Distinct IFT mechanisms contribute to the generation of ciliary structural diversity in C. elegans

Abstract

Individual cell types can elaborate morphologically diverse cilia. Cilia are assembled via intraflagellar transport (IFT) of ciliary precursors; however, the mechanisms that generate ciliary diversity are unknown. Here, we examine IFT in the structurally distinct cilia of the ASH/ASI and the AWB chemosensory neurons in Caenorhabditis elegans, enabling us to compare IFT in specific cilia types. We show that unlike in the ASH/ASI cilia, the OSM-3 kinesin moves independently of the kinesin-II motor in the AWB cilia. Although OSM-3 is essential to extend the distal segments of the ASH/ASI cilia, it is not required to build the AWB distal segments. Mutations in the fkh-2 forkhead domain gene result in AWB-specific defects in ciliary morphology, and FKH-2 regulates kinesin-II subunit gene expression specifically in AWB. Our results suggest that cell-specific regulation of IFT contributes to the generation of ciliary diversity, and provide insights into the networks coupling the acquisition of ciliary specializations with other aspects of cell fate.

Figure 1

Effect of mutations in IFT genes and IFT motors on AWB ciliary morphology. (A) Diagrammatic representation of channel and wing cilia morphologies (left) and their location in the C. elegans head (right). (Adapted from Ward et al, 1975). (B–G) AWB cilia visualized via expression of a str-1p∷gfp transgene in wild-type (panel B), osm-5(p813) (panel C), che-3(e1124) (panel D), osm-3(p802) (panel E), kap-1(ok676) (panel F) and kap-1; osm-3 (panel G) mutant animals. Zones corresponding to ultrastructurally defined segments (Figure 2) are indicated in panel B; M, middle segment; D, distal segment; FD, far-distal segment. Arrowheads indicate accumulations at the cilia tips or base (panels C, D). Average lengths of the long and short ciliary branches were 8.0±1.3 and 6.2±1.3 μm (wild-type; n=43), and 7.8±1.3 and 6.2±1.2 μm (osm-3; n=50), respectively. (H) Expression of a functional osm-3∷gfp fusion gene in an AWB neuron of an osm-3 animal. AWB neurons were identified by filling with DiI. Images in panels B and E–G were acquired using a confocal microscope. Scale bars are as follows: 5 μm (panel A); 4 μm (panels B, E–G); 10 μm (panels C, D); 15 μm (panel H). Anterior is at left in all images.

Figure 2

Ultrastructure of the AWB cilia in wild-type (A), kap-1(ok676); klp-11(tm324) (B) and osm-3(p802) (C) mutant animals. The cilium is outlined by a dashed line. Images were acquired at 60,000-fold magnification. Lengths of the middle, distal and far-distal segments were approximately 2, 5 and 1 μm, respectively. Arrowheads indicate singlet microtubules, closed and open arrows indicate doublet microtubules in the AWB, and channel cilia, respectively. Note the absence of an axoneme in the far-distal segment. Scale bars, 0.2 μm.

Figure 3

Movement of GFP-tagged KAP-1 in the ASH/ASI (A, C) and AWB (B, D) cilia. For each panel, still images (left) and kymographs (right) from representative movies of KAP-1∷GFP transport are shown. Histograms of KAP-1∷GFP velocities are shown below in each panel. For each still image, the genetic background is indicated in the top left corner, the imaged cilia in the top right corner and the fusion protein examined in the bottom left corner. Dashed lines on the still images indicate the continuous lines used to generate the kymographs. The osm-3(p802) allele was used. Scale bars in still images are 5 μm. Scale bars in kymographs are as follows: horizontal bar, 5 μm; vertical bar, 5 s. M, middle segment; D, distal segment. No ASH/ASI distal segments are formed in osm-3 mutants. Statistical analyses are shown in Supplementary Tables 2 and 3.

Figure 4

Movement of GFP-tagged OSM-3 in the ASH/ASI (A, C) and AWB (B, D) cilia. For each panel, still images (left) and kymographs (right) from representative movies of OSM-3∷GFP transport are shown. Histograms of OSM-3∷GFP velocities are shown below in each panel. Velocities in the middle (M) and distal segments (D) are indicated by black and gray bars, respectively, in each histogram. Panels are labeled as in Figure 3. The kap-1(ok676) allele was used. Scale bars in still images are 5 μm. Scale bars in kymographs are as follows: horizontal bar, 5 μm; vertical bar, 5 s. Statistical analyses are shown in Supplementary Tables 2 and 3.

Figure 5

Movement of GFP-tagged OSM-6 in the ASH/ASI (A, C, E) and AWB (B, D, F) cilia. For each panel, still images (left) and kymographs (right) from representative movies of OSM-6∷GFP transport are shown. Histograms of OSM-6∷GFP velocities are shown below in each panel. Panels are labeled as in Figure 3. Velocities in the middle (M) and distal (D) segments are indicated by black and gray bars, respectively, in each histogram. Scale bars in still images are 5 μm. Scale bars in kymographs are as follows: horizontal bar, 5 μm; vertical bar, 5 s. M, middle segment; D, distal segment. No ASH/ASI distal segments are formed in osm-3 mutants. Statistical analyses are shown in Supplementary Tables 2 and 3.

Figure 6

Mutations in fkh-2 result in dendritic extension and ciliary defects in the AWB neurons. (A) A rescuing fkh-2∷gfp fusion gene is expressed postembryonically in an AWB neuron (arrow) of a wild-type animal. AWB was identified by filling with the lipophilic dye DiD. The genomic organization of fkh-2 with the location of inserted gfp-encoding sequences is shown at top. Arrows indicate the two predicted transcription initiation sites. Sequences encoding the forkhead domain are shaded. A dashed line indicates the extent of the ok683 deletion. (B) Shown is the cell body and dendrite of an AWB neuron of a wild-type (upper panel) and fkh-2 mutant (lower panel) adult animal expressing str-1p∷gfp. Arrowheads indicate the dendrite ends. (C) Confocal images of AWB cilia from fkh-2 mutant animals. Observed ciliary defects include truncated cilia (upper panel) or loss of a ciliary branch (lower panel). (D) Localization of a GFP-tagged ODR-10 protein to the AWB neuron cilia in wild-type (upper panel) or fkh-2 mutants (lower panel). AWB neurons are visualized via expression of an srd-23p∷dsRed fusion gene. (E) AWB neurons (arrow) fill with DiI in a wild-type pattern (upper panel), but fail to fill in a fraction of fkh-2 mutants (lower panel). Scale bars are as follows: 15 μm (panels A, B, D, E); 4 μm (panel C). Anterior is at left in all images.

Figure 7

FKH-2 regulates kap-1 expression in the AWB neurons. (A) Still image (left panel) and kymograph (right panel) from a representative movie of OSM-6∷GFP in an AWB cilia of a fkh-2(ok683) animal. Histograms of OSM-6∷GFP velocities are shown below. Dashed lines on the still images indicate the continuous lines used to generate the kymographs. Velocities in the middle (M) and distal (D) segments are indicated in black and gray bars, respectively. Scale bar in the still image is 5 μm. Scale bars in kymographs are as follows: horizontal bar, 5 μm; vertical bar, 5 s. (B) Shown are the percentages of the indicated chemosensory neuron types expressing kap-1p∷dsRed in wild-type or fkh-2(ok683) animals. Numbers shown are from two independent transgenic lines expressing kap-1p∷dsRed from extrachromosomal arrays. Asterisk indicates values that are different from WT at P<0.001 using a chi-square test. (C) A fkh-2∷gfp (IeIs916) fusion construct is expressed in the AWB (arrowheads) and additional neurons in wild-type animals (left panel) but not in daf-19(m86) mutants (right panel). A total of 100% of daf-19 mutants failed to express fkh-2∷gfp (n=119) compared with 0% of wild-type animals (n=31) in the L1 developmental stage. Scale bars, 15 μm. Anterior is to the left in both images.

Figure 8

Cell-specific modulation of IFT may generate ciliary diversity. (A) Models comparing IFT in the channel and AWB cilia. Number of arrows are directly proportional to measured velocity. Note that distal segments are absent in channel but not in AWB cilia in osm-3 mutants. In the AWB middle segments, OSM-3 is proposed to function partly independently of kinesin-II. We detected IFT events and motor movement in the AWB distal segments rarely in wild-type or osm-3 mutant cilia, suggesting that the majority of motor proteins and IFT particles may undergo turnaround (represented by a curved arrow) at the middle/distal segment border in AWB cilia. See text for additional details. (B) Model of FKH-2 function in fate specification of the AWB neurons. FKH-2 may link a ciliogenic module (left box) with an AWB-specific developmental module (right box) to generate AWB-specific morphology and AWB fate. Dashed lines indicate postulated regulatory relationships.