Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit

Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, we found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. Our findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit.


Supplementary Note 1. The soma position of larval LNs.
Larval LNs form two clusters in the larval AL, one in the lateral region and the other in the ventral region (Fig. 2a, Supplementary Fig. 1a). Our data show that all 189Y-positive larval LNs (in the ventral cluster) and NP3056-positive larval LNs (in both lateral and ventral clusters) reintegrate into the adult circuit. Furthermore, the reintegrated cells all have somas that are located in the lateral LN cluster of adult brains (Fig. 4c,  Previous studies have used clonal analyses to demonstrate that LNs are born from embryonic to late pupal stages [1][2][3] . Therefore the previous studies provided only the birth time of distinct types of LNs and no information about how and when those LNs develop and integrate to the circuit. In this work, we found a certain time delay between the birth time of a given LN and the emergence and integration of the LN to the circuit. In addition, not counting those LNs born at the embryonic stage, at least 48 lateral LNs are born during the larval stage and remain in the adult brains 2 . However we found that approximately 22 larval LNs survive metamorphosis and remain in the AL of 24 h APF pupae. In other words, it is likely that more than 26 adult-specific LNs (after deducting 22 LNs from 48 LNs born at the larval stage plus embryonic born LNs) in the lateral cluster emerge and integrate to the circuit after 24 h APF.
The first and second types of 189Y-positive larval LNs were reported previously 4 . Four types of NP3056-larval LNs in late 3rd instar larval brains were also identified in this work . They either exhibit processes restricted to and covering the entire AL ( Fig. 4d 1 , dashed circle, n = 21/86 SCCs), covering a part of the AL and innervating the SEZ (Fig. 4d 2 , contoured by white dashed line, n = 24/86 SCCs), covering the entire AL and innervating the SEZ (Fig. 4d 3 , n = 25/86 SCCs), or with most processes in the AL and a few processes to dorsal brain regions (arrows) and the SEZ (Fig. 4d   . Middle panels show a presumably regional LN, and right panels show a presumably oligo-glomeruli LN in the right AL, with 1-2 bilateral LNs (magenta arrowheads) in the ipsi-and/or contralateral AL. ALs are indicated by yellow dashed contours. Yellow arrowheads indicate LN soma. Red arrowheads indicate the processes of presumably regional LNs or oligo-glomerular LNs. Red arrow indicates glomerulus V that is likely innervated by the regional LN. Supplementary Fig. 11 Schematic diagram of a brain 3D reconstruction by Imaris.
The surface of individual glomeruli, AL and MB medial lobe were manually outlined (grey) based on neuropil marker (red). The origin for the axes was set at the tip of MB peduncle (orange ball). The Y-Z plane was set such that the two brain hemispheres were evenly divided, with Y-axis parallel to the MB dorsal lobe. Accordingly, Z-axis was perpendicular to the MB dorsal lobe. The X-axis was set perpendicular to the Y-Z plane. Scale bar, 30 μm. Fig. 12 The relative distance between any two glomeruli in the ALs of flies with genetically ablated larval LNs. a The relative distance between any two glomeruli in GAL4 and UAS-DTI controls and LN>DTI ALs was estimated as in Fig. 6h.

Supplementary
Results for NP3056-GAL4 and NP3056>DTI were reproduced from Fig. 6h. b The differences between a given pair of glomeruli in GAL4 control and LN>DTI ALs were shown. Results of NP3056 and 670 were reproduced from Fig. 6h, i. c The differences between a given pair of glomeruli in UAS-DTI control and LN>DTI ALs were shown. d The relative distance between any two glomeruli in NP3056-GAL4 controls and NP3056>DTI ALs was estimated and realigned by the glomerulus index used in the yposition shift analysis (Fig. 6j). The result demonstrates no clear correlation between changes of glomerulus-glomerulus distance and y-position shifts. The information of glomerular index is provided in Supplementary Note 4.

Supplementary Table 1 Number of LNs labeled by individual GAL4 lines and their neurotransmitters.
* N.D., not determined.