Javier Morante

Dissection of the Peripheral Motion Channel in the Visual System of Drosophila melanogaster

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

The Color-Vision Circuit in the Medulla of Drosophila

BACKGROUND Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. RESULTS We reconstruct the neural network in the medulla, focusing on neurons likely to be involved in processing color vision. We identify the full complement of neurons in the medulla, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. We also examine third-order neurons and local neurons that likely modulate information from second-order neurons. Finally, we present highly specific tools that will allow us to functionally manipulate the network and test both activity and behavior. CONCLUSIONS This precise characterization of the medulla circuitry will allow us to understand how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates.

Temporal patterning of Drosophila medulla neuroblasts controls neural fates

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. Here we describe how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors—Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless—are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. We provide evidence that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, our work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts.

Perlecan controls neurogenesis in the developing telencephalon

BackgroundPerlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact.ResultsBrain atrophy in the absence of perlecan started in the ventral forebrain and extended to lateral and dorsal parts of the cortex in the following stages. First, the subpallial forebrain developed poorly in early perlecan-null embryos, because of a reduced cell proliferation: the number of cells in mitosis decreased since the early stages of development. This reduction resulted in a decreased tangential migration of interneurons to the cerebral cortex. Concomitant with the early hypoplasia observed in the medial ganglionic eminences, Sonic Hedgehog signal decreased in the perlecan-null floor plate basal lamina at E12.5. Second, neurogenesis in the pallial neuroepithelium was affected in perlecan deficient embryos. We found reductions of nearly 50% in the number of cells exiting the cell cycle at E12–E13. The labeling index, which was normal at this age, significantly decreased with advancing corticogenesis. Moreover, nestin+ or PCNA+ progenitors increased since E14.5, reaching up to about 150% of the proportion of PCNA+ cells in the wild-type at E17.5. Thus, labeling index reduction together with increased progenitor population, suggests that atrophy is the result of altered cell cycle progression in the cortical progenitors. Accordingly, less neurons populated the cortical plate and subplate of perlecan-null neocortex, as seen with the neuronal markers β-tubulin and Tbr1.ConclusionAs a component of the basal lamina, perlecan both maintains this structure and controls the response of the neuroepithelium to growth factors. Less mitotic cells in the early medial ganglionic eminences, and impaired cell cycle progression in the late neocortex, suggests insufficient recruitment and signaling by neurogenic morphogens, such as SHH or FGF2.

A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3

Brain keeps body size and shape in check Animal systems show amazing left-right symmetry—think of how our legs or arms, or the legs or wings of an insect, are matched in size and shape. Environmental insults and growth defects can challenge these developmental programs. In order to limit the resultant variation, juvenile organisms buffer variability through homeostatic mechanisms, so that the correct final size is attained. Vallejo et al. report that the Drosophila brain mediates such homeostatic control via an insulin-like peptide Dilp8 binding to the relaxin hormone receptor Lgr3. Lgr3 neurons distribute this information to other neuronal populations to adjust the hormones ecdysone, insulin, and juvenile hormone in a manner that stabilizes body and organ size. Science, this issue p. 10.1126/science.aac6767 Drosophila Lgr3 defines a neural circuit for homeostatic regulation of body size. INTRODUCTION Animals have a remarkable capacity to maintain a constant size, even in the face of genetic and environmental perturbations. Size imperfections and asymmetries have an effect on fitness, potentially decreasing competitiveness, survival, and reproductive success. Therefore, immature animals must employ homeostatic mechanisms to counteract substantial size variations and withstand developmental growth perturbations caused by genetic errors, disease, environmental factors, or injury. Such mechanisms ensure that, despite inevitable variations, the appropriate final body size is attained. A better understanding of homeostatic size maintenance will afford insights into normal organ and organismal size control, as well as the developmental origin of anomalous random left-right asymmetries. RATIONALE The Drosophila insulin-like peptide Dilp8 has been shown to mediate homeostatic regulation. When growth is disturbed, Dilp8 is strongly activated and sexual maturation is postponed until the affected elements are recomposed; simultaneously, the growth of other organs is retarded during this process. This compensatory mechanism allows the growth of the affected tissues to catch up. It maintains the synchrony between organs so that the animals achieve the correct size, preserving proportionality and bilateral symmetry. However, the Dilp8 receptor and its site of action remain uncharacterized. RESULTS We found that Dilp8 binds to andactivates the relaxin leucine-rich repeat–containing G protein–coupled receptor Lgr3 to mediate homeostatic control through a pathway dependent on adenosine 3′,5′-monophosphate. Larvae that lack lgr3 in neurons alone do not respond to Dilp8, indicating that the homeostatic system is centered in the brain. Dilp8 delays reproductive maturation by suppressing the neurons releasing the prothoracicotropic hormone (PTTH), which projects to the prothoracic gland and regulates ecdysone production for growth termination. However, this modulation alone is insufficient to adjust growth and stabilize body size. We show that Dilp8-Lgr3 balances growth against the extended growth period by dampening the production of dilp3 and dilp5 by insulin-producing cells (IPCs) in the brain and inhibiting synthesis of the juvenile hormone (JH). We also identify two pairs of dorsomedial neurons in the pars intercerebralis that are necessary and sufficient to mediate the effects of Dilp8. Simultaneous detection of pre- and postsynaptic markers revealed that the Lgr3 neurons mediating this homeostatic control have extensive axonal arborizations. Genetic and GRASP (GFP reconstitution across synaptic partners) analyses demonstrate that these neurons are connected to both the IPCs and PTTH neurons critical for adjusting growth and maturation rate, respectively. Thus, through their extensive axonal arborizations, Lgr3 neurons function like a “neuronal hub”: They route peripheral information about growth status to other neuronal populations, thereby synchronizing damaged tissues and other (undamaged) ones and allocating additional development time so that each organ attains the correct size and maintains proportionality and symmetry. CONCLUSION We identified the relaxin receptor Lgr3 as a Dilp8 receptor and defined a brain circuit for homeostatic control of organismal and organ size in the face of perturbations. Lgr3 neurons that respond to Dilp8 signals directly input on the insulin-producing cells and the PTTH-producing neurons. As Lgr3 outputs, the modulation of these neuronal populations according to Dilp8 levels is critical to delay maturation and promote growth compensation in a manner that stabilizes body size. Without adequate Dilp8-Lgr3 signaling, the brain is incapable of stabilizing size between the distinct body parts, and we see left-right asymmetries and size variations that are greater than usual, reflecting developmental instability. Dilp8-Lgr3 neural circuit and outputs for body-size homeostasis. The brain detects growth status and anomalies via Dilp8 activation of the Lgr3 receptor in a pair of symmetric neurons. These neurons distribute this information to IPCs and PTTH neurons, which then trigger the hormonal responses that regulate size. Without Dilp8-Lgr3 homeostasis, the brain cannot correct variation, and identical body parts can display imperfect symmetry and size. Body-size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Drosophila insulin-like peptide Dilp8 is responsive to growth perturbations and controls homeostatic mechanisms that coordinately adjust growth and maturation to maintain size within the normal range. Here we show that Lgr3 is a Dilp8 receptor. Through the use of functional and adenosine 3′,5′-monophosphate assays, we defined a pair of Lgr3 neurons that mediate homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and green fluorescent protein reconstitution across synaptic partners show that these neurons connect with the insulin-producing cells and prothoracicotropic hormone–producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size.

Cell migration in Drosophila optic lobe neurons is controlled by eyeless/Pax6

In the developing Drosophila optic lobe, eyeless, apterous and distal-less, three genes that encode transcription factors with important functions during development, are expressed in broad subsets of medulla neurons. Medulla cortex cells follow two patterns of cell movements to acquire their final position: first, neurons are arranged in columns below each neuroblast. Then, during pupation, they migrate laterally, intermingling with each other to reach their retinotopic position in the adult optic lobe. eyeless, which encodes a Pax6 transcription factor, is expressed early in progenitors and controls aspects of this cell migration. Its loss in medulla neurons leads to overgrowth and a failure of lateral migration during pupation. These defects in cell migration among medulla cortex cells can be rescued by removing DE-Cadherin. Thus, eyeless links neurogenesis and neuronal migration.

Dissection and Staining of Drosophila Optic Lobes at Different Stages of Development

The Drosophila visual system is composed of the retina and the optic lobes, which are the ganglia where photoreceptors project and initial processing of visual inputs occur. This protocol outlines procedures for dissecting the optic lobes from Drosophila larvae, pupae, and adults. It also describes methods for visualizing the anatomy of brain neural circuits by stainingwith fluorescent secondary antibodies and primary antibodies specific for various neuronal populations and architectural features.