Multilevel regulation of the glass locus during Drosophila eye development.

Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.

Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode.

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.

Circadian and Genetic Modulation of Visually-Guided Navigation in Drosophila Larvae.

Organisms possess an endogenous molecular clock which enables them to adapt to environmental rhythms and to synchronize their metabolism and behavior accordingly. Circadian rhythms govern daily oscillations in numerous physiological processes, and the underlying molecular components have been extensively described from fruit flies to mammals. Drosophila larvae have relatively simple nervous system compared to their adult counterparts, yet they both share a homologous molecular clock with mammals, governed by interlocking transcriptional feedback loops with highly conserved constituents. Larvae exhibit a robust light avoidance behavior, presumably enabling them to avoid predators and desiccation, and DNA-damage by exposure to ultraviolet light, hence are crucial for survival. Circadian rhythm has been shown to alter light-dark preference, however it remains unclear how distinct behavioral strategies are modulated by circadian time. To address this question, we investigate the larval visual navigation at different time-points of the day employing a computer-based tracking system, which allows detailed evaluation of distinct navigation strategies. Our results show that due to circadian modulation specific to light information processing, larvae avoid light most efficiently at dawn, and a functioning clock mechanism at both molecular and neuro-signaling level is necessary to conduct this modulation.

Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit.

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on investigating the physiological properties and behavioral relevance of larval visual interneurons. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Gαo signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system.

Two Pairs of Drosophila Central Brain Neurons Mediate Larval Navigational Strategies Based on Temporal Light Information Processing.

Some animals are attracted by sun light, others are highly repulsed by it. Especially for slowly moving animals, such as Drosophila larvae, direct sunlight may be perceived as noxious stimulus as it increases the risk of desiccation, DNA-damaging by UV-light and exposure to predators. For several reasons, model organisms like Drosophila larvae are well-suited for investigating how light cues are translated into an appropriate behavioral output. First, many of the genetic tools, which were created for use in adult fruit flies, work also in larvae. Second, the lower number of cells in Drosophila larvae compared to adults makes this system adequate for reconstructing neural circuits. Third, the relatively simple behavioral repertoire of larvae facilitates the study of basic functions like navigation with regards to light. Larvae navigate robustly away from a light source by the use of several sophisticated behavioral strategies which are based on temporal or spatial information processing. Two central brain neurons, the NP394-neurons, are highly important for larval light avoidance. It was even reported that these cells seem to play a functional role in a putative larval light preference switch right before pupation. However, the exact function of the NP394-neurons in light navigation remains unknown. We here show that the functional role of NP394-neurons in larval light navigation is restricted to behaviors based on temporal information processing, but not for spatial navigation.

GABA immunostaining in the central complex of dicondylian insects.

The central complex is a group of midline-crossing neuropils in the insect brain involved in head direction coding, sky compass navigation, and spatial visual memory. To compare the neuroarchitecture and neurochemistry of the central complex in insects that differ in locomotion, ways of orientation, time of activity (diurnal, nocturnal), and evolutionary history, we studied the distribution of γ-aminobutyric acid (GABA) immunostaining in the central complex of 29 species, ranging from Zygentoma to Diptera. In all species, the lower division of the central body was densely innervated by GABA-immunoreactive tangential neurons. These neurons had additional arborizations in the bulb, a distinct region of synaptic complexes in the lateral complex, and somata in a cell cluster mediodorsally to the antennal lobe. Differences in the appearance of GABA immunostaining in the lower division of the central body corresponded to differences in neuropil architecture, such as transformation of the lower division into a toroid in certain Diptera and Heteroptera. In nearly all species two additional systems of tangential neuron of the upper division of the central body were GABA-immunoreactive. One of these systems diffusely invaded a superior layer, while the second system showed fan-like projections in an inferior layer. Sparse immunostaining in the protocerebral bridge was detected in cockroaches, a cricket, and two hemipteran species. The data show that three systems of GABA-immunoreactive tangential neurons of the central body are highly conserved and suggest that the layered organization of the upper division of the central body is, likewise, largely maintained from basal to advanced species.

Dedicated photoreceptor pathways in Drosophila larvae mediate navigation by processing either spatial or temporal cues.

To integrate changing environmental cues with high spatial and temporal resolution is critical for animals to orient themselves. Drosophila larvae show an effective motor program to navigate away from light sources. How the larval visual circuit processes light stimuli to control navigational decision remains unknown. The larval visual system is composed of two sensory input channels, Rhodopsin5 (Rh5) and Rhodopsin6 (Rh6) expressing photoreceptors (PRs). We here characterize how spatial and temporal information are used to control navigation. Rh6-PRs are required to perceive temporal changes of light intensity during head casts, while Rh5-PRs are required to control behaviors that allow navigation in response to spatial cues. We characterize how distinct behaviors are modulated and identify parallel acting and converging features of the visual circuit. Functional features of the larval visual circuit highlight the principle of how early in a sensory circuit distinct behaviors may be computed by partly overlapping sensory pathways.

Age- and Wavelength-Dependency of Drosophila Larval Phototaxis and Behavioral Responses to Natural Lighting Conditions.

Animals use various environmental cues as key determinant for their behavioral decisions. Visual systems are hereby responsible to translate light-dependent stimuli into neuronal encoded information. Even though the larval eyes of the fruit fly Drosophila melanogaster are comparably simple, they comprise two types of photoreceptor neurons (PRs), defined by different Rhodopsin genes expressed. Recent findings support that for light avoidance Rhodopsin5 (Rh5) expressing photoreceptors are crucial, while Rhodopsin6 (Rh6) expressing photoreceptors are dispensable under laboratory conditions. However, it remains debated how animals change light preference during larval live. We show that larval negative phototaxis is age-independent as it persists in larvae from foraging to wandering developmental stages. Moreover, if spectrally different Rhodopsins are employed for the detection of different wavelength of light remains unexplored. We found that negative phototaxis can be elicit by light with wavelengths ranging from ultraviolet (UV) to green. This behavior is uniquely mediated by Rh5 expressing photoreceptors, and therefore suggest that this photoreceptor-type is able to perceive UV up to green light. In contrast to laboratory our field experiments revealed that Drosophila larvae uses both types of photoreceptors under natural lighting conditions. All our results, demonstrate that Drosophila larval eyes mediate avoidance of light stimuli with a wide, ecological relevant range of quantity (intensities) and quality (wavelengths). Thus, the two photoreceptor-types appear more likely to play a role in different aspects of phototaxis under natural lighting conditions, rather than color discrimination.

Successive requirement of Glass and Hazy for photoreceptor specification and maintenance in Drosophila.

Development of the insect compound eye requires a highly controlled interplay between transcription factors. However, the genetic mechanisms that link early eye field specification to photoreceptor terminal differentiation and fate maintenance remain largely unknown. Here, we decipher the function of 2 transcription factors, Glass and Hazy, which play a central role during photoreceptor development. The regulatory interactions between Glass and Hazy suggest that they function together in a coherent feed-forward loop in all types of Drosophila photoreceptors. While the glass mutant eye lacks the expression of virtually all photoreceptor genes, young hazy mutants correctly express most phototransduction genes. Interestingly, the expression of these genes is drastically reduced in old hazy mutants. This age-dependent loss of the phototransduction cascade correlates with a loss of phototaxis in old hazy mutant flies. We conclude that Glass can either directly or indirectly initiate the expression of most phototransduction proteins in a Hazy-independent manner, and that Hazy is mainly required for the maintenance of functional photoreceptors in adult flies.