Altering the Temporal Regulation of One Transcription Factor Drives Evolutionary Trade-Offs between Head Sensory Organs.

Size trade-offs of visual versus olfactory organs is a pervasive feature of animal evolution. This could result from genetic or functional constraints. We demonstrate that head sensory organ size trade-offs in Drosophila are genetically encoded and arise through differential subdivision of the head primordium into visual versus non-visual fields. We discover that changes in the temporal regulation of the highly conserved eyeless/Pax6 gene expression during development is a conserved mechanism for sensory trade-offs within and between Drosophila species. We identify a natural single nucleotide polymorphism in the cis-regulatory region of eyeless in a binding site of its repressor Cut that is sufficient to alter its temporal regulation and eye size. Because eyeless/Pax6 is a conserved regulator of head sensory placode subdivision, we propose that its temporal regulation is key to define the relative size of head sensory organs.

Evolutionary changes in transcription factor coding sequence quantitatively alter sensory organ development and function.

Animals are characterized by a set of highly conserved developmental regulators. Changes in the cis-regulatory elements of these regulators are thought to constitute the major driver of morphological evolution. However, the role of coding sequence evolution remains unresolved. To address this question, we used the Atonal family of proneural transcription factors as a model. Drosophila atonal coding sequence was endogenously replaced with that of atonal homologues (ATHs) at key phylogenetic positions, non-ATH proneural genes, and the closest homologue to ancestral proneural genes. ATHs and the ancestral-like coding sequences rescued sensory organ fate in atonal mutants, in contrast to non-ATHs. Surprisingly, different ATH factors displayed different levels of proneural activity as reflected by the number and functionality of sense organs. This proneural potency gradient correlated directly with ATH protein stability, including in response to Notch signaling, independently of mRNA levels or codon usage. This establishes a distinct and ancient function for ATHs and demonstrates that coding sequence evolution can underlie quantitative variation in sensory development and function.

Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex.

The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body.

Transcriptional control of cell fate specification: lessons from the fly retina.

It is now widely recognized that as cells of developing tissues transition through successive states of decreasing pluripotency into a state of terminal differentiation, they undergo significant changes in their gene expression profiles. Interestingly, these successive states of increasing differentiation are marked by the spatially and temporally restricted expression of sets of transcription factors. Each wave of transcription factors not only signals the arrival of a given stage in cellular differentiation, but it is also necessary for the activation of the next set of transcription factors, creating the appearance of a smooth, directed, and deterministic genetic program of cellular differentiation. Until recently, however, it was largely unknown which genes, besides each other, these transcription factors were activating. Thus, the molecular definition of any given step of differentiation, and how it gave rise to the following step remained unclear. Recent advances in transcriptomics, bioinformatics, and molecular genetics resulted in the identification of numerous transcription factor target genes (TGs). These advances have opened the door to using similar approaches in developmental biology to understand what the transcriptional cascades of cellular differentiation might be. Using the development of the Drosophila eye as a model system, we discuss the role of transcription factors and their TGs in cell fate specification and terminal differentiation.

Genetically encoded dendritic marker sheds light on neuronal connectivity in Drosophila.

In recent years, Drosophila melanogaster has emerged as a powerful model for neuronal circuit development, pathology, and function. A major impediment to these studies has been the lack of a genetically encoded, specific, universal, and phenotypically neutral marker of the somatodendritic compartment. We have developed such a marker and show that it is effective and specific in all neuronal populations tested in the peripheral and central nervous system. The marker, which we name DenMark (Dendritic Marker), is a hybrid protein of the mouse protein ICAM5/Telencephalin and the red fluorescent protein mCherry. We show that DenMark is a powerful tool for revealing novel aspects of the neuroanatomy of developing dendrites, identifying previously unknown dendritic arbors, and elucidating neuronal connectivity.

Architecture of the primary taste center of Drosophila melanogaster larvae.

A simple nervous system combined with stereotypic behavioral responses to tastants, together with powerful genetic and molecular tools, have turned Drosophila larvae into a very promising model for studying gustatory coding. Using the Gal4/UAS system and confocal microscopy for visualizing gustatory afferents, we provide a description of the primary taste center in the larval central nervous system. Essentially, gustatory receptor neurons target different areas of the subesophageal ganglion (SOG), depending on their segmental and sensory organ origin. We define two major and two smaller subregions in the SOG. One of the major areas is a target of pharyngeal sensilla, the other one receives inputs from both internal and external sensilla. In addition to such spatial organization of the taste center, circumstantial evidence suggests a subtle functional organization: aversive and attractive stimuli might be processed in the anterior and posterior part of the SOG, respectively. Our results also suggest less coexpression of gustatory receptors than proposed in prior studies. Finally, projections of putative second-order taste neurons seem to cover large areas of the SOG. These neurons may thus receive multiple gustatory inputs. This suggests broad sensitivity of secondary taste neurons, reminiscent of the situation in mammals.

DmOAZ, the unique Drosophila melanogaster OAZ homologue is involved in posterior spiracle development.

In this paper, we study DmOAZ, the unique Drosophila melanogaster homologue of the OAZ zinc finger protein family. We show partial conservation of the zinc finger organization between DmOAZ and the vertebrate members of this family. We determine the exon/intron structure of the dmOAZ gene and deduce its open reading frame. Reverse transcriptase-polymerase chain reaction analysis shows that dmOAZ is transcribed throughout life. In the embryo, strongest DmOAZ expression is observed in the posterior spiracles. We suggest that dmOAZ acts as a secondary target of the Abd-B gene in posterior spiracle development, downstream of cut and ems. In a newly created loss-of-function mutant, dmOAZ ( 93 ), the "filzkörper" part of the posterior spiracles, is indeed structurally abnormal. The dmOAZ ( 93 ) mutant is a larval lethal, a phenotype that may be linked to the spiracular defect. Given the dmOAZ ( 93 ) mutant as a new tool, the fruit fly may provide an alternative model for analyzing in vivo the functions of OAZ family members.

Glomerular maps without cellular redundancy at successive levels of the Drosophila larval olfactory circuit.

BACKGROUND: Drosophila larvae possess only 21 odorant-receptor neurons (ORNs), whereas adults have 1,300. Does this suggest that the larval olfactory system is built according to a different design than its adult counterpart, or is it just a miniature version thereof? RESULTS: By genetically labeling single neurons with FLP-out and MARCM techniques, we analyze the connectivity of the larval olfactory circuit. We show that each of the 21 ORNs is unique and projects to one of 21 morphologically identifiable antennal-lobe glomeruli. Each glomerulus seems to be innervated by a single projection neuron. Each projection neuron sends its axon to one or two of about 28 glomeruli in the mushroom-body calyx. We have discovered at least seven types of projection neurons that stereotypically link an identified antennal-lobe glomerulus with an identified calycal glomerulus and thus create an olfactory map in a higher brain center. CONCLUSIONS: The basic design of the larval olfactory system is similar to the adult one. However, ORNs and projection neurons lack cellular redundancy and do not exhibit any convergent or divergent connectivity; 21 ORNs confront essentially similar numbers of antennal-lobe glomeruli, projection neurons, and calycal glomeruli. Hence, we propose the Drosophila larva as an "elementary" olfactory model system.

The Drosophila metabotropic glutamate receptor DmGluRA regulates activity-dependent synaptic facilitation and fine synaptic morphology.

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased ( approximately 10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

Integration of complex larval chemosensory organs into the adult nervous system of Drosophila.

The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis in a third larval pharyngeal organ. Hence, our experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis.

Developmental origin of wiring specificity in the olfactory system of Drosophila.

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. We have previously shown that second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of approximately 50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? Here, we examine the cellular and developmental events that lead to this wiring specificity. We find that, before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. We propose instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites.

Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development.

The tactile bristles of the fly comprise four cells that originate from a single precursor cell through a fixed lineage. The gene tramtrack (ttk) plays a crucial role in defining the fates of these cells. Here we analyse the normal pattern of expression of ttk, as well as the effect of ttk overexpression at different steps of the lineage. We show that ttk is never expressed in cells having a neural potential, and that in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. Furthermore, this expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. Our results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice.