If Nervy serves to tether PKA to the PlexA receptor, then type II PKA should associate in a complex with PlexA. An antibody specific for PKA RII decorates embryonic Drosophila CNS and motor axons, and PKA RII coimmunoprecipitated (co-IP) with HA-PlexA expresses in neurons, showing that type II PKA is associated with the PlexA receptor complex. pka RII LOF mutant embryos also exhibit highly penetrant axon guidance defects that closely resemble the guidance defects observed in nervy LOF, PlexA GOF, and MICAL GOF mutants. In addition, pka RII LOF mutants, like nervy LOF mutants, enhance the repulsive effects of Sema-1a, which suggests that type II PKA antagonizes Sema-1a repulsive axon guidance (Terman, 2004).
To test the necessity of nervy-type II PKA interactions in regulating Sema-1a-PlexA signaling, a single amino acid substitution of a proline for a valine residue was made in Nervy (nervyV523P) that was analogous to a mutation that disrupts MTG16-PKA RII interactions. Transgenic flies were generated expressing epitope (myc)-tagged nervyV523P, but unlike neuronal expression of wild-type nervy in a nervy LOF mutant background, neuronal nervyV523P failed to rescue the nervy LOF mutant phenotypes. Therefore, it was reasoned that nervyV523P might function in a dominant-negative manner by retaining its ability to bind to PlexA but blocking the coupling of PKA to PlexA. Indeed, expression of myc-nervyV523P in all neurons in a wild-type background results in axon guidance phenotypes similar to those seen in nervy or pka RII LOF mutants. These phenotypes are the opposite of those seen when wild-type Nervy is expressed in all neurons and are indicative of increased Sema-1a-PlexA repulsion because they resemble MICAL and PlexA GOF mutants. These results suggest that nervy's ability to bind type II PKA is critical for the modulation of Sema-1a-PlexA repulsive guidance (Terman, 2004).
In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. A subtractive hybridization procedure was used to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, a set of mutant genotypes was developed that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, it was demonstrated that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. In abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes, nervy, whose expression is confined to the nervous system, predicts a protein that is similar to a human proteoncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes (Feinstein, 1995).
Two nervy loss-of-function (LOF) mutants, nervy PDFKG1 and nervy PDFKG38 were generated; they exhibit highly penetrant axon guidance phenotypes consistent with increased axonal repulsion. Motor axons within the intersegmental nerve b (ISNb) pathway require Sema-1a-PlexA repulsive signaling to selectively defasciculate from the intersegmental nerve (ISN) and normally innervate muscles 6/7 and 12/13. In nervy LOF mutants, motor axons within the ISNb pathway often exit the ISN and ISNb in abnormal locations, are excessively defasciculated, and project incorrectly within the ventral musculature. Motor axons within other pathways such as segmental nerve a (SNa) are also abnormally defasciculated in nervy LOF mutants and project to inappropriate areas. CNS projections are also abnormal in nervy LOF mutants. In wild-type embryos, three evenly spaced and uniformly thick longitudinal axon bundles are detected on each side of the CNS with an antibody to Fasciclin II (FasII). In nervy LOF mutants, axons within the third, most lateral, longitudinal bundle are less tightly fasciculated and often extend away from the CNS in inappropriate bundles. A full-length nervy transgene expressed in all neurons rescues these axon guidance defects in nervy LOF mutants, demonstrating that these phenotypes result from a lack of neuronal Nervy. nervy LOF mutant phenotypes are qualitatively and quantitatively similar to those phenotypes observed following increased expression in all neurons [gain of function (GOF)] of PlexA or its downstream signaling partner MICAL, which suggests that Nervy may antagonize Sema-1a-PlexA repulsive axon guidance (Terman, 2004).
In contrast to nervy LOF phenotypes, overexpression of Nervy in all neurons in a wild-type background (nervy GOF) decreases the ability of motor axons to defasciculate and innervate their muscle targets. These phenotypes are consistent with the absence of, or inability to respond to, an axonal repellent and are identical to those seen in Sema1a, PlexA, MICAL, and Off-track (OTK, part of the Sema-1a signaling cascade) LOF mutants. ISNb axons often fail to defasciculate from each other, or even from the ISN, in nervy GOF mutants and bypass their muscle targets. Likewise, axons within the SNa pathway fail to defasciculate in nervy GOF mutants and often stall along their trajectory. Sema1a, PlexA, MICAL, and OTK LOF-like phenotypes are also seen in the CNS of nervy GOF mutants: The outermost 1D4-positive longitudinal connective was thinner in some segments, discontinuous in others, and often fused with the middle Fas2-positive fascicle. These results support a role for nervy in antagonizing Sema-1a-PlexA repulsive axon guidance (Terman, 2004).
If nervy antagonizes Sema-1a signaling, then reducing nervy expression should increase the repulsive effects of Sema-1a. Very few axon guidance defects were observed when low levels of Sema1a were expressed in all muscles. Expression of low levels of Sema-1a in all muscles in a nervy heterozygous background, however, results in an increase in Sema-1a repulsion and leads to the inability of motor axons to defasciculate and innervate their muscle targets. These phenotypes are identical to those seen when high levels of Sema-1a are expressed in all muscles. This dominant enhancement of a weak Sema1a GOF phenotype by nervy, together with dominant suppression of a weak Sema1a LOF phenotype by nervy, suggest that Nervy and Sema1a function in the same signaling pathway but have opposing effects, and that Nervy acts downstream of Sema-1a to regulate repulsive guidance (Terman, 2004).
Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).
To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).
To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).
In addition to genes with functions in promoting dendrite arborization, 20 group A genes were identified that regulate dendrite arborization by limiting dendrite growth and/or branching. Consistent with recent reports that loss of function of the BTB/POZ domain TF abrupt (ab) causes an increase in dendritic branching and altered distribution of branches, it was found that ab(RNAi) altered the arborization of class I dendrites. ab(RNAi) caused an increase in the number and length of lateral branches, expanding the coverage field most noticeably along the anteroposterior (AP) axis. In addition to these defects, ab(RNAi) also caused frequent cell death, consistent with the phenotype observed for a hypomorphic allele of ab (Parrish, 2006).
Increased dendritic branching also resulted from RNAi of several genes known to affect nervous system development, including Adh transcription factor 1 (Adf1), the zinc finger TF nervy (nvy), the basic helix–loop–helix (bHLH) TF deadpan (dpn), as well as genes not previously known to affect neuronal function, such as the putative transcription elongation factor Elongin c. Both Adf1 and dpn mutants have defects in larval locomotion and, in light of recent findings suggesting that da neurons may regulate aspects of larval locomotion, it is possible that dendrite defects underlie these behavioral defects. Consistent with its role in class I dendrite development, dpn is expressed in all PNS neurons. Likewise, nervy has been implicated in regulation of axon branching in motorneurons and is apparently expressed in most neurons. Thus, nervy likely regulates multiple aspects of neuronal differentiation. Finally, Elongin C may regulate transcriptional elongation but also likely functions as a component of a multimeric protein complex that includes the von Hippel-Lindau (VHL) tumor suppressor and targets specific proteins for poly-ubiquitination and degradation. Moreover, BTB/POZ domain proteins (such as cg1841 and ab) function as substrate adaptors for cullin E3 ligases. Interestingly, RNAi of a Drosophila homolog (tango) of a known VHL substrate (HIF-1) also affected dendrite arborization. It thus appears that protein degradation pathways regulate dendrite arborization (Parrish, 2006).
Reference names in red indicate recommended papers.
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date revised: 10 April 2008
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