Activity and stress during a critical period regulate olfactory sensory neuron differentiation

Here, we reveal that the regulation of Drosophila odorant receptor (OR) expression during the pupal stage is permissive and imprecise. We found that olfactory sensory neuron activity directly after hatching both directs and refines OR expression. We demonstrate that, as in mice, dLsd1 and Su(var)3-9 balance heterochromatin formation to direct OR expression. Neuronal activity induces dLsd1 and Su(var)3-9 expression, linking neuronal activity to OR expression. OR expression refinement shows a restricted duration, suggesting a gene regulatory critical period brings olfactory sensory neuron differentiation to an end. Consistent with a change in differentiation, stress during the critical period represses dLsd1 and Su(var)3-9 expression and makes the early permissive OR expression permanent. This induced permissive gene regulatory state makes OR expression resilient to stress later in life. Hence, during a critical period, OR activity feedback similar to in mouse OR selection, defines adult OR expression in Drosophila.


Introduction
Olfactory sensory neurons (OSNs) in most vertebrates and insects are specified to express a 27 single odorant receptor (OR) from a large repertoire of OR genes in the genome (Couto et al.,28 2005; Fishilevich and Vosshall, 2005; Mombaerts et al., 1996;Ressler et al., 1994). Two OR 29 gene regulatory models have been described: the vertebrate probabilistic selection model and 30 the invertebrate predetermined instructive model. 31 The vertebrate OR regulatory model depends on chromatin state changes-from a repressed in some ways similar that found in vertebrates also seems to exist in Drosophila. 63 Here, we further address the role of heterochromatin in Drosophila OR regulation. We first 64 demonstrate that OR gene regulation stringency increases after a restricted time of heightened 65 plasticity and stress-sensitive period of early fly development. We show that, as they do for 66 vertebrate OR selection, dLsd1 and su(var) 3-9 initiate and maintain OR expression stringency 67 in Drosophila. OSN activity regulates dLsd1 and su(var)3-9 expression, creating a feedback 68 loop that restricts and balances OR expression. Stress during this period inhibits the feedback 69 loop and produces permanent changes in OR expression.   Figure 1B, table S1), but stopped increasing after day four. The 83 expression of the ionotropic receptors (IRs) and gustatory receptors (GRs) also increased 84 during the first four days post-eclosion ( Figure 1B, table S1). We found that 13 of 22 antennal 85 IRs and eight of ten GRs expressed in OSNs increased one or more fold. As with the ORs, any 86 changes in IR and GR expression after day four were minor without any discernible pattern 87 ( Figure 1B, table S1). Thus, chemoreceptor expression in general seems to mature during the 88 first four days post-eclosion.   (Sanchez et al., 2016). Interestingly, we found increasing expression of the IFT and BB genes 99 during the first four days post-eclosion and no further change after the fourth day ( Figure 1D, 100 table S1). OSNs also express high levels of another auxiliary set of olfactory proteins required 101 for specific odor responses the odorant binding proteins (OBPs) (Larter et al., 2016). The 102 expression of most OBP genes either remains steady or increases from day one to day four 103 ( Figure 1E, table S1), lending further support to the idea that OSNs continue to develop and 104 sensory transduction continues to change after the pupal stage.  The line is the reference at which gene expression is the same between conditions. Statistics 116 for the figure in Table S1. Stress modulates the maturation of OR expression 120 We have previously observed that starvation and thermal stress increases OR expression 121 plasticity (Jafari and Alenius, 2015) and that cooperative transcription factor interactions in the 122 cis regulatory region stabilize the OR expression. In these studies, we focused on adult (5-7 123 DPE) flies but the stress induced plasticity suggested that stress could change also the OR 124 expression maturation process. To visualize stress-induced modulation of OR expression at all 125 stages, we used the Or59b minimal enhancer (Or59bME), an Or59b reporter that lacks the 126 cooperative regulation region required to resist stress-induced changes (Jafari and Alenius,  If stress modulates terminal OSN differentiation, the ectopic expression phenotypes we 139 observed with early temperature shifts could be expected to become permanent. Indeed, when 140 we returned Or59bME flies that underwent early temperature shifts to room temperature, we 141 found that the stress-induced ectopic Or59bME reporter expression pattern persisted 142 throughout a seven-day recovery period (Figure 2A). It even remained similar after a prolonged 143 18-day recovery period (Figure 2A). If the process of OR expression maturation is the final 144 stage of OSN differentiation, then temperature shifts after maturation is complete should be 145 reversible. Consistent with this hypothesis, we found that shifts back to room temperature for 146 those exposed to thermal stress after day three led to a restoration of the expression pattern in 147 only a single OSN class ( Figure 2B). This indicates that the OR expression state was already 148 fixed when the flies were subjected to the temperature shift. To address this further, we 149 subjected flies carrying Or59bME to two cold shifts, one during the critical period and another 150 after a four-day recovery period. As expected, the resulting Or59bME ectopic expression 151 pattern for flies subjected to shifts was similar to those subjected to a single early shift ( Figure  152 2A). Together, these results indicate that stress during the maturation phase switches adult OR 153 expression from a stress-sensitive refined expression pattern to a potentially less refined but 154 stress-resilient expression pattern.  The percentages show the fraction of the phenotype that is presented in that panel. Scale bar 163 denotes 3.5 m. Note the persistent ectopic expression after 18-day recovery or a second 164 exposure to low temperature (2x). The loss phenotype reverted to single OSN class expression 165 after 14-day recovery at room temperature. have low spontaneous activity (Hallem and Carlson, 2006). We found that about half the 173 ectopic Or47b expression flies lost the Or59b reporter expression ( Figure 3A and B), indicating 174 that high spontaneous OR activity can suppress OR expression. Consistent with the hypothesis 175 that it is OR activity rather than OR expression driving this repression of OR expression, we 176 found that ectopic expression of Or42b, an OR with low spontaneous activity, lost Or59b 177 reporter expression in 11% of the resulting flies ( Figure 3A and B). To determine whether odor 178 responses induce this negative feedback, we exposed flies to ethyl propionate (EP diluted 10 -  (Table S2 and Figure S1), suggesting that activity establish trichoid-related OR 193 expression during the pupal stage and restricts basiconic-related OR expression post-eclosion.

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To further address whether OSN activity is required for OR expression, we performed an RNA-   Table S2.  Table S2.

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The balance between dLsd1 and Su(var)3-9 refines OR expression 227 The similarity of the OR activity feedback we observed to the vertebrate OR choice mechanism 228 suggested a conserved OR regulatory mechanism. In mouse OSNs, Lsd1 catalyzes the    Figure 6A). Kdm4b expression, in contrast, decreased over the same period ( Figure 6B), suggesting that the maturation of OR expression involves a 297 reduction in the initiation and manifestation of OR expression. A more detailed dLsd1 and 298 Su(var)3-9 expression analysis showed that the main increase of these two enzymes occurred 299 during the first hours post-eclosion ( Figure 6C), suggesting that OSN activity may induce 300 dLsd1 and Su(var)3-9 expression. Consistent with this hypothesis, Orco mutant flies that lack 301 OR activity show reduced dLsd1 and Su(var)3-9 mRNA levels ( Figure 6D). Interestingly, we 302 found Kdm4b mRNA levels also increased in Orco mutants three days post-eclosion compared 303 to controls ( Figure 6D). This suggests OR expression initiation likely increases in the absence 304 of OSN activity. Next, we over expressed Or47b in a heterozygous Su(var)3-9 mutant 305 background. We found, consistent with the hypothesis that OSN activity induces 306 heterochromatin formation, that the heterozygote Su(var)3-9 mutant balanced the ectopic 307 expression of the highly active Or47b and rescued the loss of Or59b reporter expression 308 ( Figure 6E).  showed no significant change (Figure 7). The resulting imbalance between Su(var)3-9 and 336 dLsd1 is consistent with the loss of Or59b expression we observed when we exposed adult 337 heterozygous dLsd1 mutant flies to stress ( Figure 4C).   The mechanism that establishes OR expression was first identified in mice (Dalton and other ORs. Our result proposes that high OR activity suppress OR expression. We do not 386 explore the art of the activity but that the ORs are ionotropic channels (Benton et al., 2006) 387 suggest a calcium signal to induce the Su(var)3-9 and dLsd1. 388 After maturation, the OR expression mechanisms differ between Drosophila and mice. In 389 Drosophila, both OR alleles are expressed in each OSN (Dobritsa et al., 2003). In mice, one 390 OR allele is selected and expressed continuously by what is likely a separate mechanism.

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Another difference in Drosophila is that dLsd1 activity balances su(var)3-9 activity after 392 maturation, whereas in mice, Lsd1 is down-regulated after maturation (Dalton and Lomvardas, 393 2015). We found that it is only Lsd1 that is supressed after maturation, suggesting the memory 394 mechanism that maintains single OR allelic expression is an inflexible su(var)3-9 expression 395 that produces a defined heterochromatin level and sets the OR expression baseline. It remains 396 unclear if such a memory mechanism is conserved given the differences in regulation after OR 397 expression maturation.  images were collected on an LSM 700 (Zeiss) and analyzed using the LSM Image Browser.

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The numbers of OSNs co-expressing BP104 and GFP for the different constructs were counted 490 in these images. Adobe Photoshop CS4 (Adobe Systems) was used for image processing. Illumina NextSeq 500 next-generation sequencing system in mid-output mode via 1X100 bp 517 paired-end sequencing. 519 The RNA read counts were estimated with Kallisto (version 0.45.1). Differentially expressed 520 genes were estimated by DESeq2 (version 1.26.0) after counts had been rounded to the 521 nearest integer count. The linear model was simply one group vs the other group, e.g., WT 522 day 1 vs day 4, or WT day 1 vs Treatment day 1. Plots were made using ggplot2 and R, 523 showing log10 size-factor normalized read counts.