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The insect antennal lobe (AL) contains the first synapses of the olfactory system, where olfactory sensory neurons (OSNs) contact second order projection neurons (PNs). In Drosophila melanogaster, OSNs expressing specific receptor genes send stereotyped projections to one or two of about 50 morphologically defined glomeruli[1–3]. The mechanisms for this precise matching between OSNs and PNs have been studied extensively in D. melanogaster, where development is deterministic and independent of neural activity[4–6]. However, a number of insect lineages, most notably the ants, have receptor gene repertoires many times larger than D. melanogaster and exhibit more structurally complex antennal lobes[7–12]. Moreover, perturbation of OSN function via knockout of the odorant receptor (OR) co-receptor, Orco, results in drastic AL reductions in ants[13,14], but not in Drosophila[15]. Here, we characterize AL development in the clonal raider ant, Ooceraea biroi. We find that, unlike in Drosophila, ORs and Orco are expressed before the onset of glomerulus formation, and Orco protein is trafficked to developing axon terminals, raising the possibility that ORs play a role during ant AL development. Additionally, ablating ant antennae at the onset of pupation results in AL defects that recapitulate the Orco mutant phenotype. Thus, early loss of functional OSN innervation reveals latent structure in the AL that develops independently of peripheral input, suggesting that the AL is initially pre-patterned, and later refined in an OSN-dependent manner. This two-step process might increase developmental flexibility and thereby facilitate the rapid evolution and expansion of the ant chemosensory system.

Social experience and pheromone signaling in olfactory neurons affect neuronal responses and male courtship behaviors in Drosophila. We previously showed that social experience and pheromone signaling modulate chromatin around behavioral switch gene fruitless, which encodes a transcription factor necessary and sufficient for male behaviors. Fruitless drives social experience dependent modulation of courtship behaviors and pheromone responses in sensory neurons, however, the molecular mechanisms underlying this neuromodulation remain less clear. To identify the molecular mechanisms driving social experience-dependent neuromodulation, we performed RNA-seq from antennal samples of mutants in pheromone receptors and fruitless, as well as grouped or isolated wild-type males. We found that loss of pheromone detection differentially alters the levels of fruitless exons suggesting changes in splicing patterns. In addition, many Fruitless target neuromodulatory genes, such as neurotransmitter receptors, ion channels, ion and membrane transporters, and odorant binding proteins are differentially regulated by social context and pheromone signaling. Recent studies showed that social experience and juvenile hormone signaling coregulate fru chromatin to modify pheromone responses in olfactory neurons. Interestingly, genes involved in juvenile hormone metabolism are also misregulated in different social contexts and mutant backgrounds. Our results suggest that modulation of neuronal activity and behaviors in response to social experience and pheromone signaling likely arise due to large-scale changes in transcriptional programs for neuromodulators downstream of behavioral switch gene function.

Summary Critical to evolutionary fitness, animals regulate social behaviors by integrating signals from both their external environments and internal states. Here, we find that population density modulates the courtship behavior of male Drosophila melanogaster in an age-dependent manner. In a competitive mating assay, males reared in a social environment have a marked advantage in courting females when pitted against males reared in isolation. Group housing promotes courtship in mature (7-day) but not immature (2-day) males; this behavioral plasticity requires the Or47b pheromone receptor. Using single-sensillum recordings, we find that group housing increases the response of Or47b olfactory receptor neurons (ORNs) only in mature males. The effect of group housing on olfactory response and behavior can be mimicked by chronically exposing single-housed males to an Or47b ligand. At the molecular level, group housing elevates Ca2+ levels in Or47b ORNs, likely leading to CaMKI-mediated activation of the histone-acetyl transferase CBP. This signaling event in turn enhances the efficacy of juvenile hormone, an age-related regulator of reproductive maturation in flies. Furthermore, the male-specific Fruitless isoform (FruM) is required for the sensory plasticity, suggesting that FruM functions as a downstream genomic coincidence detector in Or47b ORNs—integrating reproductive maturity, signaled by juvenile hormone, and population density, signaled by CBP. In all, we identify a neural substrate and activity-dependent mechanism by which social context can directly influence pheromone sensitivity, thereby modulating social behavior according to animals’ life-history stage.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe. Despite the identification of cell surface receptors regulating axon guidance, how ORN axons sort to form 50 stereotypical glomeruli remains unclear. Here we show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. DIP/Dpr profiles are dynamic during development and correlate with sensilla type lineage for some ORN classes. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. Our results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli., Author summary In the human brain there are over 80 billion neurons that form approximately 100 trillion specific connections. How the brain organizes the axon terminals of these neurons into distinct synaptic units on such a large scale is largely unknown. In Drosophila, 50 classes of olfactory receptor neurons (ORNs) connect to 50 class-specific and uniquely positioned glomeruli in the antennal lobe, providing a complex yet workable model to understand the organization of glomerular structures and morphology. Here we show that the heterophilic cell adhesion proteins, DIPs and Dprs, are expressed in ORNs during glomerular formation. Many ORN classes express a unique combination of DIPs/dprs, with neurons of the same class expressing interacting partners, suggesting a role in class-specific self-adhesion between ORN axons. Analysis of DIP/Dpr expression revealed that ORNs that target neighboring glomeruli have different combinations, and ORNs with very similar DIP/Dpr combinations can project to distant glomeruli in the antennal lobe. Perturbations of DIP/dpr gene function result in local projection defects of ORN axons and glomerular positioning, without altering correct matching of ORNs with their target neurons. Our results suggest that context-dependent differential adhesion through DIP/Dpr combinations regulate self-adhesion and sort ORN axons into uniquely positioned glomeruli.

Detection of a broad range of chemosensory signals is necessary for the survival of multicellular organisms. Chemical signals are the main facilitators of foraging, escape, and social behaviors. To increase detection coverage, animal sensory systems have evolved to create a large number of neurons with highly specific functions. The olfactory system, much like the nervous system as a whole, is astonishingly diverse. The mouse olfactory system has millions of neurons with over a thousand classes, whereas the more compact Drosophila genome has approximately 80 odorant receptor genes that give rise to 50 neuronal classes and 1300 neurons in the adult.(4) Understanding how neuronal diversity is generated remains one of the central questions in developmental neurobiology. Here, we review the current knowledge on the development of the adult Drosophila olfactory system and the progress that has been made toward answering this central question.

Detection of a broad range of chemosensory signals is necessary for the survival of multicellular organisms. Chemical signals are the main facilitators of foraging, escape, and social behaviors. To increase detection coverage, animal sensory systems have evolved to create a large number of neurons with highly specific functions. The olfactory system, much like the nervous system as a whole, is astonishingly diverse.1‐3 The mouse olfactory system has millions of neurons with over a thousand classes, whereas the more compact Drosophila genome has approximately 80 odorant receptor genes that give rise to 50 neuronal classes and 1300 neurons in the adult.4 Understanding how neuronal diversity is generated remains one of the central questions in developmental neurobiology. Here, we review the current knowledge on the development of the adult Drosophila olfactory system and the progress that has been made toward answering this central question. WIREs Dev Biol 2015, 4:609–621. doi: 10.1002/wdev.197 For further resources related to this article, please visit the WIREs website.

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.

Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1–4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. Here we use transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. We surveyed four critical developmental stages of the olfactory system: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. We focused on the expression profiles of olfactory receptor genes and transcription factors—the two main classes of genes that regulate the sensory identity of ORNs. We identify distinct clusters of genes that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. We show that the expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Carbon dioxide is an important environmental cue for many insects, regulating many behaviors including some that have direct human impacts. To further improve our understanding of how this system varies among closely related insect species, we examined both the behavioral response to CO2 as well as the transcriptional profile of key developmental regulators of CO2 sensory neurons in the olfactory system across the Drosophila genus. We found that CO2 generally evokes repulsive behavior across most of the Drosophilids we examined, but this behavior has been lost or reduced in several lineages. Comparisons of transcriptional profiles from the developing and adult antennae for subset these species suggest that behavioral differences in some species may be due to differences in the expression of the CO2 co-receptor Gr63a. Furthermore, these differences in Gr63a expression are correlated with changes in the expression of a few genes known to be involved in the development of the CO2 circuit, namely dac, an important regulator of sensilla fate for sensilla that house CO2 ORNs, and mip120, a member of the MMB/dREAM epigenetic regulatory complex that regulates CO2 receptor expression. In contrast, most of the other known structural, molecular, and developmental components of the peripheral Drosophila CO2 olfactory system seem to be well-conserved across all examined lineages. These findings suggest that certain components of CO2 sensory ORN development may be more evolutionarily labile, and may contribute to differences in CO2-evoked behavioral responses across species.

It is estimated that there are billions of neurons in the human brain. Each of these neurons is thought to play a distinct role in nervous system function. It is the emergent functional properties of these neurons that allow organisms to detect and process information about the complex sensory environment, and successfully navigate through these environments. To perform this task, many neural circuits representing different modalities of sensory information are organized to optimally encode and discriminate the nature of the stimuli. Diversity of neuronal fates that constitute these circuits is generated during development, as cells with high differentiation potentials, such as neural stem/progenitor/precursor cells, divide, communicate and make fate decisions that gradually diversify and restrict the type of cells they can generate. Sensory systems are a particularly interesting place to understand mechanisms of neural diversity as functional types of sensory neurons are under the constraints brought about by the complexity of the sensory world. In this chapter, we will review our current understanding of mechanisms generating neuronal diversity in the visual and olfactory systems (Van Bortle and Corces in Cell 152(6):1213–1217, 2013; Henikoff in Neuron 86(6):1319–1321, 2015; Mo et al. in Neuron 86(6):1369–1384, 2015).

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., The fruitless (fru) gene regulates sexual behavior in flies, but what regulates fru? This study shows that the chromatin modulator Alhambra restricts fru expression to specific neurons in the developing Drosophila olfactory system. In adults, olfactory receptor signaling and the histone acetyltransferase p300 are required to maintain fru expression., Author Summary How do individual neurons know what type of a circuit they must integrate into? To correctly assemble neural circuits during development, the identities of neurons must be coordinated with the identities of the circuits into which they will be integrated. How is this process regulated? We have used the olfactory circuits that regulate sex-specific behaviors in Drosophila to answer this question. In Drosophila, the fruitless (fru) gene, which encodes a transcription factor, acts as a molecular marker that labels circuits regulating sex-specific behaviors. Fru is both a necessary and sufficient regulator of sex-specific behaviors like courtship and aggression and is expressed in only about 2,000 interconnected neurons in the fly nervous system. Even though fru expression and function is so critical to sex-specific behavior, the mechanisms regulating its expression in each of the neurons within the circuit are not known. Here, we revealed two different modes of transcriptional regulation of fru during the development of the olfactory receptor neurons that are involved in sex-specific behaviors. In the first mode, the putative chromatin modulator Alhambra (Alh) coregulates both olfactory receptors and fru expression in two olfactory receptor neuron classes during development. In adult flies, the second mode maintains fru expression through the activity of olfactory receptors, calcium signaling, and chromatin modulatory proteins in these olfactory receptor neurons. These two genetic programs separate the developmentally hardwired and the olfactory receptor activity-mediated regulation of fru expression in olfactory circuits, and might represent the molecular mechanisms that mediate the innate and adaptable aspects of odor-guided social behaviors.

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., Author Summary Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes to decipher a complex chemical environment. Each sensilla subtype houses 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. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), is part of a conserved proximodistal (PD) gene regulatory network module that patterns the antennal disc into seven concentric rings and diversifies SOP identities. Each ring expresses a unique combination of the aforementioned transcription factors, and encodes the differentiation potentials for a limited number of sensilla subtypes. Genetic perturbations of the network lead to predictable changes in ORN diversity. These data suggest that the diversification of precursor fields by the prepatterning network is the first step to neuronal diversification, followed by SOP selection by proneural genes, and Notch-mediated neurogenesis. As each step operates in a context-dependent manner, deployment of the same transcription factor network module may regulate neuronal diversity in parallel systems with completely different fate outputs.

Individual members of the E2F/DP protein family control cell cycle progression by acting predominantly as an activator or repressor of transcription. In Drosophila melanogaster the E2f1, E2f2, Dp, and Rbf1 genes all contribute to replication control in ovarian follicle cells, which become 16C polyploid and subsequently undergo chorion gene amplification late in oogenesis. Mutation of E2f2, Dp, or Rbf1 causes ectopic DNA replication throughout the follicle cell genome during gene amplification cycles. Here we show by both reverse transcription-PCR and DNA microarray analysis that the transcripts of prereplication complex (pre-RC) genes are elevated compared to the wild type in E2f2, Dp, and Rbf1 mutant follicle cells. For some genes the magnitude of this transcriptional derepression is greater in Rbf1 than in E2f2 mutants. These differences correlate with differences in the magnitude of the replication defects in follicle cells, which attain an inappropriate 32C DNA content in both Rbf1 and Dp mutants but not in E2f2 mutants. The ectopic genomic replication of E2f2 mutant follicle cells can be suppressed by reducing the Orc2, Orc5, or Mcm2 gene dose by half, indicating that small changes in pre-RC gene expression can affect DNA synthesis in these cells. We conclude that RBF1 forms complexes with both E2F1/DP and E2F2/DP that cooperate to repress the expression of pre-RC genes, which helps confine DNA synthesis to sites of gene amplification. In contrast, E2F1 and E2F2 repressors function redundantly for some genes in the embryo. Thus, the relative functional contributions of E2F1 and E2F2 to gene expression and cell cycle control depends on the developmental context.

E2F transcription factors are thought to influence the G1–S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis. But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase.

To increase detection of a complex chemical environment, vertebrates and insects express an extraordinary number of distinct olfactory receptor neuron (ORN) classes, each functionally specialized to detect a set of odorants. This is achieved as the olfactory system develops and each of these ORN classes makes developmental decisions defining the olfactory receptor genes they will express and their class-specific connections in the brain. In addition to this high level of ORN diversity, olfactory systems are also very dynamic evolutionarily, with both the number and functionality of olfactory receptor genes as well as the requirement for certain ORN circuits being under ecological constraints. In this review, we will discuss molecular and developmental strategies underlying ORN diversity and evolutionary plasticity as well as present the insect olfactory system as a model for evo-devo research in light of recent findings.

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 .

Many insects are equipped with neurons that detect carbon dioxide. The use to which they are put varies according to the insect's ecology: hawk-moths use it as a measure of the quality of datura flowers. Closer to home, mosquitoes and other blood-feeders are attracted to the carbon dioxide emitted by us. Drosophila too has these sensors: two receptors, called Gr21a and Gr63a, have now been identified in the fruit fly. Flies with only one of the two are insensitive to carbon dioxide, but when both genes are expressed the neurons are sensitive to the gas. Similar genes are present in the malaria mosquito, so they may be useful targets for drugs aimed at reducing the ability of mosquitoes to find humans to bite. Identification of two CO2 receptors in Drosophila antennae, Gr21a and Gr63a, determines that their co-expression is necessary and sufficient for CO2 sensing. Flies lacking one of the two are insensitive to CO2, and confer CO2 responsivity when both receptors are expressed in heterologous neurons. Blood-feeding insects, including the malaria mosquito Anopheles gambiae, use highly specialized and sensitive olfactory systems to locate their hosts. This is accomplished by detecting and following plumes of volatile host emissions, which include carbon dioxide (CO2)1. CO2 is sensed by a population of olfactory sensory neurons in the maxillary palps of mosquitoes2,3 and in the antennae of the more genetically tractable fruitfly, Drosophila melanogaster4. The molecular identity of the chemosensory CO2 receptor, however, remains unknown. Here we report that CO2-responsive neurons in Drosophila co-express a pair of chemosensory receptors, Gr21a and Gr63a, at both larval and adult life stages. We identify mosquito homologues of Gr21a and Gr63a, GPRGR22 and GPRGR24, and show that these are co-expressed in A. gambiae maxillary palps. We show that Gr21a and Gr63a together are sufficient for olfactory CO2-chemosensation in Drosophila. Ectopic expression of Gr21a and Gr63a together confers CO2 sensitivity on CO2-insensitive olfactory neurons, but neither gustatory receptor alone has this function. Mutant flies lacking Gr63a lose both electrophysiological and behavioural responses to CO2. Knowledge of the molecular identity of the insect olfactory CO2 receptors may spur the development of novel mosquito control strategies designed to take advantage of this unique and critical olfactory pathway. This in turn could bolster the worldwide fight against malaria and other insect-borne diseases.

Item does not contain fulltext Fibroblast growth factors (FGFs) are important regulators of retinal development and survival. We examined the expression and distribution of FGF9 and its preferred receptors FGFR2IIIc and FGFR3IIIc in this tissue. FGF9 transcripts in whole rat retina were detected by RT-PCR but were not present in purified cultured Muller glia. Transcripts appeared as 3.2-kb and 4.0-kb bands on Northern blots, and Western blotting of whole retina revealed FGF9-immunoreactive bands at 30 and 55 kDa. FGF9 mRNA demonstrated a biphasic expression profile, elevated at birth and adulthood, but relatively decreased during terminal retinal differentiation (4-14 days postnatal). Antibody labeling broadly reflected these findings: staining in vivo was observed mainly in the inner retina (and outer plexiform layer in adults) whereas FGF9 was not detectable in cultured Muller glia. In adults, FGF9 in situ hybridization also showed a detectable signal in inner retina. FGFR2IIIc and FGFR3IIIc were detected by RT-PCR, and Western blotting showed both FGFRs existed as multiple forms between approximately 100-200 kDa. FGFR2 and FGFR3 antibodies showed prominent labeling in the inner retina, especially in proliferating cultured Muller glia. Exogenous FGF9 elicited a dose-dependent increase in Muller glial proliferation in vitro. These data suggest a role for FGF9 in retinal differentiation and maturation, possibly representing a neuronally derived factor acting upon glial (and other) cells.

Drosophila contains two members of the E2F transcription factor family (E2f and E2f2), which controls the expression of genes that regulate the G1-S transition of the cell cycle. Previous genetic analyses have indicated that E2f is an essential gene that stimulates DNA replication. We show that loss of E2f2 is viable, but causes partial female sterility associated with changes in the mode of DNA replication in the follicle cells that surround the developing oocyte. Late in wild-type oogenesis, polyploid follicle cells terminate a program of asynchronous endocycles in which the euchromatin is entirely replicated, and then confine DNA synthesis to the synchronous amplification of specific loci, including two clusters of chorion genes that encode eggshell proteins. E2f2 mutant follicle cells terminate endocycles on schedule, but then fail to confine DNA synthesis to sites of gene amplification and inappropriately begin genomic DNA replication. This ectopic DNA synthesis does not represent a continuation of the endocycle program, as the cells do not complete an entire additional S phase. E2f2 mutant females display a 50% reduction in chorion gene amplification, and lay poorly viable eggs with a defective chorion. The replication proteins ORC2, CDC45L and ORC5, which in wild-type follicle cell nuclei localize to sites of gene amplification, are distributed throughout the entire follicle cell nucleus in E2f2 mutants, consistent with their use at many genomic replication origins rather than only at sites of gene amplification. RT-PCR analyses of RNA purified from E2f2 mutant follicle cells indicate an increase in the level of Orc5 mRNA relative to wild type. These data indicate that E2f2 functions to inhibit widespread genomic DNA synthesis in late stage follicle cells, and may do so by repressing the expression of specific components of the replication machinery.