Intracellular trafficking of Notch orchestrates temporal dynamics of Notch activity in the fly brain.

While Delta non-autonomously activates Notch in neighboring cells, it autonomously inactivates Notch through cis-inhibition, the molecular mechanism and biological roles of which remain elusive. The wave of differentiation in the Drosophila brain, the 'proneural wave', is an excellent model for studying Notch signaling in vivo. Here, we show that strong nonlinearity in cis-inhibition reproduces the second peak of Notch activity behind the proneural wave in silico. Based on this, we demonstrate that Delta expression induces a quick degradation of Notch in late endosomes and the formation of the twin peaks of Notch activity in vivo. Indeed, the amount of Notch is upregulated and the twin peaks are fused forming a single peak when the function of Delta or late endosomes is compromised. Additionally, we show that the second Notch peak behind the wavefront controls neurogenesis. Thus, intracellular trafficking of Notch orchestrates the temporal dynamics of Notch activity and the temporal patterning of neurogenesis.

DWnt4 and DWnt10 Regulate Morphogenesis and Arrangement of Columnar Units via Fz2/PCP Signaling in the Drosophila Brain.

Columns are structural and functional units of the brain. However, the mechanism of column formation remains unclear. The medulla of the fly visual center shares features with the mammalian cerebral cortex, such as columnar and layered structures, and provides a good opportunity to study the mechanisms of column formation. Column formation is initiated by three core neurons in the medulla, namely, Mi1, R8, and R7. The proper orientation of neurons is required for the orientation and arrangement of multiple columns. Their orientations may be under the control of planar cell polarity (PCP) signaling, because it is known to regulate the orientation of cells in two-dimensional tissue structures. In this study, we demonstrate that the ligands DWnt4 and DWnt10 expressed specifically in the ventral medulla and dorsal medulla, respectively, globally regulate the columnar arrangement and orientation of Mi1 and R8 terminals through Fz2/PCP signaling in a three-dimensional space.

Dscam1 establishes the columnar units through lineage-dependent repulsion between sister neurons in the fly brain.

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.

N-Cadherin Orchestrates Self-Organization of Neurons within a Columnar Unit in the Drosophila Medulla.

Columnar structure is a basic unit of the brain, but the mechanism underlying its development remains largely unknown. The medulla, the largest ganglion of the Drosophila melanogaster visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. In this study, using N-cadherin (Ncad) as a marker, we reveal the donut-like columnar structures along the 2D layer in the larval medulla that evolves to form three distinct layers in pupal development. Column formation is initiated by three core neurons, R8, R7, and Mi1, which establish distinct concentric domains within a column. We demonstrate that Ncad-dependent relative adhesiveness of the core columnar neurons regulates their relative location within a column along a 2D layer in the larval medulla according to the differential adhesion hypothesis. We also propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.SIGNIFICANCE STATEMENT The columnar structure is a basic unit of the brain, but its developmental mechanism remains unknown. The medulla, the largest ganglion of the fly visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. We reveal that column formation is initiated by three core neurons that establish distinct concentric domains within a column. We demonstrate the in vivo evidence of N-cadherin-dependent differential adhesion among the core columnar neurons within a column along a 2D layer in the larval medulla. The 2D larval columns evolve to form three distinct layers in the pupal medulla. We propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns.

Netrin Signaling Defines the Regional Border in the Drosophila Visual Center.

The brain consists of distinct domains defined by sharp borders. So far, the mechanisms of compartmentalization of developing tissues include cell adhesion, cell repulsion, and cortical tension. These mechanisms are tightly related to molecular machineries at the cell membrane. However, we and others demonstrated that Slit, a chemorepellent, is required to establish the borders in the fly brain. Here, we demonstrate that Netrin, a classic guidance molecule, is also involved in the compartmental subdivision in the fly brain. In Netrin mutants, many cells are intermingled with cells from the adjacent ganglia penetrating the ganglion borders, resulting in disorganized compartmental subdivisions. How do these guidance molecules regulate the compartmentalization? Our mathematical model demonstrates that a simple combination of known guidance properties of Slit and Netrin is sufficient to explain their roles in boundary formation. Our results suggest that Netrin indeed regulates boundary formation in combination with Slit in vivo.

Wnt Signaling Specifies Anteroposterior Progenitor Zone Identity in the Drosophila Visual Center.

UNLABELLED: During brain development, various types of neuronal populations are produced from different progenitor pools to produce neuronal diversity that is sufficient to establish functional neuronal circuits. However, the molecular mechanisms that specify the identity of each progenitor pool remain obscure. Here, we show that Wnt signaling is essential for the specification of the identity of posterior progenitor pools in the Drosophila visual center. In the medulla, the largest component of the visual center, different types of neurons are produced from two progenitor pools: the outer proliferation center (OPC) and glial precursor cells (GPCs; also known as tips of the OPC). We found that OPC-type neurons are produced from the GPCs at the expense of GPC-type neurons when Wnt signaling is suppressed in the GPCs. In contrast, GPC-type neurons are ectopically induced when Wnt signaling is ectopically activated in the OPC. These results suggest that Wnt signaling is necessary and sufficient for the specification of the progenitor pool identity. We also found that Homothorax (Hth), which is temporally expressed in the OPC, is ectopically induced in the GPCs by suppression of Wnt signaling and that ectopic induction of Hth phenocopies the suppression of Wnt signaling in the GPCs. Thus, Wnt signaling is involved in regionalization of the fly visual center through the specification of the progenitor pool located posterior to the medulla by suppressing Hth expression. SIGNIFICANCE STATEMENT: Brain consists of considerably diverse neurons of different origins. In mammalian brain, excitatory and inhibitory neurons derive from the dorsal and ventral telencephalon, respectively. Multiple progenitor pools also contribute to the neuronal diversity in fly brain. However, it has been unclear how differences between these progenitor pools are established. Here, we show that Wnt signaling, an evolutionarily conserved signaling, is involved in the process that establishes the differences between these progenitor pools. Because ß-catenin signaling, which is under the control of Wnt ligands, specifies progenitor pool identity in the developing mammalian thalamus, Wnt signaling-mediated specification of progenitor pool identity may be conserved in insect and mammalian brains.

Formation of Neuronal Circuits by Interactions between Neuronal Populations Derived from Different Origins in the Drosophila Visual Center.

A wide variety of neurons, including populations derived from different origins, are precisely arranged and correctly connected with their partner to establish a functional neural circuit during brain development. The molecular mechanisms that orchestrate the production and arrangement of these neurons have been obscure. Here, we demonstrate that cell-cell interactions play an important role in establishing the arrangement of neurons of different origins in the Drosophila visual center. Specific types of neurons born outside the medulla primordium migrate tangentially into the developing medulla cortex. During their tangential migration, these neurons express the repellent ligand Slit, and the two layers that the neurons intercalate between express the receptors Robo2 and Robo3. Genetic analysis suggests that Slit-Robo signaling may control the positioning of the layer cells or their processes to form a path for migration. Our results suggest that conserved axon guidance signaling is involved in the interactions between neurons of different origins during brain development.

Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent.

Super-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we report an optimized optical clearing agent for high-resolution fluorescence imaging (SeeDB2). SeeDB2 matches the refractive indices of fixed tissues to that of immersion oil (1.518), thus minimizing both light scattering and spherical aberrations. During the clearing process, fine morphology and fluorescent proteins were highly preserved. SeeDB2 enabled super-resolution microscopy of various tissue samples up to a depth of >100 µm, an order of magnitude deeper than previously possible under standard mounting conditions. Using this approach, we demonstrate accumulation of inhibitory synapses on spine heads in NMDA-receptor-deficient neurons. In the fly medulla, we found unexpected heterogeneity in axon bouton orientations among Mi1 neurons, a part of the motion detection circuitry. Thus, volumetric super-resolution microscopy of cleared tissues is a powerful strategy in connectomic studies at synaptic levels.

eyeless/Pax6 controls the production of glial cells in the visual center of Drosophila melanogaster.

Pax6 is known as a neurogenic factor in the development of the central nervous system and regulates proliferation of neuronal progenitor cells and promotes neuronal differentiation. In addition to neurogenesis, Pax6 is also involved in the specification and maturation of glial cells. Here, we show that Eyeless (Ey), Drosophila homolog of Pax6, regulates the production of glial cells in the brain. In the developing fly visual center, the production of neurons and glial cells are controlled by the temporal transcription factors that are sequentially expressed in neuroblasts (NBs). Among them, NBs of the last temporal window produce astrocyte-like glial cells. Ey is strongly expressed in the middle aged NBs, whose temporal window is earlier compared with glia producing older NBs. Weak Ey expression is also detected in the glia producing NBs. Our results suggest that Ey expression in the middle aged NBs indirectly control gliogenesis from the oldest NBs by regulating other temporal transcription factors. Additionally, weak Ey expression in the NBs of last temporal window may directly control gliogenesis. Ey is also expressed in neurons produced from the NBs of Ey-positive temporal window. Interestingly, neuron-specific overexpression of Ey causes significant increase in glial cells suggesting that neuronal expression of Ey may also contribute to gliogenesis. Thus, Pax6-dependent regulation of astrocyte-like glial development is conserved throughout the animal kingdom.

A temporal mechanism that produces neuronal diversity in the Drosophila visual center.

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf), correspond to types of medulla neurons. Here, we examine the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, we searched for transcription factors that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates (Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)). The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. Our results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequentially expressed in NBs.

Brain-specific-homeobox is required for the specification of neuronal types in the Drosophila optic lobe.

The Drosophila optic lobe comprises a wide variety of neurons forming laminar and columnar structures similar to the mammalian brain. The Drosophila optic lobe may provide an excellent model to investigate various processes of brain development. However, it is poorly understood how neuronal specification is regulated in the optic lobe to form a complicated structure. Here we show that the Brain-specific-homeobox (Bsh) protein, which is expressed in the lamina and medulla ganglia, is involved in specifying neuronal identity. Bsh is expressed in L4 and L5 lamina neurons and in Mi1 medulla neurons. Analyses of loss-of-function and gain-of-function clones suggest that Bsh is required and largely sufficient for Mi1 specification in the medulla and L4 specification in the lamina. Additionally, Bsh is at least required for L5 specification. In the absence of Bsh, L5 is transformed into glial cells.

Concentric zones, cell migration and neuronal circuits in the Drosophila visual center.

The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. Here, we describe the key features of the developing medulla and report novel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.