Carbon dioxide (CO_2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO_2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO_2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO_2 receptors and form connections characteristic of CO_2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO_2

Cayirlioglu, Pelin

Gunning, Dorian

Kadow, Ilona Grunwald

Lai, Eric C.

Okamura, Katsutomo

Suh, Greg S. B.

Zhan, Xiaoli

Zipursky, S. Lawrence

Caltech Authors - Main

 
 
www.sciencemag.org/cgi/content/full/319/5867/1256/DC1 
 
 
 
Supporting Online Material for 
 
Hybrid Neurons in a MicroRNA Mutant Are Putative Evolutionary 
Intermediates in Insect CO2 Sensory Systems 
Pelin Cayirlioglu, Ilona Grunwald Kadow, Xiaoli Zhan, Katsutomo Okamura, Greg S. B. Suh, 
Dorian Gunning, Eric C. Lai, S. Lawrence Zipursky* 
 
*To whom correspondence should be addressed. E-mail: lzipursky@mednet.ucla.edu 
Published 29 February 2008, Science 319, 1256 (2008) 
DOI:  10.1126/science.1149483 
 
 
 
This PDF file includes: 
 
Materials and Methods 
Figs. S1 to S7 
References 
 
 1 
Supporting Online Material 
www.sciencemag.org 
Materials and Methods 
Figs. S1, S2, S3, S4, S5, S6, S7 
 
Materials and methods  
Genetics and Molecular Biology  
Fly stocks were maintained in standard medium. P-element mutants were 
obtained from Bloomington or Szeged stock centers and were recombined to FRT 
chromosomes by the UCLA undergrad consortium (1).  
The genetic screen was done using FRT/FLP mosaic analysis and MARCM 
analysis as described previously (2). To generate large antennal and maxillary palp (MP) 
clones eyeless-FLP was used in combination with a cell lethal mutation on 3R. Mutations 
(P-element) on the right arm of chromosome 3 were screened using the following 
genotype: w-; Or-syt-GFP/+; FRT82 mutation/FRT82 CL GMR-hid ey-GAL4 UAS-FLP. 
MARCM analysis was carried out on flies of the following genotype: eyFLP; Or-gal4 
UAS-sytGPF (or UAS-mCD8GFP)/+; FRT82 mutation/FRT82Gal80E2F. ORN labeling 
was achieved by fusing the promoter-elements to GAL4 or directly to synaptotagmin-
GFP (3). Promoters or fly stocks were generously provided by the labs of Leslie Vosshall 
(Rockefeller University) and Barry Dickson (IMP, Vienna). P-element plasmid rescue 
was performed as described previously (4). MPS-GAL4 driver is a promoter fusion of 
Or59c 5’ with GAL4. This was the original Or59c driver (3). However, our 
 2 
characterization of the wiring pattern as well as in situ experiments examining the 
odorant receptor expression of the ORNs that express this driver (5) suggest that this is 
an unfaithful driver that is expressed in additional ORNs in the MP. This driver was used 
in cell count and pupal developmental studies in the maxillary palps. The fibers labeled 
with this driver innervate two major glomeruli targeted by Or59c and Or42a ORNs. 
Occasionally, in some of the brains, a third glomerulus that corresponds to Or85d 
cognate glomerulus also is slightly labeled. Our developmental study suggests that it is 
expressed in single cells within a pb1 and pb3 sensilla in wild type MP that harbor Or42a 
and Or59c ORNs, respectively.  
 
As the S0962-07 mutation is organismal lethal, all analyses were done in mosaic 
animals where more than 50% of cells in the antenna and the MPs were homozygous 
mutant while the rest of the animal was wild. Gr21a transcriptional reporter was used for 
CO2 neurons (i.e. Gr21a-GAL4 driving membrane-bound GFP (UAS-mCD8GFP)), to 
detect cell bodies in the antenna and MP.  
 
miR-279 deletions were generated using P-element excision of two different P-
element insertions. We used standard methods to generate ~200 excisions each from 
EP(3)3626 and EP(3)3069, which are both inserted downstream of miR-279. This 
yielded a 1.9kb deletion from EP(3)3069 and 1.2kb and 0.8kb deletion alleles from 
EP(3)3626. Genomic sequencing revealed that the miR-279 locus is cleanly removed in 
the two larger deletion alleles. UAS-nerfin transgenic flies and nerfin-1 loss of function 
alleles were generously provided by Ward Odenwald. miR-279-GAL4 and miR-279 
genomic transgenes were generated using these primer sequences: MIR279FOR1: 5’ 
 3 
TAT TTT TGC GCC TGC CAA TAA GCG 3’ MIR279REV1: 5’ TAG ATC AGT GAC TCA 
GCT GGC AAC 3’ MIR279FOR2: 5’ GTA TTC AAC GCG TGT TTT CTG 3’ 
MIR279REV2: 5’ AAT CGG AAT CGG AAT CAG AAT CGC 3’ MIR279FOR3: 5’ TGA 
AAA TAC GCG TAT GGA AAT GCC 3’ MIR279REV3: 5’ CAG CTC CAG TCC CAA TTC 
C 3’  
 
miR-279 mutant phenotype is 100% penetrant. Genomic rescue experiments 
were carried out such that eyflp; +/[miR-279];FRT82 S0962-07/ TM6B males were 
crossed to Gr21-Gal4,UAS-mCD8GFP/CyO; FRT82Gal80E2F/TM2 virgin females. Only 
half of the progeny that was dissected was expected to have the rescue transgene. Our 
quantifications show that more than half of the dissected progeny show no phenotype 
suggesting complete rescue of the mutant phenotype. The rescue observed for the 
wiring phenotype in the brain, and the generation of ectopic Gr21a cells in the MP were, 
60% (n=25) and 67% (n=30) of all analyzed progeny, respectively.  
 
miR-279-GAL4 transcriptional reporter comprised the entire 5’ upstream 
sequence of miR-279 from the genomic rescue fragment fused to the transcriptional 
activator GAL4. 
 
Genotypes for mutant analysis:  
Control: 
eyflp;Gr21-Gal4,UAS-mCD8GFP/+;FRT82/FRT82Gal80E2F.  
 4 
eyflp;Gr21-sytGFP/+;FRT82/FRT82Gal80E2F  
eyflp;MPS-GAL4UAS-mCD8GFP/+;FRT82/FRT82Gal80E2F 
eyflp;Or-Gal4,UAS-mCD8GFP/+; FRT82/FRT82Gal80E2F  
Mutant: 
eyflp; Gr21-Gal4,UAS-mCD8GFP/+;FRT82S0962-07/FRT82Gal80E2F   
eyflp;Gr21- sytGFP/+;FRT82S0962-07/FRT82Gal80E2F   
eyflp;MPS-GAL4UAS-mCD8GFP/+;FRT82S0962-07/FRT82Gal80E2F  
eyflp; Or-Gal4,UAS-mCD8GFP/+;FRT82S0962-07/FRT82Gal80E2F   
eyflp; Gr21-Gal4,UAS-sytGFP/+; FRT82 miR-279 Δ0.8/FRT82Gal80E2F  
miR-279 rescue: 
eyflp; Gr21-Gal4,UAS-sytGFP/[miR-279];FRT82 S0962-07/FRT82Gal80E2F 
Nerfin-1 genetic interactions: 
eyFLP;Gr21a-GAL4,UAS-mCD8GFP/+;Df(2)nerfin-1154FRT82S0962-
07/FRT82GAL80E2F (nerfin154 miR-279P).  
eyFLP;Gr21a-GAL4,UAS-mCD8GFP/+;FRT82S0962-07/FRT82GAL80E2F (miR-279P). 
Electrophysiology: 
Mutant: 
eyflp; Gr21-Gal4,UAS-mCD8GFP/+;FRT82S0962-07/FRT82Gal80E2F   
 5 
Control: 
Canton S. 
Electrophysiology 
 
Flies were mounted on a glass slide and viewed with an Olympus BX51WI 
microscope at 1000X magnification. Electrical signals were recorded with a Multiclamp 
700B amplifier and Digi-data 1322A at the sampling rate of 10kHz, stored in a PC with 
pClamp 9.2 software, and plotted with Clampfit 9.2 and SigmaPlot 9 software as 
described(6). Recordings were performed by placement of a glass electrode onto the 
patch of GFP+ cells on the lateral side of the palp with a reference electrode inserted in 
the eye.  
 
 Ectopic neurons showed a weak but significant response to CO2. By contrast, 
wild type MPs showed no respone (see Fig.1). These data suggest that there are 
differences between these neurons and wild type CO2 neurons in the antenna. These 
differences in the hybrid neurons could include lower levels of receptor expression, 
inhibitory effects of MP olfactory receptors that are co-expressed, or the lack of certain 
co-factors downstream or upstream Gr21a/Gr63a.  
 
Immunohistology  
Staining was performed as previously described (3). Primary antibodies were 
used in the following dilutions: Guinea pig anti-Nerfin-1 antibody (provided by the 
Odenwald lab), 1:1000; mouse anti-NC82 (DSHB), 1:20; mouse anti-prospero (DSHB), 
 6 
1:20; mouse and rat anti-elav (DSHB), 1:100; and rabbit and mouse anti-GFP, 1:2000 
and 1:500, respectively. All secondary antibodies were used at 1:200.  
 
Whole mount in situ hybridization  
Adult probiscises were fixed in 4% Paraformaldehyde, 0.05% Tween-20 for 30 
min at room temperature. Prehybridization and hybridization were done at 55˚C in 50% 
Formamide, 5xSSC (pH 4.5), 0.1mg/ml yeast tRNA, 0.05 mg/ml Heparin, 0.1% Tween20 
for 1 hr and overnight, respectively. Probes were generated using digoxigenin-RNA 
labeling kit. Probe templates were kindly provided by Leslie Vosshall. After hybridization 
proboscises were washed 5 times in hybridization buffer at 55˚C, followed by 3 times 5 
min washes in 0.1M Tris pH7.5, 0.15M NaCl, and 0.05% Tween-20. Samples were 
blocked in 0.1M Tris pH7.5, 0.15M NaCl, 0.05% Tween-20, and 0.5% blocking reagent 
TSA Biotin kit and incubated overnight in anti-dig Peroxidase (1:200) and anti-GFP-
alexa488 antibodies (1:200). To amplify the RNA probe signal, samples were incubated 
in biotinyl tyramide (1:100) for 2 hrs at room temperature, followed by incubation in 
streptavidin-cy3. After subsequent washes in 0.1M Tris pH7.5, 0.15M NaCl, and 0.05% 
Tween-20, samples were mounted with Vectastain and analyzed using a confocal 
microscope.  
 
PicTar sequence comparison  
All the computational analysis and sequence comparison was done using the 
PicTar program that can be accessed via: http://genome.ucsc.edu/cgi-
bin/hgTrackUi?db=hg17&g=picTar or http://pictar.bio.nyu.edu/. 
 7 
 
Luciferase assay  
To generate luciferase targets, we amplified a 1.8kb nerfin fragment (including 
the entire 3' UTR and 220bp of downstream sequence), and a 550bp nerfin 3'UTR 
fragment starting at position 680 of the 3' UTR ("nerfin 680", including three miR-279 
sites) and cloned these downstream of the renilla luciferase coding region in 
psiCHECK2; this vector contains an internal firefly luciferase gene that serves as an 
internal control. The inclusion of downstream genomic sequences in the full-length 
constructs permits the evaluation of miRNA-mediated regulation of endogenously-
terminated transcripts. For the miR-279 expression construct, we cloned 415 bp of 
genomic sequence, centered on the miR-279 hairpin, into the 3' UTR of UAS-DsRed (7). 
Different 3’UTRs were fused to a luciferase reporter construct. psiCheck, a control 
3’UTR; nerfin, the entire nerfin-1 3’UTR containing four 8mer (blue boxes) and two 7mer 
(light blue boxes) miR-279 binding sites (8); nerfin680, a shorter piece containing the 
major cluster of three 8mer miR-279 sites. Primer sequences are available upon 
request. We then transfected 25 ng target, 12.5 ng ub-Gal4 and 25 ng UAS-DsRed-miR-
279 plasmids into 1x10[5] S2 cells in 96 well format. Three days later, we lysed the cells 
and subjected them to dual luciferase assay and analyzed these on a plate luminometer. 
Quadruplicate transfections were performed on two different batches of cells on different 
days, and the data were pooled in the figure shown.   
 
Luciferase sensor assays were performed as previously described (9). We 
performed quadruplicate transfections of 25 ng target, 12.5 ng ub-Gal4 and 25 ng UAS-
DsRed-miRNA plasmids into 1x105 S2 cells in 96 well format. For 2’Omethyl antisense 
 8 
mediated de-silencing assay, we introduced 25ng target plasmid and 5 pmol (50nM) or 
10pmol (100nM) of 2’Ome oligonucleotides for each well (miR-279ASO: 
GTTATTAATGAGTGTGGATCTAGTCACAAAT, miR-124ASO: 
GCTCTTGGCATTCACCGCGTGCCTTATGGTG). Three days after transfection, we 
lysed the cells and subjected them to dual luciferase assay and analyzed these on a 
Veritas plate luminometer. 
 
 
Fig. S1: Characterization of ectopic neurons in S0962-07 mutants. 
(A) Average number of Elav-positive cells in wild type and mutant pupal MPs. ** p < 
0.001. MPs scored: wild type, n = 7; mutant, n = 8. (B) Mutant and control MPs labeled 
with Or83b-GAL4, UAS-mCD8GFP. Single neurons or clusters of up to 2 cells were 
observed in the control, while also clusters of up to 4 cells were found in the mutant 
palps. (C) Quantification of Or83b-positive cells in mutant and control palps. 
Approximately 10 clusters containing 3-4 positive cells were observed in the mutant 
compared to controls (n = 11 (control), n = 14 (mutant), ** p < 0.0001).  The number of 1-
2 cell clusters was reduced in the mutant compared to control, while the number of total 
cell clusters was unchanged compared to control palps. This suggests that additional 
neurons are added to existing sensilla. (B,C) Genotypes: eyflp; Or83b-Gal4,UAS-
mCD8GFP/+; FRT82 / FRT82Gal80E2F (control). eyflp; Or83b-Gal4,UAS-
mCD8GFP/+;FRT82 S0962-07/ FRT82Gal80E2F (miR-279P) 
 
Fig.S2: The adult Drosophila olfactory system.  
 9 
Neurons expressing the CO2 receptors, Gr21a/Gr63a (orange) reside in the antenna (an) 
and project to the V-glomerulus in the antennal lobe (al). Or59c and Or42a expressing 
neurons (green) in the MP innervate two medial glomeruli. 
 
Fig.S3: S0962-07 gene encodes a microRNA.  
(A) A P-element (green triangle) insertion maps 1kb upstream of miR-279 in S0962-07. 
Deletion mutants, red lines; 3kb genomic region used in the rescue experiments and 
miR-279-GAL4 reporter construct, blue lines. (B, C) Gr21a targeting to the medial 
antennal lobe. (B) and ectopic Gr21a cells in the MP (C) are rescued by the miR-279 
rescue transgene. Both antennae and MP are present in these studies. Genotypes: (B) 
(from top to bottom) eyflp; Gr21-Gal4,UAS-sytGFP/+; FRT82 / FRT82Gal80E2F 
(control). eyflp; Gr21-Gal4,UAS-sytGFP/+;FRT82 S0962-07/ FRT82Gal80E2F (miR-
279P). eyflp; Gr21-Gal4,UAS-sytGFP/+; FRT82 miR-279 Δ0.8 / FRT82Gal80E2F (miR-
279Δ0.8). eyflp; Gr21-Gal4,UAS-sytGFP/[miR-279];FRT82 S0962-07/ FRT82Gal80E2F 
(miR-279 rescue). (C) eyflp; Gr21-Gal4,UAS-mCD8GFP/+;FRT82 S0962-07/ 
FRT82Gal80E2F (miR-279P). eyflp; Gr21-Gal4,UAS-mCD8GFP/[miR-279];FRT82 
S0962-07/ FRT82Gal80E2F (miR-279 rescue). 
 
Fig.S4: Elav, 22c10 and Prospero expression in miR-279 positive cells.  
miR-279 is expressed in big cells that appear to be similar to sensory organ precursors 
(SOP),  which give rise to the components of individual sensilla through rounds of cell 
divisions. However, these precursors appear different from previously described 
antennal SOPs (10). 
 10 
A) miR-279-GAL4, UAS-mCD8GFP (green) expressing MPs co-stained with anti-Elav 
antibody (red) at 40-50 hrs and 50-60 hrs APF. Lower panel is a higher magnification of 
a cell cluster at 50-60 hrs APF. Similar to the observations in the antennal precursors  
(10), Elav protein is detected in the precursors before neuronal differentiation. Arrows 
denote unequal distribution of Elav within miR-279 expressing clusters. Arrowheads 
point to ORNs that have undergone neuronal differentiation, and no longer express miR-
279. (B) miR-279-GAL4, UAS-mCD8GFP (green) and 22C10 (red) on pupal MPs (50-60 
hrs APF). 22C10 labels Futsch protein, which is expressed in postmitotic neurons. (C) 
anti-Prospero staining (red) on miR-279-GAL4, UAS-mCD8GFP (green) pupal MPs (50-
60 hrs APF). All these neuronal markers are initially expressed in the SOP. Later, they 
become unequally distributed as multiple cell clusters form through presumed cell 
divisions from the original SOP. The identity of these cells as they divide is unknown, 
due to lack of information on the development of MPs, and of developmental markers 
that label different cell types within the cluster.  
 
Fig.S5: Nerfin-1 and miR-279 expression in developing MP. 
Pie charts showing quantification of (A) miR-279-GAL4,UAS-mCD8GFP and Nerfin-1 
protein distribution in developing MP and (B) of Nerfin-1 protein expression in ectopic 
Gr21a cells in mutant MPs. (** p < 0.001). 
 
Fig.S6: Suppression of Nerfin-1 expression requires specific miR-279 target 
sequences. 
 11 
(A) miR-279 binding sites within nerfin-1 3’UTR are conserved in different Drosophilids. 
(B) Antisense oligos directed against the core recognition sequence of miR-279 or miR-
124 were co-transfected with the nerfin-1 3’UTR luciferase reporter into S2 cells. miR-
279 specific antisense oligos relieved the repression on Nerfin-1 expression monitored 
with the 3’UTR nerfin-1 luciferase reporter. miR-124 antisense oligos had no effect on 
nerfin-1 reporter repression. This also shows that miR-279 specifically counteracts 
endogenous miR-279 that is expressed in S2 cells, and further strongly suggests that 
the predicted miR-279 core sequences in the 3’UTR are required for suppression by 
miR-279. We also note that Odenwald and colleagues recently reported complementary 
evidence for direct repression of nerfin-1 by miR-279 in the Drosophila embryo (11).  
 
 
Fig.S7: Effect of Nerfin-1 on the number of Gr21a cells.  
(A) The number of antennal Gr21a-positive ORNs is not affected by removal of one copy 
of nerfin-1. Gr21a expression is not seen in wild type palps, or wild type palp 
heterozygous for the Nerfin154 allele. Genotypes: Gr21a-Gal4,UAS-
mCD8GFP/+;TM2/TM6B (wild type), Gr21a-Gal4,UAS-mCD8GFP/+;nerfin154/TM6B 
(Nerfin154/+). (B) Overexpression of Nerfin-1 in ey-flp clones using the miR-279-GAL4 
driver construct did not result in a miR-279-like phenotype. Genotype: eyflp; UAS-nerfin-
1/miR-279-GAL4;FRT82Gr21a-sytGFP/FRT82Gal80E2F. (C) Miswiring of Gr21a 
neurons still persists in flies where the dose of Nerfin-1 in miR-279 mutant background is 
reduced to half. These fibers presumably originate from the cell bodies that are still 
present in the MP (see Figure 3G). Genotype: eyflp; Gr21a-GAL4,UAS-mCD8GFP/+; 
nerfin154FRT82 miR-279P/FRT82Gal80E2F. 
 12 
References 
1. J. Chen et al., PLoS Biol 3, e59 (2005). 
2. T. Lee, L. Luo, Trends Neurosci 24, 251 (2001). 
3. T. Hummel et al., Neuron 37, 221 (Jan 23, 2003). 
4. W. Sullivan, M. Ashburner, R. S. Hawley, Cold Spring Harbor Laboratory Press  
(2000). 
5. J. R. Carlson,  (personal communication). 
6. G. S. Suh et al., Curr Biol 17, 905 (May 15, 2007). 
7. A. Stark, J. Brennecke, R. B. Russell, S. M. Cohen, PLoS Biol 1, E60 (Dec, 
2003). 
8. B. P. Lewis, C. B. Burge, D. P. Bartel, Cell 120, 15 (Jan 14, 2005). 
9. K. Okamura, J. W. Hagen, H. Duan, D. M. Tyler, E. C. Lai, Cell 130, 89 (Jul 13, 
2007). 
10. A. Sen, G. V. Reddy, V. Rodrigues, Dev Biol 254, 79 (2003). 
11. A. Kuzin, M. Kundu, T. Brody, W. F. Odenwald, Dev Biol 310, 35 (Oct 1, 2007). 
 
 
control mutant mutant 
0
10
20
30
40
50
60
mutant control
# of cell cluster of 3-4 cells
# of cell cluster of 1-2 cells
total # of cell cluster 
# 
of
 c
el
l c
lu
st
er
 (+
/- 
S
E
M
)
**
**
Figure S1
B
C
A
0
20
40
60
80
100
120
140
P
M/sllec vale fo #
**
mutwt
ant
mp
al
Figure S2.
AmiR-279∆1.9
miR-279∆0.8
miR-279∆1.2
genomic rescue construct
miR-279-GAL4
miR-279 CG31044
1kb
P
B
control miR-279∆0.8
 miR-279P+genomic rescuemiR-279P
GAL4
miR-279 deletion 
mutants
C miR-279P  miR-279P+genomic rescue
Figure S3.
miR-279-GAL4 Elav merge
 
40
-5
0 
hr
s 
A
P
F
50
-6
0 
hr
s 
A
P
F
50
-6
0 
hr
s 
A
P
F
Figure S4.
A
B
miR-279-GAL4
22C10
merge
miR-279-GAL4 prospero mergeC
Figure S5.
MAXILLARY PALP GR21A CD8-GFP IN MIR279 
MUTANTS
Nerfin-1 positive
Nerfin-1 negative
p<0.005
NERFIN EXPRESSION IN MIR279-GAL4 UAS-CD8GFP 
EXPRESSING CELLS IN THE MAXILLARY PALPS
HIGH MIR-279 LOW
NERFIN
HIGH NERFIN LOW
MIR-279
HIGH NERFIN HIGH
MIR-279
A
B
00.5
1
1.5
2
2.5
100nM 50nM
anti-miR-124
anti-miR-279
lu
ci
fe
ra
se
 u
ni
ts
 (n
er
fin
-3
’U
TR
)
**
** p<0.001
**
nerfin-1 3’UTR
D. melanogaster
D. sechellia
D. yakuba
D. pseudoobscura
D. virilis
A
B
Figure S6.
AmiR-279::nerfin-1 
B
0
5
10
15
20
25
30
35
40
MPantenna
w
t
Figure S7.
C
nerfin154 miR-379P
+        miR-379P
N
ER
FI
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15
4 /
+
N
ER
FI
N
15
4 /
+
w
t


Hybrid Neurons in a MicroRNA Mutant Are Putative Evolutionary Intermediates in Insect CO_2 Sensory Systems

http://authors.library.caltech.edu/51951/7/Cayirlioglu.SOM.pdf

Hybrid Neurons in a MicroRNA Mutant Are Putative Evolutionary Intermediates in Insect CO_2 Sensory Systems

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