Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, we used the mosaic analysis with a repressible cell marker (MARCM) technique to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. We found that individual neurons in the peripheral nervous system (PNS) developed clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster developed a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis revealed that Flamingo did not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limited the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype was partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Our molecular analysis also indicated that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons.     

Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons

To cover the receptive field completely but without redundancy, neurons of certain functional groups exhibit tiling of their dendrites via dendritic repulsion. Here we show that two evolutionarily conserved proteins, the Tricornered (Trc) kinase and Furry (Fry), are essential for tiling and branching control of Drosophila sensory neuron dendrites. Dendrites of fry and trc mutants display excessive terminal branching and fail to avoid homologous dendritic branches, resulting in significant overlap of the dendritic fields. Trc control of dendritic branching involves regulation of RacGTPase, a pathway distinct from the action of Trc in tiling. Timelapse analysis further reveals a specific loss of the ability of growing dendrites to turn away from nearby dendritic branches in fry mutants, suggestive of a defect in like-repels-like avoidance. Thus, the Trc/Fry signaling pathway plays a key role in patterning dendritic fields by promoting avoidance between homologous dendrites as well as by limiting dendritic branching.     

Different levels of the homeodomain protein cut regulate distinct dendrite branching patterns of Drosophila multidendritic neurons

Functionally similar neurons can share common dendrite morphology, but how different neurons are directed into similar forms is not understood. Here, we show in embryonic and larval development that the level of Cut immunoreactivity in individual dendritic arborization (da) sensory neurons correlates with distinct patterns of terminal dendrites: high Cut in neurons with extensive unbranched terminal protrusions (dendritic spikes), medium levels in neurons with expansive and complex arbors, and low or nondetectable Cut in neurons with simple dendrites. Loss of Cut reduced dendrite growth and class-specific terminal branching, whereas overexpression of Cut or a mammalian homolog in lower-level neurons resulted in transformations toward the branch morphology of high-Cut neurons. Thus, different levels of a homeoprotein can regulate distinct patterns of dendrite branching.     

Branching-pattern analysis of the dendritic arborization in the thalamic nuclei of the rat brain

We investigated the dendritic patterns of rapid Golgi-impregnated, highly similar multipolar neurons from two functionally different thalamic regions of the rat brain: two dorsal nuclei (the nucleus laterodorsalis thalami, pars dorsomedialis and the nucleus laterodorsalis thalami, pars ventrolateralis), and two ventral nuclei (the nucleus ventrolateralis thalami and the nucleus ventromedialis thalami). The analysis involved conventional morphometric parameters (height and size) and a new parameter derived from graph theory, the relative imbalance (RI), derived from the branching patterns of the dendrites, which permits quantitative characterization of the dendritic arborization of a neuron. On this basis, neurons can be grouped into three fundamentally different types: type A, or highly-polarized (imbalanced) neurons (RI values close to 1); type B, or medium-polarized neurons (RI values around 0.5); and type C, or balanced neurons with low polarization (RI values close to 0). The orientations of the dendritic arbor, and thus the receptive fields, of the dorsal and ventral thalamic neurons, were mutually perpendicular. The H and S values indicated that the neurons in the dorsal and ventral thalamic nuclei differed significantly. However, their RI values demonstrated that they were similar neurons of type B. Our data reveal that 1 ) the dendritic arbor cannot be reliably characterized purely on the basis of height and size, and 2) RI is a valuable morphometric parameter that identifies the true nature of the dendritic arborization.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.     

Statistical evaluation of dendritic growth models

A mathematical model (Kliemann, W. 1987.Bull. math. Biol. 49, 135–152.) that predicts the quantitative branching pattern of dendritic tree was evaluated using the apical and basal dendrites of rat hippocampal neurons. The Wald statistics for χ²-test was developed for the branching pattern of dendritic trees and for the distribution of the maximal order of the tree. Using this statistic, we obtained a reasonable, but not excellent, fit of the mathematical model for the dendritic data. The model's predictability of branching patterns was greatly enhanced by replacing one of the assumptions used for the original model “splitting of branches for all dendritic orders is stochastically independent”, with a new assumption “branches are more likely to split in areas where there is already a high density of branches”. The modified model delivered an excellent fit for basal dendrites and for the apical dendrites of hippocampal neurons from young rats (30–34 days postpartum). This indicates that for these cells the development of dendritic patterns is the result of a purely random and a systematic component, where the latter one depends on the density of dendritic branches in the brain area considered. For apical dendrites there is a trend towards decreasing pattern predictability with increasing age. This appears to reflect the late arrival of afferents and subsequent synaptogenesis proximal on the apical dendritic tree of hippocampal neurons.     

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is due to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field.     

Development of the Drosophila mushroom bodies: elaboration,remodeling and spatial organization of dendrites in the calyx

One Drosophila mushroom body (MB) is derived from four indistinguishable cell lineages, development of which involves sequential generation of multiple distinct types of neurons. Differential labeling of distinct MB clones reveals that MB dendrites of different clonal origins are well mixed at the larval stage but become restricted to distinct spaces in adults. Interestingly, a small dendritic domain in the adult MB calyx remains as a fourfold structure that, similar to the entire larval calyx, receives dendritic inputs from all four MB clones. Mosaic analysis of single neurons demonstrates that MB neurons, which are born around pupal formation, acquire unique dendritic branching patterns and consistently project their primary dendrites into the fourfold dendritic domain. Distinct dendrite distribution patterns are also observed for other subtypes of MB neurons. In addition, pruning of larval dendrites during metamorphosis allows for establishment of adult-specific dendrite elaboration/distribution patterns. Taken together, subregional differences exist in the adult Drosophila MB calyx, where processing and integration of distinct types of sensory information begin.     

A Golgi study of anterior dorsal ventricular ridge in the alligator,Alligator mississippiensis

The anterior dorsal ventricular ridge was examined in the American alligator, Alligator mississippiensis, with cresyl violet and Golgi-Kopsch preparations. Four cytoarchitectonic areas (lateral dorsolateral, medial dorsolateral, intermediolateral, and lateral) can be distinguished by variations in the density of neurons and their tendency to form clusters of neurons with apposed somata. Three distinct types of neurons are distributed throughout these areas. Juxtaependymal neurons lie near the ventricular surface and have dendritic fields paralleling the ependymal layer. Their dendrites bear a moderate density of spines. Spiny neurons all have stellate shaped dendritic fields and dendrites that bear dendritic spines, but they vary greatly in the density of spines and the thickness of their dendrites. A very spiny variety has a high spine density and relatively thick dendrites. A moderately spiny variety has a moderate spine density and thin dendrites. A sparsely spiny variety has a low spine density and thick dendrites. Aspiny neurons have a relatively large number of dendrites that form a gnarled dendritic field and lack spines.     

Dendritic projections of different types of octopaminergic unpaired median neurons in the locust metathoracic ganglion

Octopaminergic dorsal unpaired median (DUM) neurons of locust thoracic ganglia are important components of motor networks and are divided into various sub-populations. We have examined individually stained metathoracic DUM neurons, their dendritic projection patterns, and their relationship to specific architectural features of the metathoracic ganglion, such as longitudinal tracts, transverse commissures, and well-defined sensory neuropils. The detailed branching patterns of individually characterized DUM neurons of various types were analyzed in vibratome sections in which architectural features were revealed by using antibodies against tubulin and synapsin. Whereas DUM3,4,5 and DUM5 neurons (the group innervating leg and "non-wing-power" muscles) had many ventral and dorsal branches, DUM1 and DUM3,4 neurons (innervating "wing-power" muscles) branched extensively only in dorsal areas. The structure of DUM3 neurons differed markedly from that of the other DUM neurons examined in that they sent branches into dorsal areas and had differently structured side branches that mostly extended laterally. The differences between the branching patterns of these neurons were quantified by using currently available new reconstruction algorithms. These structural differences between the various classes of DUM neurons corresponded to differences in their function and biophysical properties.     

Tiling of the Drosophila epidermis by multidendritic sensory neurons

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.     

A new approach to reconstruction models of dendritic branching patterns

Quantitative models for characterising the detailed branching patterns of dendritic trees aim to explain these patterns either in terms of growth models based on principles of dendritic development or reconstruction models that describe an existing structure by means of a canonical set of elementary properties of dendritic morphology, which when incorporated into an algorithmic procedure will generate samples of dendrites that are statistically indistinguishable in both canonical and emergent features from those of the original sample of real neurons. This article introduces a conceptually new approach to reconstruction modelling based on the single assumption that dendritic segments are built from sequences of units of constant diameter, and that the distribution of the lengths of units of similar diameter is independent of location within a dendritic tree. This assumption in combination with non-parametric methods for estimating univariate and multivariate probability densities leads to an algorithm that significantly reduces the number of basic parameters required to simulate dendritic morphology. It is not necessary to distinguish between stem and terminal segments or to specify daughter branch ratios or dendritic taper. The procedure of sampling probability densities conditioned on local morphological features eliminates the need, for example, to specify daughter branch ratios and dendritic taper since these emerge naturally as a consequence of the conditioning process. Thus several basic parameters of previous reconstruction algorithms become emergent parameters of the new reconstruction process. The new procedure was applied successfully to a sample of 51 interneurons from lamina II/III of the spinal dorsal horn.     

The torus semicircularis in a gekkonid lizard

The cytoarchitecture and neuromorphology of the torus semicircularis in the tokay gecko, Gekko gecko, were examined in Nissl-stained, fiber-stained, and Golgi-impregnated tissues. From a superficial position, the torus semicircularis extends rostrally under the caudal half of the optic tectum. Caudally, the two tori abut upon one another; rostrally, they diverge. The torus semicircularis consists of central, laminar, and superficial nuclei. The central nucleus consists of fusiform, spherical and triangular neurons. Their dendrites are highly branched, with numerous dendritic spines, and are oriented mediolaterally, dorsoventrally, and rostrocaudally. Fusiform and spherical neurons display two dendritic patterns: “single axis,” ramifying in one axis, and “dual axis,” exhibiting higher-order branches perpendicular to the primary dendrites. Triangular neurons exhibit a “radiate” dendritic pattern. In the rostral half of the torus semicircularis, the laminar nucleus caps the central nucleus. The laminar nucleus encircles the central nucleus in the caudal torus semicircularis. The neurons of the laminar nucleus have dendritic arrays oriented parallel to the border of the central nucleus. These dendrites exhibit a paucity of dendritic spines and higher-order branches. Fusiform and spherical neurons exhibit “single axis” and “dual axis” dendritic patterns. Triangular neurons display “radiate” patterns. The caudal superficial nucleus lies dorsal and dorsolateral to the central nucleus. The superficial nucleus is sparsely populated by small fusiform and spherical neurons with moderately branched dendrites and moderate numbers of dendritic spines. These neurons display “single axis” (fusiform neurons) as well as “dual axis” and “radiate” (spherical neurons) dendritic patterns. They are oriented either parallel to or perpendicular to the boundary of the laminar nucleus.     

The ankyrin repeat protein Diego mediates Frizzled-dependent planar polarization.

During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. This work identifies a tissue polarity gene, diego, that comprises six ankyrin repeats and colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. Our data suggest that Frizzled acts through Diego to promote local clustering of Flamingo, and that clustering of Diego and Flamingo in one cell nonautonomously propagates to others.     

The shape and arrangement of the cholinergic neurons in the rabbit retina

The acetylcholine-synthesizing neurons of the rabbit retina were selectively stained by intraocular injection of the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI). Retinas were then isolated from the eye, fixed for 10-30 min with 4% paraformaldehyde, and mounted flat on the stage of a fluorescence microscope. The acetylcholine-synthesizing cells were penetrated under visual control by microelectrodes filled with lucifer yellow CH. When the dye was electrophoretically injected into the cells, complete filling of their dendrites often occurred. Cells were successfully injected as long as one month after fixation of the tissue. Complete or nearly complete filling of 281 cells was accomplished, at retinal locations systematically covering the retinal surface. The cells stained with DAPI were found to form a single morphological population. They have two to seven primary dendrites, which branch repeatedly within a narrow plane and form a round or slightly oval dendritic tree. The branching becomes very fine for the distal one third of the dendritic tree, and the dendrites there are studded with small swellings. The distal dendritic tree lies mainly within one of the two thin strata of the inner plexiform layer where acetylcholine is present. The shape and size of the dendritic tree are continuously graded across the retina, the dendritic tree is narrower and the branching denser in the central retina, wider and sparser in the periphery. From knowledge of the population density and the shape of the neurons, one can reconstruct the array of dendrites that exists within the inner plexiform layer. The overlap of the dendritic fields is an order of magnitude greater than of any other retinal neuron previously described. Because the cells not only overlap widely but branch quite profusely, a very dense plexus of cholinergic dendrites is created.     

Volumetric and golgi studies of the corpus geniculatum laterale pars dorsalis of Alticola stoliczkanus barakshin and Alticola argentatus semicanus

The dorsal lateral geniculate bodies (dLGB) in Alticola stoliczkanus barakshin, the Gobi-Altai-Mountain vole, and in Alticola argentatus semicanus, the silver grey mountain vole, and investigated using the nissl- and the golgi method. The geniculo-cortico-relay neurons (GCR neurons) of both species have 5 primary dendrites (D1), a dendritic field of about 100 micron, about 17 free dendritic distal parts (FDE), 10 branching points (VZP) and a average of the perikaryon of 10 micron. All tufted neurons are small and topographically distinctly localised. The dLGB's volume of Alticola stoczkanus, barakshin is 0.16 mm3, the dLGB's volume of Alticola argentatus semicanus is 0.23 mm3.