A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity

Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1–4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity

Patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species

Organisms have evolved strikingly parallel phenotypes in response to similar selection pressures suggesting that there may be shared constraints limiting the possible evolutionary trajectories. For example, the behavioral adaptation of specialist Drosophila species to specific host plants can exhibit parallel changes in their adult olfactory neuroanatomy. We investigated the genetic basis of these parallel changes by comparing gene expression during the development of the olfactory system of two specialist Drosophila species to that of four other generalist species. Our results suggest that the parallelism observed in the adult olfactory neuroanatomy of ecological specialists extends more broadly to their developmental antennal expression profiles, and to the transcription factor combinations specifying olfactory receptor neuron (ORN) fates. Additionally, comparing general patterns of variation for the antennal transcriptional profiles in the adult and developing olfactory system of the six species suggest the possibility that specific, non-random components of the developmental programs underlying the Drosophila olfactory system harbor a disproportionate amount of interspecies variation. Further examination of these developmental components may be able to inform a deeper understanding of how traits evolve

Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons.

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru). The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a) involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh). The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors

<i>fru</i> expression in adult Or47b ORNs requires Or47b function.

<p><b>(A)</b> Heterozygous <i>Or47b</i> mutant antennae (3–5 d old) expressing <i>fruGal4 40XUASCD8GFP</i> <b>(A)</b> and <i>OR47b-CD2</i> <b>(A’)</b>. <b>(A”)</b> shows the merge of two images. <b>(B)</b> Homozygous <i>Or47b</i> mutant antennae (3–5 d old). <b>(C)</b> Overexpression of <i>UAS-Or47b</i> under the control of <i>fruGal4</i> in <i>Or47b</i> mutants (14 d old). <b>(D)</b> Overexpression of <i>UAS-Or88a</i> under the control of <i>fruGal4</i> in <i>Or47b</i> mutants (14 d old).</p> <p>GENOTYPES:</p> <p>A–A”: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/+;fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/+</i></p> <p>B–B”: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/+</i></p> <p>C: <i>Or47b</i>^<i>2</i><i>/Or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/UAS-Or47b</i></p> <p>D: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/Or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/UAS-Or88a</i></p

qPCR primers.

<p>qPCR primers.</p

<i>fru</i>-positive OR expression in at4 sensilla expands to developmentally related <i>fru</i>-negative ORNs in <i>alh</i> mutants.

<p><b> A)</b> Adult antennae and brains labeled with <i>Or47bGal4 UAS-CD8GFP</i> (green) in wild type and <i>alh</i> mutant clones. Magenta staining in brains is against N-cadherin, a neuropil marker. <b>B)</b> Adult antennae and brains labeled with <i>Or88aGal4 UAS-CD8GFP</i> in wild-type and <i>alh</i> mutant clones. <b>C)</b> Adult antennae and brains labeled with <i>Or65aGal4 UAS-CD8GFP</i> in wild-type and <i>alh</i> mutant clones. <b>D–G)</b> Quantification of cell bodies observed in the adult antennae of WT and <i>alh</i> mutant clones. For all graphs, asterisks indicate significant (<i>p</i> < .05) differences from wild type. Error bars represent standard error of the mean (SEM). An ANOVA was performed for each cell type and followed with Tukey’s Honest Significant Difference (HSD)—see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#sec015" target="_blank">Materials and Methods</a>. <b>D)</b> Total <i>Or47b</i>-positive cells. Wild type flies were significantly different from both <i>alh</i> conditions (<i>p</i> < .0001). <i>n</i> = 10–40. All count data may be found in the Supporting Information as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s001" target="_blank">S1 Data</a>. <b>E)</b> Total <i>Or88a</i>-positive cells. Both <i>alh</i> conditions were significantly different from wild-type males (<i>p</i> < .0001). <i>n</i> = 27 − 57. <b>F)</b> Total <i>Or65a</i>-positive cells. Both <i>alh</i> conditions were significantly different from wild-type males (<i>p</i> < .05). <i>n</i> = 9–27. <b>G)</b> Total <i>or47b</i>-positive clusters, normalized by total <i>Or47b</i>-positive cells. Wild type flies were significantly different from all <i>alh</i> conditions (<i>p</i> < .0001). <i>n</i> = 10–40. <b>H)</b> Model: in <i>alh</i> mutants, the <i>Or47b</i> odorant receptor expression is expanded to the other ORNs in the at4 sensilla, at the expense of their native OR expression, but the axons of these ORNs continue to target their original locations in the antennal lobe.</p> <p>GENOTYPES:</p> <p>A) <i>eyflp</i>; <i>Or47bGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or47bGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or47bGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p> <p>B) <i>eyflp</i>; <i>Or88aGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or88aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or88aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p> <p>C) <i>eyflp</i>; <i>Or65aGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or65aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or65aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p

<i>fru</i> expression expands together with <i>Or47b</i> expression in <i>alh</i> mutants.

<p><b>(A/B)</b> Antennae labeled with <i>fru</i>^<i>GAL4</i> <i>UAS-RedStinger</i> (magenta), and <i>Or47bCD8GFP</i> (green) in wild type and <i>alh</i> mutant clones. Right panels represent higher magnification images. Arrows label <i>Or47b/fru</i>-positive nuclei in wild type images. In <i>alh</i> mutants, arrows point to sensilla with 2–3 Or47b ORNs that are also <i>fru</i>-positive. <b>(C)</b> Antennal lobes labeled with fruGal4 UAS-sytGFP (Z-stack, anterior sections of antennal lobe). <b>(D)</b> Antennal lobes labeled with fruGal4 UAS-CD8GFP (Z-stack, posterior sections of antennal lobe). Asterisks denote <i>fru</i>-labeled glomeruli thought to be innervated by neurons from the antennal sacculus.</p> <p>GENOTYPES:</p> <p>(A) wild type: <i>eyFLP/+;Or47bCD8GFP/UAS-RedStinger; FRT82 fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p> <p>(B) <i>alh</i> mutant: <i>eyFLP/+;Or47bCD8GFP/UAS-RedStinger; FRT82 alh</i>^<i>1353</i> <i>fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p> <p>(C) wild type: <i>eyFLP/+; UAS-syTGFP/+; FRT82 fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p> <p><i>alh</i> mutant: <i>eyFLP/+; UAS-syTGFP/+; FRT82 alh</i>^<i>1353</i> <i>fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p> <p>(D) wild type: <i>eyFLP/+; UAS-CD8GFP/+; FRT82 fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p> <p><i>alh</i> mutant: <i>eyFLP/+; UAS-CD8GFP/+; FRT82 alh</i>^<i>1353</i> <i>fru</i>^<i>GAL4</i><i>/FRT82Gal80E2F</i></p

Maintenance of <i>fru</i> expression in adult ORNs requires CamK signaling and p300/CBP.

<p><b>(A)</b> Wild-type antennae expressing <i>fruGal4 UAS-40XUASGFP</i> (green) and <i>Or47b-CD2</i> (magenta). <b>(B–D)</b> Antennae expressing <i>fruGal4 UAS-40XUASGFP</i> (green) and <i>Or47b-CD2</i> (magenta) as well as <i>fruGal4 CamKI</i> RNAi <b>(B)</b>, <i>UAS-creb</i> <b>(C)</b>, and <i>UAS-p300</i> RNAi <b>(D)</b>. <b>(E)</b> Quantification of antennal <i>fru</i>-positive ORN cell counts for experiments in Figs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.g005" target="_blank">5</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.g006" target="_blank">6</a> and 8. Data shown represents the fraction of <i>Or47b</i>-positive cells that are also <i>fru</i>-positive. For all graphs, asterisks indicate significant (<i>p</i> < .01) differences from <i>fru</i>^<i>Gal4</i>. Error bars represent SEM. A one-way ANOVA was performed and followed with Tukey’s HSD—see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#sec015" target="_blank">Materials and Methods</a>. Cell count data also graphed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s011" target="_blank">S10 Fig</a>. All raw count data may be found in the Supporting Information as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s001" target="_blank">S1 Data</a>.</p> <p>GENOTYPES:</p> <p>(A) <i>Or47b-CD2</i> /<i>+; fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP</i></p> <p>(B) UAS-CamKI RNAi/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP</p> <p>(C) UAS-CREB/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP</p> <p>(D) UAS-p300RNAi/+; Or47b-CD2/+; fru^GAL4 UAS-40XCD8GFP</p

<i>fru</i>-positive OR expression in ac4 sensilla expands to developmentally related <i>fru</i>-negative ORNs in <i>alh</i> mutants.

<p><b>A)</b> Adult antennae and brains labeled with <i>Ir84aGal4 UAS-CD8GFP</i> (green) in wild type and <i>alh</i> mutant clones. Magenta staining in brains is against N-cadherin, a neuropil marker. <b>B)</b> Total <i>Ir84a</i>-positive cells. Asterisks indicate significant (<i>p</i> < .05) differences from wild type. Error bars represent SEM. ANOVAs were performed and followed with Tukey’s HSD—see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#sec015" target="_blank">Materials and Methods</a>. Wild type flies were significantly different from all <i>alh</i> conditions (<i>p</i> < .0001). <i>n</i> = 24–50. All count data may be found in the Supporting Information as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s001" target="_blank">S1 Data</a>. <b>C) Model:</b> In <i>alh</i> mutants, the <i>Ir84a</i> odorant receptor identity is expanded to other coeloconic ORNs as observed through glomerular innervation. <i>Ir84a</i> expression is expanded to <i>ir75a</i> and <i>ir76a</i> ORNs. <b>D)</b> Adult antennae and brains labeled with <i>Or67dGal4 UAS-CD8GFP</i> (green) in wild type and <i>alh</i> mutant clones in <i>Drosophila</i>. Magenta staining in brains is against N-cadherin, a neuropil marker. <b>E)</b> Total <i>Or67d</i>-positive cells. An ANOVA for this data was not significant. <i>n</i> = 20–30. All count data may be found in the Supporting Information as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s001" target="_blank">S1 Data</a>. <b>F)</b> Model: In <i>alh</i> mutants, the expression and axonal targeting patterns of <i>or67d</i>-positive ORNs are unchanged.</p> <p>GENOTYPES:</p> <p>A) <i>eyflp</i>; <i>Ir84aGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Ir84aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Ir84aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p> <p>D) <i>eyflp</i>; <i>Or67dGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or67dGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i></p