 'Branching' phenotypes have trees with an anterior-posterior extent comparable to controls (Additional file 2) but have an altered pattern of branching: fewer branching events and more segments that are longer (>5 μm). *P < 0.01, **P < 0.005, t-test, N = 5. Error bars indicate the standard error. Arrows in (b,o,p) point to medial branches present in controls (b) and absent/reduced in experiments (o,p). Black asterisks in (e,p) indicate the cell body of the contralateral RP2 neuron. Scale bar: 10 μm.

Figure 5

Dendrite growth and branching are distinctly affected by gene misexpression. Proportional Venn diagrams to show degree of overlap among lines with effects on dendrite growth and/or branching.

Figure 6

Dendritic targeting relative to the ventral midline.(a) Control and (b,c,e,f) experiments showing confocal images (maximal Z-projections) of RP2 neurons at 25–31 hours AEL, visualised with UAS-mCD8::GFP (green) in the context of a set of axon tracts visualised by anti-FasciclinII staining (magenta). Dendrites between the lateral and central intermediate Fasciclin II fascicle are defined as 'lateral'; dendrites located between the central intermediate fascicle and the midline as 'medial'. Misexpression lines are indicated in the bottom left hand corner of each panel. (b) Misexpression of commissureless (comm) leads to aberrant midline crossing of dendritic branches (arrowhead), though no apparent increase of dendrites targeted towards the midline between the intermediate and medial FascilinII tracts. The high variability in phenotype is partly due to the varying lengths the dendritic tree mis-routed across the ventral midline. (c) Misexpression of frazzled (fra) causes increased targeting of dendrites into the medial neuropile (arrowhead). Black asterisk indicates the cell body of the contralateral RP2 neuron. (d,d') Ventral (d) and lateral (d') views of stage 13 embryos driving expression of GSd433 with engrailed-GAL4 and stained by in situ hybridisation using an anti-sense probe against robo2. The staining shows the segmentally repeated stripes characteristic for engrailed. The reaction had to be terminated before the endogenous robo2 expression pattern appeared (see Additional file 3) due the high levels of expression. (e,f) Misexpression of robo2 by GSd433 (e) or robo (f) leads to a reduction to near absence (robo) of branches innervating the medial neuropile (arrowheads), and some dendritic branches positioned aberrantly lateral of the lateral Fasciclin II axon tract (arrows). (g) Quantification showing ratios of medial/lateral dendrites; *P = 0.04, **P < 0.001, t-test, N = 5; error bars indicate the standard error. Anterior is left. Scale bars: (a-c,e,f) = 10 μm; (d,d') = 140 μm.

Figure 7

Displacement of dendritic branches from medial anterior to posterior lateral regions. RP2 neurons at 25–31 hours AEL and visualised with UAS-mCD8::GFP in the context of FascicilinII positive axon bundles (magenta) demarcating the medial and lateral neuropile (maximal Z-projections of confocal image stacks). (a) Control. (b) Misexpression of UAS-robo-Y-F (activated robo) leads to a lack of dendritic innervation of the medial neuropile (normally located anterior to the axon (arrowhead in (a)) and a concomitant expansion of dendrites in the lateral neuropile posterior to the axon (arrowhead in (b))). Dendritic extent anterior or posterior to the axon is indicated by brackets. (c) Quantification of anterior, posterior and total (combined) maximal dendritic extent for controls (green, N = 10) and UAS-robo-Y-F expression RP2 neurons (magenta, N = 8). The significance of pair-wise comparisons using Student's t-test is indicated. Anterior is left and the ventral midline is down. Scale bar: 20 μm.

Figure 8

Dendritic growth, branching and targeting are regulated independently. (a,b) Three-dimensional reconstructions from confocal image stacks of RP2 neurons at 25–31 hours AEL and visualised with UAS-mCD8::GFP generated with AMIRA software. (a) Control. (b) Misexpression of GSd421 causes aberrant dendritic targeting to the posterior. Brackets in (a) indicate the dendritic tree. (a',b') Dendrograms derived from the reconstructions with branch points highlighted in magenta and the cell body and axon offset from the dendritic tree by green. (c) Quantification of the dendritic architectures for controls (green, N = 4) and GSd421 expressing RP2 neurons (magenta, N = 4). The significance of pair-wise comparisons using Student's t-test is indicated. Error bars indicate the standard error. Anterior is left and the ventral midline is down. Scale bar: 10 μm.

Figure 9

Overlap of RP2 and da screens, classified by sites of gene activity. Proportional Venn diagrams to describe the degree of overlap among genes that emerged from both screens. The total is shown at top left, and then broken down by the predicted site of gene product activity.

Figure 10

The EcR pathway is required for peripheral dendrite development.(a) Control class IV ddaC MARCM clone. (b) usp² MARCM clone showing reduced ddaC dendrite branches. (c) Quantification of the mean number of branch ends per neuron, comparing wild-type (WT) to usp² MARCM clones. The asterisk indicates significant reduction (t-test, P < 0.000001). (d) Control ddaC neuron (genotype: UAS-mCD8::GFP/+;;ppk1.9-GAL4/+). (e) Expression of RNAi-inducing UAS-IR-EcR, targeting all EcR isoforms. (f) Expression of a dominant-negative EcR (EcR-DN). (g) The graph on left shows the mean number of branch ends per neuron for all genotypes tested, including those co-expressing UAS-Dicer2 (Dcr2), a component of the RNAi machinery that can potentiate the RNAi effect [88]. The graph on right shows the mean branch density in ddaC class IV neurons. Pairwise comparisons (ANOVA, Tukey, P < 0.0001) determined that EcR RNAi significantly reduced both branch number per neuron and branch density (single asterisks). EcR-DN further reduced branch number and density to levels lower than both controls and RNAi (double asterisks). The analysis revealed that the RNAi-induced reduction of branch density (right graph) was not enhanced by coexpression of Dcr2. (h) Control ddaC neuron (same genotype as (d)) in first instar larva (28–30 hours AEL). (i) Expression of EcR-DN (same genotype as (f)). (j) EcR-DN reduced branch number in first instar larvae (asterisk, t-test, P < 1e^-7), but did not influence the field area (control = 11,349.7 ± 324.6 μm² versus EcR-DN = 12,261.0 ± 372.7 μm², t-test, P = 0.07). Error bars in (c,g,j) indicate standard error. Anterior is left and ventral is down. Scale bars: (a,b,d-f) = 100 μm; (h,i) = 25 μm.