Figure 5. GAL4-dHNF4 can be activated by starvation and exogenous long chain fatty acids.

(A) Fed larvae carrying both the GAL4-dHNF4 and UAS-nlacZ transgenes were heat-treated, allowed to recover for ~6 hours, and dissected organs were stained with X-gal for β-galactosidase activity. Low levels of ligand sensor activity are seen in the fat body, midgut, and epidermis of late second instar (late L2) or early third instar larvae (early L3), while high levels of activity are detected in mid-third instar larvae (mid L3). GAL4-dHNF4 activity in the mid-third instar midgut is restricted to the anterior region, adjacent to the proventriculus (arrow). No ligand sensor activity is detected in the oenocytes, although negative results with the ligand sensor system are difficult to interpret (Palanker et al., 2006). (B) Late second instar GAL4-dHNF4; UAS-nlacZ larvae were fasted or fed for 3 hrs, heat-treated, and fasted or fed for another 3– 4 hrs, after which dissected organs were stained with X-gal for β-galactosidase activity. Starvation leads to strong activation of GAL4-dHNF4 in the fat body and epidermis of early third instar larvae compared to fed controls (A, early L3). (C) Late second instar GAL4-dHNF4; UAS-nlacZ larvae were heat-treated to induce GAL4-dHNF4 expression, allowed to recover, and then bisected, inverted, and cultured overnight in either control medium or medium supplemented with fatty acids as shown. Palmitic acid (C16:0), oleic acid (C18:1), and the very long chain fatty acid, lignoceric acid (C24:0), activate the ligand sensor in epidermis. This activation, however, is not seen in animals that express either the L279Q or R285G GAL4-dHNF4 mutant ligand sensors. Activation is also seen in the trachea, as shown in the C16:0-treated control tissue.