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doi: 10.1242/10.1242/dev.00413

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RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia

Department of Zoology, University of British Columbia, Vancouver V6T 1Z4, Canada

View larger version (93K): [in a new window]   Fig. 1. Profile of peripheral glial actin-GFP during wild-type embryonic development. repo::actin-GFP embryos were fixed and stained with anti-GFP, then viewed with laser scanning confocal microscopy. Anterior is towards the top, CNS is to the left of vertical broken lines. For a detailed characterization of wild-type glial migration in relation to motor and sensory neurons, see also Sepp et al. (Sepp et al., 2000). (A) Stage 12. Peripheral glia arise and proliferate at the lateral edge of the CNS (arrows). At this early stage, they are compact, and the glial cluster forms a cone shaped array in each hemisegment. (B) Stage 13. The peripheral glia begin to migrate into the periphery. The leading glial cells extend small actin-filled projections (concave arrow). (C) Stage 14. As the leading glia move further peripherally, their cell bodies (nuclei are oval shaped and lacking actin-GFP staining) can travel just behind the leading cytoplasmic edge (solid arrow). Alternatively, the cell body can be found on the trailing region of the cell, while a long process with lamellar-like structures (arrowhead) can be found at the leading edge. Filopodia-like protrusions extend from the leading glial cells in each hemisegment (concave arrows). (D) Stage 15. The phase of glial migration is almost complete. The ventral peripheral glia (vPG, solid arrow), which initially migrates in the cone-shaped glial cluster, separates its processes from the other glia to ensheathe the ventral cluster sensory neurons. The lateral chordotonal glia (concave arrow), lateral bipolar dendritic glia (asterisk) and the lateral line glia (arrowhead) arise from the periphery and are also labeled with actin-GFP. (E) Stage 16. In the mature embryo, the overall actin-GFP profile is smoother, with no visible spike-like protrusions as in the earlier migratory phase. The vPG cell has fully resolved from the main (anterior fascicle) nerve tract (arrow). The lateral chordotonal glia (concave arrow), which associate with the cell bodies of the lateral chordotonal neurons, are interconnected with the peripheral glia.

View larger version (119K): [in a new window]   Fig. 3. Profile of actin-GFP and tau-lacZ in third instar larval peripheral glia. (A) Fluorescence of actin-GFP in peripheral glial processes. The actin-GFP distribution is mesh-like with dense concentrations interspersed (arrow). The image was collected from a live third instar larva using a disk scanning confocal microscope. (B) Tau-lacZ (green) and anti-HRP staining (red) to show peripheral glial microtubule network and peripheral neurons, respectively. The glial microtubule network consists of long, rope-like structures (arrow) extending along the length of the cell. The image was collected on a laser-scanning confocal microscope.

View larger version (80K): [in a new window]   Fig. 2. Comparison of Actin-GFP and Tau-lacZ profiles in mature embryonic peripheral glia. Embryos expressing UAS-actin-GFP and UAS-tau-lacZ using the repo:GAL4 driver were fixed and stained with anti-GFP and anti-ß-galactosidase, then viewed with laser scanning confocal microscopy. Anterior is towards the top, CNS is towards the left. Embryos are stage 16. (A) Actin-GFP channel (green). (B) Tau-lacZ channel (red). (C) Merge of A and B shows that within the cell, actin-GFP and tau-lacZ profiles show little direct overlap. However, at the leading edge of the growing vPG cell, both actin-GFP and tau-lacZ labeling is observed (arrows).

View larger version (148K): [in a new window]   Fig. 4. Glial expression of transgenic Rho1 disrupts migration and nerve ensheathement. repo:GAL4 embryos carrying the UAS-actin-GFP marker were used as the wild type (A-C). The repo::actin-GFP flies were also crossed to lines carrying UAS-RhoAV14 (D-F), UAS-RhoAwt (G-I) and UAS-RhoAN19 (J-L). Embryos were stained with anti-GFP (green) and mAb 22C10 (red). (A,D,G,J) Green channels. (B,E,H,K) Red channels. (C,F,I,L) Merge. Anterior is to the top, CNS is to the left. Embryos are stage 16. (A) Wild-type peripheral glial actin-GFP staining includes vPG cell (concave arrow), lateral line glia (arrowheads) and lateral chordotonal glia (asterisk). (D) RhoAV14 expression in glia results in glial stalling at the CNS/PNS transition zone (compare with A). Large tracts of PNS nerves are not ensheathed by the glia (arrow). The lateral line glia fail to extend processes to interconnect across hemisegments (arrowheads). Long spike-like projections emanate from the glia (concave arrows). The lateral chordotonal glia are small and rounded (compare asterisk in D with those in A and G); however, the underlying sensory neurons are relatively normal (asterisk in E). (E) The sensory axons are defasciculated (arrow) as a result of glial RhoAV14 expression. (F) The spike-like projections of peripheral glia due to RhoAV14 expression do not always correspond with axonal tracts (concave arrow). (G) Ectopic wild type RhoA expression in glia disrupts nerve wrapping profile (compare with A) and results in breaks in the glial sheath (arrows). (J) Expression of RhoAN19 disrupts glial wrapping profile and prevents vPG cell (concave arrow) from separating from main nerve branch. (H,K) Sensory axon defasciculation (arrows) in RhoAwt and RhoAN19 embryos indicates that glia do not effectively wrap the axon tracts.

View larger version (170K): [in a new window]   Fig. 5. Loss-of-function mutations in RhoA and Rac disrupt peripheral glial development. Wild type and mutant embryos were labeled with anti-Neuroglian (green) and anti-HRP (red) to label peripheral glia and neurons, respectively. The anti-Neuroglian marker also labels the epidermis (asterisks). CNS is towards the left, anterior is towards the top. (A) A wild-type embryo at stage 16. The arrow shows peripheral glial sheaths extending along the peripheral nerve in the lateral region of the hemisegment. (B) A RhoAk02107a homozygous mutant embryo shows a lack of peripheral glial sheaths in the lateral region of the embryo (arrows) and loss of glia at the CNS/PNS transition zone (arrowhead, compare with A). (C) A Rac1J11 homozygous mutant shows lack of peripheral glial coverage of lateral axon tracts (arrow) and abnormal glial profiles in the ventral region (arrowhead). (D) The neuronal profile of the Rac1J11 mutant shown in C. The motor projections in the upper hemisegment are stalled (arrow), as are the lateral chordotonal sensory neurons (arrowhead). (E) A Rac2 homozygous mutant embryo shows lack of glial sheaths in the lateral region of the axonal tracts (arrow). (F) An Mtl mutant shows largely normal peripheral glial coverage of PNS axon tracts, with glial cells wrapping the lateral regions of the main ISN branch at stage 16 (arrow).

View larger version (146K): [in a new window]   Fig. 6. Glial expression of transgenic Rac1 disrupts migration and nerve ensheathement. repo:GAL4 embryos carrying the UAS-actin-GFP marker were used as the wild type (A-C). The repo::actin-GFP flies were also crossed to lines carrying UAS-Rac1V12 (D-F), UAS-Rac1wt (G-I), UAS-Rac1L89 (J-L) and UAS-Rac1N17 (M-O). Embryos were stained with anti-GFP (green) and mAb 22C10 (red). (A,D,G,J,M) Green channels. (B,E,H,K,N) Red channels. (C,F,I,L,O) Merge. Anterior is towards the top, CNS is towards the left. Embryos are stage 16. (D) Rac1V12 embryos have collapsed glial sheaths (concave arrows) as well as migration defects (solid arrow). The lateral line glia fail to connect across hemisegments (compare arrowhead with those in A). (E) The underlying sensory axon tracts are defasciculated in Rac1V12 embryos (arrows). (G) Overexpression of wild type Rac1 causes abnormal glial wrapping which is often overgrown in appearance compared with wild type (concave arrow). The lateral line glia do not consistently connect across hemisegments (arrowhead). (J) The Rac1L89 embryos show ectopic lamellar-like projections (concave arrows) as well as failed inter-connection of lateral line glia (arrowhead). (K) The underlying sensory axonal tracts are defasciculate (solid arrow). Misplacements of sensory neurons in Rac1L89 mutants (concave arrows) appear to be associated with glia (compare concave arrows in upper hemisegment in J,K). (M) Rac1N17 embryos have mildly disrupted glial wrapping profiles with occasional small gaps in the nerve sheath (arrow). (N) Sensory neurons in Rac1N17 embryos show defasciculation (arrow).

View larger version (84K): [in a new window]   Fig. 7. Ectopic lamellar-like structures on peripheral glia in Rac1L89 embryos do not always cover motor or sensory neuron projections. repo:GAL4 embryos carrying the UAS-actin-GFP marker and UAS-Rac1L89 transgenic construct are were double labeled for glial actin-GFP (green) and for motoneurons (A; red, mAb 1D4) and sensory neurons (B,C; red, mAb 22C10). Anterior is towards the top, CNS is towards the left. Stage 16 embryos are shown. (A) Lamellar-like structures of peripheral glia (arrows) do not overlap with motoneuron staining and are not found where sensory neurons normally occur. (B) Low magnification of Rac1L89 embryo with ectopic lamellar-like projections. The ectopic glial projections do not match sensory neuron patterning. (C) Higher magnification of box in B shows lamellar-like structures with fine spike-like actin-GFP formations further projecting out of the lamellae. The left lamellar-like structure appears to have a glial nucleus at its center. Peripheral glial nuclei are oval shaped and do not stain with Actin-GFP.

View larger version (108K): [in a new window]   Fig. 8. Sensory and motor axon pathfinding is relatively normal in Rac1V12 embryos. repo:GAL4 embryos carrying the UAS-actin-GFP marker are the wild type (A,B). repo::actin-GFP flies were crossed to a UAS-Rac1V12 transgenic construct line (C,D). Embryonic progeny were double labeled for glial Actin-GFP (green), sensory neurons (A,C; red, mAb 22C10) and motoneurons (B,D; red, mAb 1D4). Anterior is towards the top, CNS is towards the left. Stage 16 embryos are shown. (A) In the wild type, sensory axons (red) project into the CNS along two main fascicles (arrows). (C) In Rac1V12 embryos, the glial processes are collapsed. Sensory axons still pathfind such that they form two main fascicles as in the wild type (arrows); however, they are defasciculated (right arrow). (B) The SNa motor axon branch of a wild-type embryo is indicated (arrow). (D) In Rac1V12 embryos, pathfinding of the SNa motoneuron is essentially normal (arrow), as are other motor branches (not indicated).