The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

The ability to detect variations in humidity is critical for many animals. Birds, reptiles and insects all show preferences for specific humidities that influence their mating, reproduction and geographic distribution. Because of their large surface area to volume ratio, insects are particularly sensitive to humidity, and its detection can influence their survival. Two types of hygroreceptors exist in insects: one responds to an increase (moist receptor) and the other to a reduction (dry receptor) in humidity. Although previous data indicated that mechanosensation might contribute to hygrosensation, the cellular basis of hygrosensation and the genes involved in detecting humidity remain unknown. To understand better the molecular bases of humidity sensing, we investigated several genes encoding channels associated with mechanosensation, thermosensing or water transport. Here we identify two Drosophila melanogaster transient receptor potential channels needed for sensing humidity: CG31284, named by us water witch (wtrw), which is required to detect moist air, and nanchung (nan), which is involved in detecting dry air. Neurons associated with specialized sensory hairs in the third segment of the antenna express these channels, and neurons expressing wtrw and nan project to central nervous system regions associated with mechanosensation. Construction of the hygrosensing system with opposing receptors may allow an organism to very sensitively detect changes in environmental humidity.

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Here we present evidence that the SH3-SH2 adaptor protein Dreadlocks (Dock), the p21-activated serine-threonine kinase (Pak), and the Rac1/Rac2/Mtl small GTPases can function during Robo repulsion. Loss-of-function and genetic interaction experiments suggest that limiting the function of Dock, Pak, or Rac partially disrupts Robo repulsion. In addition, Dock can directly bind to Robo's cytoplasmic domain, and the association of Dock and Robo is enhanced by stimulation with Slit. Furthermore, Slit stimulation can recruit a complex of Dock and Pak to the Robo receptor and trigger an increase in Rac1 activity. These results provide a direct physical link between the Robo receptor and an important cytoskeletal regulatory protein complex and suggest that Rac can function in both attractive and repulsive axon guidance.

Experimental animals and transgenesAll fly lines were obtained from the Drosophila stock center except for the following. 981- Gal4 and SG18.1-Gal4 (Jhaveri et al., 2000) were kindly provided by V. Rodrigues. Or22a-Gal4, Or47a-Gal4 and Or47b-Gal4 (Vosshall et al., 2000) were gifts from L. Vosshall. MARCM (Lee and Luo, 1999) was carried out by heat-shocking thirdinstar larvae of the following genotypes: hs-FLP/+; dock Or47a-Gal4/dock (or +); FRT82 Gal80/FRT82 UAS-mCD8::GFP and hs-FLP/+; dock Or47b-Gal4/dock (or +); FRT82 Gal80/FRT82 UASmCD8::GFP at 37 o C for 40 minutes. Adult brains were dissected and processed as described below. GH146-Gal4 Clonal analysis ImmunohistochemistryAdult brains (from 1-to 2-day-old animals), pupal antennae and larval imaginal discs were dissected in phosphate buffered saline (PBS). Tissues were fixed in PLP (2% paraformaldehyde, 0.25% sodium periodate, 75 mM lysine-HCl and 37 mM sodium phosphate pH 7.4), washed with PBST (PBS with 0.5% Triton X-100) and subjected to antibody staining. nc82 mAb (1:20) (A. Hofbauer, PhD thesis, University of Wurzburg, 1991) was a gift from A. Hofbauer. Rabbit anti-Dock 1:500 (Clemens et al., 1996) was a gift from J. Dixon. Mouse 22C10 mAb (1:20) and rat anti-ELAV, 7E8A10 (1:20) were from Developmental Studies Hybridoma Bank. Rabbit anti-GFP polyclonal antibody (1:100) was from Clontech, and rat anti-CD8 α subunit mAb (1:100) was from Caltag. The secondary antibodies, FITC-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse and FITC-conjugated goat anti-rat, were purchased from Jackson Laboratories and used at 1:200 dilutions. Cuticle preparationsAdult antennae from animals expressing Or-Gal4/UAS-lacZ nuclear were fixed in 25% gluteraldehyde for 1 hour, washed in PBST and stained in 0.2% X-Gal solution (Ashburner, 1989). The antennae were then cleared in Faure's mountant (34% v/v chloral hydrate, 13% glycerol, 20 mg/ml gum Arabic) and photographed with the SPOT-RT cooled CCD camera. Sensilla on the third antennal segment were counted by projecting images on a video monitor.