Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans

Abstract

Nociceptive neurons innervate the skin with complex dendritic arbors that respond to pain-evoking stimuli such as harsh mechanical force or extreme temperatures. Here we describe the structure and development of a model nociceptor, the PVD neuron of C. elegans, and identify transcription factors that control morphogenesis of the PVD dendritic arbor. The two PVD neuron cell bodies occupy positions on either the right (PVDR) or left (PVDL) sides of the animal in posterior-lateral locations. Imaging with a GFP reporter revealed a single axon projecting from the PVD soma to the ventral cord and an elaborate, highly branched arbor of dendritic processes that envelop the animal with a web-like array directly beneath the skin. Dendritic branches emerge in a step-wise fashion during larval development and may use an existing network of peripheral nerve cords as guideposts for key branching decisions. Time-lapse imaging revealed that branching is highly dynamic with active extension and withdrawal and that PVD branch overlap is prevented by a contact-dependent self-avoidance, a mechanism that is also employed by sensory neurons in other organisms. With the goal of identifying genes that regulate dendritic morphogenesis, we used the mRNA-tagging method to produce a gene expression profile of PVD during late larval development. This microarray experiment identified>2,000 genes that are 1.5X elevated relative to all larval cells. The enriched transcripts encode a wide range of proteins with potential roles in PVD function (e.g., DEG/ENaC and Trp channels) or development (e.g., UNC-5 and LIN-17/frizzled receptors). We used RNAi and genetic tests to screen 86 transcription factors from this list and identified eleven genes that specify PVD dendritic structure. These transcription factors appear to control discrete steps in PVD morphogenesis and may either promote or limit PVD branching at specific developmental stages. For example, time-lapse imaging revealed that MEC-3 (LIM homeodomain) is required for branch initiation in early larval development whereas EGL-44 (TEAD domain) prevents ectopic PVD branching in the adult. A comparison of PVD-enriched transcripts to a microarray profile of mammalian nociceptors revealed homologous genes with potentially shared nociceptive functions. We conclude that PVD neurons display striking structural, functional and molecular similarities to nociceptive neurons from more complex organisms and can thus provide a useful model system in which to identify evolutionarily conserved determinants of nociceptor fate.

Figure 1. PVD displays an elaborate dendritic arbor that envelops the animal in a net-like array

(A). Confocal image of an adult worm (anterior to left, ventral to bottom) showing the PVD::GFP marker (arrows denotes other neurons in head and tail that express GFP). Insets show more highly magnified image (B) and schematic tracing (C) of region surrounding PVD soma. Note dendritic branches (1^O, 2^O, 3^O, and 4^O) and single ventrally projecting axon (arrowhead denotes location of ventral nerve cord). Scale bar is 15 um.

Figure 2. PVD dendrites tile with FLP dendritic branches in the anterior

Lateral view of adult from left side (anterior to left, ventral to bottom). PVD::GFP (A) with FLP neuron marker, pmec-7::RFP, (B) and merged image (C) demonstrate that PVD dendritic branches (green) do not overlap with FLP (red) in the anterior (inset). Schematic showing that PVD and FLP envelop the animal with similar dendritic branching patterns (D). Scale bar is 15 um.

Figure 3. PVD branches fasciculate with motor neuron commissures and sub-lateral nerve cords

Confocal images of PVD::GFP marker (A-D,P), panneural::dsRed (E-H,P) and merged reporters (I-L,O,P) show PVD dendritic branches, motor neuron commissures (arrow head) and sub-lateral nerve cords (arrow). PVD secondary branches lie in the same plane as motor neuron commissures as shown in rotated Z-stack from PVDR [(B,F,J)(rotated 55^O on the X-axis and 45^O on the y-axis)]. Rotated Z-stack of left side (ventral up) shows circumferential 4^O branches [(C,G,K (rotated 80^O on X-axis and 90^O on Y-axis)]. PVD 3^O branches fasciculate with dorsal and ventral sublateral nerve cords (D,H,L,O)(anterior left, ventral down). Schematic transverse section (M) shows PVD (L+R) (black) and fasciculation of some 2^O branches (left) but not others (right) with motor neuron commissures (red). Lateral view of PVDR (N,O) showing 3^O branches fasciculated with sub-lateral nerve chords (arrow). PVDR fasciculates with processes in the sub-lateral nerve cords (P, arrow) but does not contact the touch neuron, PVMR (P, arrowhead). Scale bars are 10um (A-C,E-G,I-K,O) or 15 um (D,H,L,P). See supplemental movie 1.

Figure 4. PVD dendritic architecture is defined by orthogonal branches

Confocal images (left) and schematic tracings (right) of PVD in L2 larval stage (A,B), panneural (C,D) and merged panels (E,F) demonstrate that both motor neuron commissures (arrowheads) and sub-lateral nerve cords (arrow) are established before the majority of PVD dendritic branches emerge. PVD 1^O branches arise in the L2 stage (B,D) followed by sequential orthogonal branching of 2^O and 3^O branches in L3 larval stage (G,M). A mature PVD neuron with 4^O branches is largely completed by late L4 larval stage (I,J). Scale bar is 15 um.

Figure 5. Dynamic initiation of PVD secondary branches is disrupted in mec-3 mutants

Confocal images and schematic tracings of PVD::GFP (green/black) and panneural::dsRed (red/gray) (anterior left, ventral down) show that sub-lateral nerve cords (arrow) and PVD axon (arrowhead) are not altered in mec-3 mutants (B) in comparison to WT (A). Images (C) and schematics (D) from time-lapse confocal microscopy of wt L2 larval stage demonstrate dynamic PVD 2^O branches (1–3) that initiate and retract in vicinity of established 2^O branches (*) over 30 min period. Images (E) and schematics (F) of mec-3 mutants do not show PVD 2^O branch initiation during 190 min of observation. Scale bar is 5 um. See supplemental movie 2 for wt and supplemental movie 7 for mec-3.

Figure 6. PVD dendritic branches turn 90^O to establish orthogonal pattern

Time-lapse confocal images of L3 larva depict PVDL dendritic outgrowth (anterior left, ventral down). (Top panel) PVD 2^O branch makes a 90^o turn (arrow) to fasciculate with sub-lateral nerve cord where it becomes a tertiary branch (inset, 0 min). A 3^O branch with opposite polarity emerges from the point of turning (arrow) and grows toward the posterior (60 min). 4^O branches are established by a similar mechanism (240 min) in which 4^O branches at each end of the menorah-like structure (arrowheads) are generated by 90^O turns. Additional, interstitial 4^O branches emerge from the outer edge of the 3^O branch. Scale bar is 5 um. See supplemental movie 3 for example.

Figure 7. PVD 4^O branches exhibit dynamic growth

Time-lapse confocal images and schematic tracings of L4 larval stage (anterior left, ventral down) illustrating dynamic outgrowth of 4^O dendrites from established 3^O branches. Nascent 4^O branches (0 min) continue to grow throughout the L4 stage until they produce the mature menorah-like structures observed in the adult. Arrow denotes an example of a maturing 4^O branch. Asterisk (*, 0 min) indicates a nascent 4^O branch that ultimately retracts (60 min). Scale bar indicates 25 um. See supplemental movie 5.

Figure 8. PVD tertiary branches demonstrate contact-dependent self-avoidance

Time-lapse confocal images of L3 larval stage (anterior left, ventral down) PVD 3^O branches growing toward each other (0–27.5 min, arrow indicates gap between branches), achieving contact (30 min, arrowhead) and then retracting (32.5–35 min) to leave intervening space (arrow). This spacing is preserved in the adult PVD dendritic network. Scale bar is 5 um. See supplemental movie 6.

Figure 9. Expression profile reveals transcripts for PVD/OLL-enriched gene families

(A) Anti-FLAG immunostaining of L4 larval stage animal shows specific ser-2 prom3B::FLAG::PAB-1 expression in PVD (L and R) (box, inset) and OLL (L and R). Scale bar is 25 um. (B) Genes (with Wormbase annotation) encoding transcripts with elevated expression (1.5x) in the PVD/OLL microarray data set organized according to protein families or functional groups. Numbers denote genes in each group. (Table Inset) Enrichment of axon guidance proteins, including multiple UNC-6/Netrin pathway transcripts, enriched in the PVD/OLL microarray.

Figure 10. Transcription factors enriched in PVD expression profile control dendritic morphogenesis

Confocal images (left) and schematics (right) of RNAi-treated animals expressing PVD::GFP marker (anterior left, ventral down). (A,B) Empty vector (EV)-treated negative control. Positive control, mec-3 RNAi (E-F), results in reduced 2^O and 3^O branches. lin-39 RNAi-treated animals (C-D) do not show PVD neurons (open circle indicates location of wt PVD cell body, arrow points to tail neuron that also expresses PVD::GFP marker). Mutants egl-46(gk692) shows fewer 2^O branches (G,H) and ahr-1(ju145) displays increased numbers of 2^O branches (I,J) (Supplemental File 4). Proposed temporal order of transcription factor function during PVD morphogenesis (M). (Table 2)