The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis (Kraut, 2001). In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).
Abnormal dendrite and axon outgrowth could originate from defects in the surrounding tissue, defects within the neurons themselves, or defects in both tissues. However, in sequoia mutant embryos, muscle differentiation, cuticle formation, epidermal structure, and trachea formation all appear normal (Gao, 2000). The sequoia mRNA is expressed exclusively in the developing and mature nervous system, consistent with the observed phenotype within the nervous system but no detectable phenotype outside it. sequoia RNA expression begins early (Stage 3/4) but without obvious maternal contribution. sequoia mRNA is expressed in the procephalic neurogenic head region, as well as neuroblasts and their progenitors. Two different Sequoia antisera generated a similar immunostaining pattern that reproduced the mRNA expression pattern. Sequoia is clearly localized to the nuclei in the developing nervous system. It is detected in nuclei of neurons and sheath cells but not the outer support cells in the dorsal cluster. By late stage 16, Sequoia is most abundant in neurons but not detectable in sheath cells (Brenman, 2001).
Given the homology to tramtrack, a gene shown to function in regulating cell fate in the developing nervous system, it was of interest to see whether there might be alterations in cell identity in sequoia mutants. The original Gal4 enhancer trap line, 109(2)80, is expressed in all eight md neurons, including the single bipolar dendrite neuron, the single tracheal innervating neuron, and six dendritic arborization (da) neurons, but not es neurons in the dorsal cluster. Hence, this Gal4 line would label eight neurons in each dorsal cluster in the wild-type embryo. The dorsal cluster in sequoia22 mutants contains 9.5 ± 0.5 GFP-positive neurons. Typically, 9-10 GFP-positive neurons are seen, but it is not unusual to find all 12 neurons in the dorsal cluster labeled with GFP. To further verify the generation of extra md neurons, a different lacZ enhancer trap, E7-2-36, was used that labels all six da neurons and the single bd neuron in the dorsal cluster. Similar results were observed with E7-2-36 as observed with Gal4 line109(2)80 -- extra ß-galactosidase immunostaining neurons at the expense of other es neurons in the cluster (Brenman, 2001).
Since tramtrack has been implicated in the Notch signaling pathway, it was of interest to see whether alteration of Notch activity would change the expression of sequoia. Given that sequoia mRNA expression preceded the formation of neurons, a priori it was hard to predict the relationship between Sequoia protein expression and Notch activity. Antibody staining of Sequoia in Notch mutant embryos revealed that all extra Elav-positive cells produced in the Notch neurogenic phenotype indeed expressed Sequoia. Conversely, overexpressing UAS-Notch with Hairy Gal4 resulted in decreased Elav-positive cells, which were the only cells with detectable Sequoia expression. Thus, the number of Sequoia expressing neurons depends on Notch activity (Brenman, 2001).
Is the sequoia gene product required for normal dendritic morphogenesis in the adult? sequoia mutants are embryonic lethal. To circumvent this problem, sequoia function was examined in the adult by making mitotic clones doubly mutant for sequoia and yellow. On both the notum and scutellum, external microchaetae and macrochaetae were found with yellow hair and socket in these mitotic sequoia loss-of-function mutant clones. There was no cell fate transformation of these external cells, in contrast to tramtrack clones that show loss of hair and socket. Remarkably, in these sequoia mutant clones marked with yellow bristles, the accompanying sensory neurons often show a range of morphological defects. The wild-type es neuron projects a single unbranched dendritic process that innervates the external hair. In sequoia mutant clones, however, some neurons have thick dendrites and laterally protruding processes and other neurons fail to extend dendrites to innervate the accompanying hair (Brenman, 2001).
Sequoia is present in a pan-neural nuclear pattern in the adult head, including photoreceptors. To determine whether sequoia is also required for proper axon morphogenesis in the visual system, the effects of loss of sequoia function in ommatidia were examined. The axon projections from photoreceptor retinal neurons to the brain optic lobe are well characterized; it is easy to identify aberrant axonal projections. Clones of sequoia mutant cells were generated. sequoia mutant ommatidia appear largely indistinguishable from wild-type when viewed by examining the R1-R7 cell numbers and arrangement in toluidine blue-stained tangential sections. This indicates that the photoreceptor cell fate specification is normal. In order to look at the projection of photoreceptor axons, cryostat sections of fly heads homozygous for either the parental the chromosome or sequoia mutant chromosome were stained with mAb24B10, which marks the axons of all photoreceptor cells, R1-R8. The Drosophila photoreceptor neurons project their axons to the optic lobe of the brain, producing a stereotyped retinotopic map. The R1-R6 growth cones terminate in the lamina, forming a dense layer of immunoreactivity, the lamina plexus. The R7 and R8 neurons project their axons through the lamina and instead terminate in the next layer, the medulla. Axons from the various photoreceptors interweave and bundle together, forming cartridges that are particularly evident in the lamina and medulla, where alternating columns of stained axon bundles and unstained areas intersperse. These cartridges in the lamina and medulla are roughly parallel to each other. In sequoia mutant heads, the regular cartridge appearance is either malformed or completely absent, indicative of either failure to extend axons fully or failure to project them appropriately (Brenman, 2001).
Insect dendritic arborization (da) neurons (one of three classes of multiple dendritic neurons) provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. The 15 da neurons in hemisegments A2-A6 are arranged in four clusters (ventral, ventral', lateral and dorsal). The nomenclature for the da neurons identifies their position within one of these four clusters with the prefix v, v', l or d, their status as a da neuron and an alphabetic suffix that orders the cells from ventral to dorsal within each cluster. Three neurons (vpda, v'ada and v'pda) do not conform to this naming scheme. In this study, each neuron has been named according to its typical position within a cluster; however, the primary criterion for identifying each cell is its peripheral dendritic morphology. Four neurons were identified grouped together in a ventral cluster (vdaA-D), one lone ventral neuron (vpda), the previously identified v'ada and v'pda neurons, the two lateral neurons ldaA and ldaB and six dorsal da neurons (ddaA-F) (Grueber, 2002).
The morphologies of Drosophila da neurons have been examined by using the MARCM (mosaic analysis with a repressible cell marker) system. Each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. These neurons were placed into four distinct morphological classes (termed class I, II, III and IV neurons) distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. The 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 (for a more complete treatment of the meaning of the word tiling, see The Tilings Around Us). In contrast, the dendritic domains of cells in different classes can overlap extensively. The cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling were examined. 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, these 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 (Grueber, 2002).
Morphological characterization of Drosophila da neurons indicates that they are similar to the da neurons of the moth Manduca sexta, of which there are at least three distinct morphological classes. In Manduca, the alpha, beta and gamma da neurons show morphological similarities to the class I/II, IV and III da neurons of Drosophila, respectively. Manduca alpha neurons appear to function as proprioceptors, whereas gamma neurons probably function as touch receptors. Whether the Drosophila da neurons are functionally similar to Manduca da neurons remains to be determined. At least two lines of evidence, however, suggest that the morphological classes that have been identified in Drosophila represent functionally distinct types of neurons. (1) pickpocket (ppk), a degenerin/epithelial sodium channel subunit, appears to be expressed only in the class IV neurons ddaC, v'ada and vdaB. Since ppk may have a physiological role in mechanotransduction, its expression in class IV neurons could underlie a functional specialization of these cells. (2) Drosophila da neurons have dichotomous axonal projections, which probably reflect their functional distinctions. Most da neurons project into the ventral neuropil, a characteristic of tactile projections. vpda and an unidentified neuron in the dorsal cluster, by contrast, have more dorsal projections, which is similar to proprioceptive neurons. The ventral-projecting neurons appear to correspond to the class II, III and IV neurons, whereas the dorsal projections belong to at least a subset of the class I neurons (Grueber, 2002).
An important issue arising from this characterization of the Drosophila da system is how the morphological properties of each neuron relate to their genetic specification. Previous studies have shown that the Drosophila da system consists of genetically distinct subgroups of neurons. Most da neurons require proneural genes in the achaete-scute complex (ASC) arise as components of external bristle lineages, and express the Cut protein. The only da neurons that do not share these characteristics are vpda and two dorsal neurons. vpda remains in ASC-mutant embryos but is lost in animals mutant for the proneural gene, atonal. One dorsal da neuron requires a third proneural gene, amos. Finally, vpda and two dorsal da neurons fail to express Cut (Grueber, 2002 and references therein).
Reconciling these accumulated data with a cell-by-cell characterization of the da system, it appears that the class II, III and IV neurons are those that require ASC genes and express Cut. In contrast, Class I neurons do not express detectable levels of Cut and could correspond to the da neurons that require atonal and/or amos (although only one ASC-independent neuron has been described in the dorsal cluster). Consistent with these assignments, during mosaic analysis class III and class IV neurons were often observed co-labeled with es neurons, suggesting that these cells could have arisen from a common precursor. The class II neurons are likely also es related because all four neurons in the ventral cluster are lineally related to es organs and two class II neurons reside here. Understanding how the genes required for early da specification are linked to the activation of distinct programs of dendritic morphogenesis is an important goal for future studies (Grueber, 2002).
Tiling is a principle of dendrite organization in which functionally similar neurons completely fill available receptive territories with little or no redundancy. Two independent tilings of the Drosophila epidermis by da neurons bearing similar morphologies have been identifed. A similar type of tiling has recently been identified in Manduca (Grueber, 2001), suggesting that this is an evolutionarily conserved plan for organizing the da sensory system. In Drosophila, tiling occurs between class III da neurons and between class IV da neurons, which each partitions the body wall into a collection of non-overlapping territories. Furthermore, class III and IV neurons each provide a nearly complete segmental coverage. Dendrites with distinct morphologies, by contrast, can cross extensively (Grueber, 2002).
The tiling of the Drosophila epidermis by class III and IV neurons appears analogous to the tiling among physiologically alike vertebrate retinal ganglion cells. In this system, ON-center and OFF-center, four ON-OFF direction-selective classes and several other cell types, including amacrine cells, provide independent tilings of the retina. Such an arrangement ensures that each region of the visual field is 'viewed' by each physiological type of ganglion cell. Additionally, as visual information is distributed to the appropriate centers of the brain, the location of its origin is unambiguous, thereby maintaining a coherent representation of sensory space. The same rules of organization applied to the insect da system would likewise be advantageous. Mechanical and thermal stimuli often necessitate rapid and finely directed behavioral responses, particularly when they could lead to damage to the cuticle. If future studies show that the da neurons comprising each tiling class are united by their physiology, as is suspected, then these modalities would have the capacity to provide accurate spatial resolution of stimuli landing anywhere on the body wall (Grueber, 2002).
Although the cellular mechanisms that control dendritic tiling are not yet understood, several developmental scenarios can be envisioned. Individual dendrites could repel like dendrites where they meet. Alternatively, neurons could be endowed with a limited capacity for dendritic growth (depending, for example, on cell size) and form their territories without influence from neighboring neurons. Finally, in the case of the da neurons, interactions with the epidermis or surrounding tissue, such as muscle, could provide permissive or restrictive growth signals. These mechanisms are not mutually exclusive and could conceivably act in concert. In the da system, however, dendritic boundaries could not always be correlated with physical boundaries, such as muscle insertion sites, and terminal dendrites typically turned abruptly where they met like dendrites. Thus, these data do not provide strong support for the latter two mechanisms (limited growth capacity and physical boundaries) but do suggest that branch interactions could contribute to tiling. Experimental studies of the effects of adding neurons to, and removing neurons from, the da system will provide essential tests of the importance of these mechanisms. Additionally, because the data are taken from mature da neurons, a crucial question still to be addressed is how dendrites of like neurons behave during development as their territories are established. Dendrites could show exclusion throughout their development or, alternatively, refine their boundaries as a maturational step (Grueber, 2002).
Regulation of tiling by dendritic branch interactions is a likely scenario in the vertebrate retina, where contact-mediated avoidance signals appear to operate in a cell-type-specific manner. Furthermore, morphological data from mammalian retinal neurons show that dendro-dendritic contacts are made between like neurons but not between unlike neurons. Such contacts could provide an opportunity for these neurons to signal to each other by their activity or cell surface composition. Similarly, typically single apparent dendritic contacts are observed between tiling class IV neurons. Whether these contacts are important for exclusion among the remaining branches remains to be determined (Grueber, 2002).
The molecular mechanisms of dendritic tiling in the vertebrate retina have not been established. Mutant screens of the second chromosome in Drosophila might be informative in this regard, since several candidate loci have been identified that cause early overextension of dendrites. Alleles of two of these genes, stan and seq, have been tested for possible roles in tiling. In seq22 and stan72 mutant embryos, dendrites show an overextension phenotype and exhibit abnormal crossing of the dorsal midline. MARCM analysis using the seq22 and stanE59 alleles suggests that such overextension might not reflect a widespread defect in dendritic exclusion, because a majority of the dendrites terminate or turn where contact with an adjacent like neuron occurs or would be expected. Importantly, however, one or two sparsely branched processes were seen extending beyond the normal boundary of the cell in 18% of the class IV stan-mutant neurons (Grueber, 2002).
If branch recognition and exclusion are required for tiling, which appears to be the case for the class IV neurons, one interpretation of the stan phenotype is that the dendrite is overextending because it does not receive or transduce a repulsive signal that requires Stan function. Alternatively, because exclusion occurs among terminal dendritic branches in wild-type neurons, a lack of exclusion in stan-mutant neurons could arise if the overextended processes are equivalent to primary trunks and thus lack the machinery for tiling. However, without information about the fields of surrounding like neurons, the possibility remains that exclusion is intact in stan-mutant neurons. By extending earlier, or more rapidly, than the rest of the dendritic field, these single processes could have successfully invaded an uninnervated region of the body wall. This latter scenario seems to provide a reasonable explanation for why a dorsal branch from ddaC is observed overextending along the dorsal midline (one of the last regions of the body wall to become innervated). Whether a similar 'invasion' scenario could account for the overextended processes from vdaB might ultimately depend on the timing and pattern of outgrowth of its class IV neighbors (i.e. how far can a dendrite of a stan- vdaB neuron extend before encountering like dendrites?). Because MARCM experiments suggest that stan acts cell autonomously in the dendritic arborization neurons, future studies might be conducted using cell-type specific markers of the class IV neurons in a stan (and seq) mutant background. Such markers would allow the visualization of all neurons together and, as a result, provide a better indication of the relationship between early dendritic overextension phenotypes and tiling (Grueber, 2002).
In addition to the dendritic exclusion that occurs between like neurons, exclusion between dendrites that belong to the same neuron is frequently observed. Such 'self-avoidance' has been identified in Manduca sensory neurons (Grueber, 2001) and characterized experimentally in leech sensory axons; however the underlying mechanisms are not understood. In theory, self-avoidance and tiling might not require distinct signals or signaling pathways (among like neurons) because isoneuronal dendrites could be developmentally identical to 'like' heteroneuronal dendrites. It will therefore be of special interest to compare the mechanisms of exclusion by isoneuronal and heteroneuronal branches during development. Ultimately, an understanding of the distinction between these two processes will require the elucidation of their molecular underpinnings (Grueber, 2002).
Brenman, J. E., Gao, F.-B., Jan, L. Y. and Jan, Y. N. (2001). Sequoia, a Tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1: 667-677. 11709187
Gao F.B., Kohwi M., Brenman J.E., Jan L.Y. and Jan Y.N. (2000). Control of dendritic field formation in Drosophila: The roles of flamingo and competition between homologous neurons. Neuron, 28: 91-101. 11086986
Grueber, W. B., Graubard, K. and Truman, J. W. (2001). Tiling of the body wall by multidendritic sensory neurons in Manduca sexta. J. Comp. Neurol. 440: 271-283. 11745623
Grueber, W. B., Jan, L. Y. and Jan, Y. N. (2002). Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development 129: 2867-2878. 12050135
Kraut, R., Menon, K. and Zinn, K. (2001). A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr. Biol. 11: 417-430. 11301252
date revised: 20 December 2001
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