Figure 6. OR42a mediated blood progenitor maintenance and its physiological relevance.

All lymph gland and brain images shown are obtained from wandering 3^rd instar larvae. TOPRO3 (blue) marks nuclei. Scale bar: white, 50 μm; yellow, 10 μm. n= # of lymph glands used for statistical analyses. Error bars in the graphs represent standard. deviation. See also Figure S6.

(A–H) Or42a function is essential for progenitor maintenance via regulating GABA.

(A) Control: Or42a-Gal4 (Pxn, green; n=10).

(B) Or42a-Gal4, UAS-Shi^ts (n=10; p=4E-08)

(C) Or42a^f04305/Df[Or42a] (n=9; p=1.4E-06)

(D) Quantitation of the percentage of Pxn positive cells in A–C,.

(E) Control: Or42a-Gal4 (GABA shown in red).

(F) Or42a-Gal4; UAS-Shi^ts (loss of GABA)

(G) Or42a^f04305/Df[Or42a] (loss of GABA).

(H) Quantitation of hemolymph GABA levels. n= # of Elisa assays; 10 animals per assay.

Or42a^f04305/CyO (control; 22 ± 1 ng of GABA/larva; n= 2)

Or42a^f04305/Df[Or42a] (11.3 ± 2 ng of GABA/larva; p= 1E-02, n= 3).

(I–L) Odor sensing is essential for progenitor maintenance in wild-type flies.

(I) Wild-type larva grown in odorless food shows dramatic loss of GABA (red) in the usually Kurs6⁺GABA⁺ neurons (demarcated with white dotted line).

I′ shows these cells in the red channel to highlight loss of GABA (red) staining.

(J) Wild-type larva grown on odorless medium shows severe reduction of GABA (red) staining in the lymph gland.

(K) Growth on odorless medium also induces extensive progenitor differentiation (Pxn, green).

(L) GH146-Gal4; UAS-NaChBac (activated PN) raised on odorless media restores GABA (red) in the MZ progenitors.

(M–R) Odor supplementation rescues lymph gland progenitor differentiation and GABA phenotypes.

(M) Schematic representation of odor deprivation and rescue experiment. The three vials depict the different food conditions: brown represents wild type larvae reared on normal cornmeal/ molasses agar food; blue represents larvae reared in odorless food. The third vial contains a dialysis-tubing pouch sealed in with normal food inserted into the minimal odor medium (referred to below as the “odor rescue condition”).

(N) Kurs6⁺GABA⁺ cells are identified (yellow) in larvae grown under “odor rescue condition”. N′ shows these cells in the red channel to highlight GABA expression. Compare with I′ which shows loss of GABA (red) expression on odorless medium.

(O) Lymph gland GABA staining is restored under “odor rescue” growth. Compare with J.

(P) Rescue of lymph gland progenitor differentiation (Pxn, green). Compare with K.

(Q) Quantitation of hemolymph GABA levels. 5 Elisa assays each genotype, 10 animals per assay.

Normal food (control; 5± 1.9 ng GABA/larva); Odorless food: (1.9 ± 1 ng GABA/larva; p=3E-03); Odor rescue condition: (4.2 ± 2.8 ng GABA/larva; p=0.5 i.e., not different from control).

(R) Quantitation of Pxn positive cells. n= # of lymph glands.

wild type (in A; n=10)

Wild type grown on odorless food (K; p=8.9E-08; n=10)

Wild-type grown in odorless food with “odor rescue” (P; p= 0.18 i.e., not significantly different compared with (A) and p=2.9E-06 compared with (K))

(S) Schematic representation of olfaction mediated control of blood progenitor maintenance via regulation of systemic GABA levels (see Discussion section for details).