The dynamics of nerfin-1 expression were assessed in embryos and in larval tissues by in situ hybridization. nerfin-1 transcript localizations were also sequentially followed with immunostaining for known neuronal precursor proteins to confirm the identity of nerfin-1 positive cells and to more accurately assess nerfin-1 expression dynamics during embryonic development. nerfin-1 expression is first detected in delaminating ventral cord neuroblasts (NBs) at late stage 7. However, the onset of nerfin-1 expression in the early delaminating NBs is not synchronous and the apparent levels of NB expression also vary. During early sublineage formation, nerfin-1 expression is detected in all Hunchback (Hb) immuno-positive NBs and in their first born GMCs, but no expression is detected in Hb positive neurons. During early CNS sublineage development, the apparent steady state levels of nerfin-1 expression varied among NBs. In situ signal intensity differences were observed between segmental homologue NBs and between different NBs (Stivers, 2000).
NB expression of nerfin-1 mRNA is transient. Starting at stage 9, there is a progressive loss of nerfin-1 in situ hybridization signal in both ventral cord and cephalic lobe NBs. Coincident with the loss of nerfin-1 NB expression is the onset of its expression in nascent GMCs. During the temporal phase of NB sublineage formation marked by castor (cas) expression, Cas positive NBs lack detectable nerfin-1 expression; however, their Cas positive GMCs express nerfin-1. nerfin-1 expression reaches its maximum during stages 11 though 12. However, starting at embryonic stage 13, there is a progressive decline in the number of nerfin-1 positive GMCs. This reduction in nerfin-1 expression correlates with the overall completion of embryonic NB lineage formation. nerfin-1 mRNA is also expressed in larval GMCs during adult CNS development. nerfin-2 spatial/temporal expression dynamics are different from those of nerfin-1; nerfin-2 transcripts were detected only in a subset of cephalic lobe neurons. No nerfin-2 expression was observed in the larval brain or imaginal discs (Stivers, 2000).
Nerfin-1 protein expression was studied using polyclonal antibodies raised against unique N- and C-terminal regions of the predicted 469 amino acid protein. The specificity of each antiserum was confirmed by the absence of Nerfin-1 immunostaining in embryos homozygous for nerfin-1null mutations. During embryonic stages 7 through 9, Nerfin-1 encoding transcripts were found in all early delaminating ventral cord NBs, albeit at differing levels (Stivers, 2000). In marked contrast, immunostaining with both Nerfin-1-specific antisera identified only four ventral cord NBs per segment that expressed Nerfin-1 protein. These NBs, the unpaired midline MP1 and MP3 and the lateral MP2 pair, are unique: Unlike other ventral cord NBs, they do not undergo multiple asymmetric GMC producing divisions during CNS development but rather divide just once to generate interneurons. The identity of the MP2 NB as the sole Nerfin-1-positive lateral NB was established by first determining that one of its medial row NB neighbors, on its posterior flank, was the 5–2 NB; this identification was subsequently confirmed by co-nuclear localization of Nerfin-1 and the Prospero (Pros) homeodomain protein; except for its nuclear localization in the MP2, Pros is excluded from the nucleus in all other ventral cord lateral NBs. Following the MP2 NB division, Nerfin-1 was detected in both the vMP and dMP nascent interneurons. Shortly after the onset of Nerfin-1 expression in the MP2 NBs, the unpaired midline MP1 and MP3 NBs and their nascent neurons also transiently express Nerfin-1 (Kuzin, 2005).
By stage 12, nerfin-1 mRNA expression is activated in most newly formed CNS GMCs and nascent neurons (Stivers, 2000). This appears to be de novo activation of gene expression, since at stage 11, nerfin-1 mRNA is absent from NBs (Stivers, 2000). Nerfin-1 and Prospero protein co-localization studies revealed that many, but not all, Prospero-positive cells express Nerfin-1. By early stage 13, many newborn neurons during the initial phase of their axon development express Nerfin-1, as judged by double immunolabeling with anti-Nerfin-1 and the neuron-specific anti-Elav antibody. However, both nerfin-1 mRNA and protein expression in neurons is transient. Starting at late stage 13, there is a progressive reduction in the number of neurons that express nerfin-1 mRNA or protein, such that by late stage 14, only a small subset of cells throughout the CNS has detectable levels of expression (Kuzin, 2005).
In the developing PNS chordotonal and external sensory (ES) organs, Nerfin-1 is detected only transiently in nascent neurons. ES organs form via a stereotypic series of asymmetric divisions and each cell, precursor, or terminally differentiated cell, can be distinguished by a unique set of protein markers. Two ES organ precursors, the 2B and 3B, give rise, respectively, to the multidendritic (MD) and ES neuron. However, neither gives rise to neurons exclusively. Nerfin-1 is transiently expressed transiently in both MD and ES neurons but not in their precursors or in any other cell types in the ES organ lineage (Kuzin, 2005).
Although approximately a third of all early delaminating nerfin-1 mRNA-positive ventral cord NBs give rise to glia, albeit in varying numbers, Nerfin-1 protein was not detected in glia as judged by co-staining with Nerfin-1 and glial specific markers. In addition, no defects were identified in glial development due to the loss of nerfin-1 function, indicating that nerfin-1 is most likely required only for neuronal development and/or function. Taken together, this analysis of nerfin-1 mRNA and protein expression in the developing embryo reveals that while its message is expressed in many neural precursors and in many nascent neurons, its encoded protein is detected only transiently in a subset of young neurons and in those precursor cells that will undergo a single final division to generate neurons (Kuzin, 2005).
Loss-of-function nerfin-1 mutations were generated by the 'ends-in' homologous recombination gene knockout technique of Rong and Golik (2001). DNA sequence analysis of the targeted nerfin-1 locus, after the initial 'ends-in' homologous recombination event, revealed that one of the tandem copies of nerfin-1 had suffered a 593-bp deletion in the transcribed region. This deletion was most likely caused by exonuclease digestion of the targeting vector after the Sce1 endonuclease-induced double-stranded break but before its integration into the nerfin-1 chromosomal locus. Deletions covering the minimal promoter and 5′ transcribed leader sequence of both the Df(3L)nerfin-154 and Df(3L)nerfin-1159 alleles (hereafter referred to as nerfin-1null alleles) were detected after the allelic substitution step and were most likely the result of illegitimate recombination between micro-homologies present in the minimal promoter of one copy of the nerfin-1 duplication and the transcribed region of the tandem copy. Using conventional X-ray and di-epoxybutane mutagenesis procedures, additional nerfin-1 mutant alleles were generated from the mini-white gene tagged nerfin-1 locus obtained from the first phase of the knockout targeting technique. Genomic DNA PCR analysis of these larger deletions revealed that both the proximal promoter region and transcribed sequence of nerfin-1 were removed (Kuzin, 2005).
The targeted gene knockout and classical mutagenesis screens resulted in the isolation of five independent embryonic recessive lethal alleles. Although late-stage homozygous mutant embryos appeared normal, with no detectable gross morphological or segmentation defects, they failed to hatch from their egg chambers. Whole-mount Nerfin-1 immunostaining using both N- and C-terminal directed antisera and mRNA localization revealed that all of the alleles were embryonic nulls for nerfin-1 expression. To confirm that the lethality and cellular phenotype observed in the mutant embryos was due to the loss of nerfin-1, two independent 2nd chromosome P-element insertions that contained an 11,154 bp nerfin-1 genomic DNA fragment were used to rescue the viability and cellular phenotype and to restore the nerfin-1 wild-type expression level (Kuzin, 2005).
To determine if there was any alteration in either neural lineage development or whether the expression of a known neural- or glial-identity genes was altered in nerfin-1null embryos, and the expression of 19 genes that have been demonstrated to play important roles in these early developmental events was examined. The analysis of all tested cell-identity markers revealed that the developmental processes that give rise to the correct numbers and identities of neurons and glia in both the CNS and PNS were not significantly affected by the loss of nerfin-1 function. For example, the spatial and temporal expression dynamics of Elav (neuronal) and Wrapper/Slit/Repo (glial) identity markers were not altered in nerfin-1null embryos. In addition, expression of the transcription factors Hunchback, Kruppel, Pdm-1, Castor, Pros, Engrailed, Eve, and Odd-skipped were indistinguishable between wild-type and mutant embryos. Although the possibility that more subtle changes in neuronal identities have occurred as a result of loss of nerfin-1 cannot be ruled out, this analysis indicates that neurons and glia have not suffered major changes in their identities (Kuzin, 2005).
Given the absence of any detectable alteration in NB-lineage development in nerfin-1null embryos, attempts were made to determine if Nerfin-1 played a more restricted role in neuronal maturation, such as axon outgrowth and/or pathfinding. To assess if axon patterning was altered in nerfin-1null embryos, a battery of antibody markers was used to identify many axons or to decorate specific subsets of axons in the CNS and PNS. Immunostains of nerfin-1null embryos revealed significant alterations in axon projections within the embryonic CNS but not in the PNS. For example, within the ventral nerve cord of stage 13 and older nerfin-1null embryos, the longitudinal connective axon fascicles were disrupted between segments, and both the anterior and posterior commissures of each ventral cord ganglia were malformed. Axons that normally project through fascicles that make up the intersegmental longitudinal connectives appeared to either stall or randomly turn at or near segmental boundaries, creating disorganized tangles. In addition, the organization of longitudinal connectives within each of the segments was abnormal with misrouted axons projecting laterally away from the longitudinal tracks. Immunostains also revealed that the overall axon fascicle organization and apparent axon density of the ventral cord commissures was affected by the loss of nerfin-1 function. In addition, BP102 immunostaining of stage 14 and older embryos showed that the diameters of both the supra- and sub-esophageal commissures of the brain were significantly reduced in loss-of-function mutants. In stage 15 and older mutants, the medial, intermediate, and lateral Fasciclin2 (Fas2) positive longitudinal tracks were disrupted along the entire length of the ventral cord (Kuzin, 2005).
In contrast to the axon fascicle organization defects observed in the ventral cord and brain, no significant patterning defects were detected in the motoneuron nerve tracts that exit the CNS. In addition, the axon patterning of PNS neurons, outside the CNS, also appeared normal in nerfin-1null embryos (Kuzin, 2005).
To better understand the axon misrouting in the CNS of nerfin-1null embryos, analysis focused on the axonal development of three ventral cord neurons, the pCC interneuron, and the aCC and RP2 motoneurons. To accomplish this, a CD8GFP expressing Gal4/UAS transformant line was employed that prominently marks cell bodies and axons of these neurons (Fujioka, 2003). Nerfin-1 and Eve expression transiently overlap in these neurons, as judged by co-nuclear localization; however, Eve expression was not altered by loss of nerfin-1 (Kuzin, 2005).
In the absence of nerfin-1 function, significant patterning defects were observed in the pCC axons, which normally send their pioneering ipsilateral axons anteriorly to establish the medial fascicle of the longitudinal connective tracks. In stage 13 nerfin-1null embryos, the pCC interneurons failed to send their axons anteriorly and instead projected their axons either in a lateral or posterior direction and many of the posterior projecting pCC axons crossed the ventral midline in adjacent posterior segments. By stage 14, all pCC interneurons in nerfin-1null embryos had misguided axons and many of these axons had extensive side branches. In contrast, when compared to the significant axon misguidance phenotype of the pCC interneurons, the overall development and patterning of the aCC and RP2 motoneuron axon tracts did not appear to be adversely affected in nerfin-1null embryos. For example, in nerfin-1null embryos, the aCC and RP2 axons project to the dorsal muscle field, which contains the synaptic targets of these motoneurons. It should be noted that although nerfin-1 function does not appear to be required for these motoneurons to project their axons to the appropriate synaptic target field, the role of Nerfin-1 in synaptic target choice has not yet been fully assessed (Kuzin, 2005).
The transient nature of Nerfin-1 expression in nascent neurons suggests its role may be restricted to a specific phase of early axon guidance and that its presence in the nuclei of neurons undergoing subsequent phases of axon guidance and/or maturation may interfere with these processes. To determine the significance of the temporally restricted expression, the effects of Nerfin-1 misexpression outside of its normal wild-type expression boundaries was studied. Targeted misexpression of Nerfin-1 was accomplished by the Gal4-UAS system. Using different Gal4 driver lines (scabrous-, pros-, or castor-Gal4 to activate the expression of UAS-linked nerfin-1 during different stages of NB-lineage development, misexpression was observed not to alter neuronal or glial development nor did it affect axon fascicle patterning. In addition, the ectopic expression of Nerfin-1 in mesodermal-derived tissues, outside of the nervous system, via a twist-Gal4 driver, had no detectable effect on muscle development or embryonic viability (Kuzin, 2005).
However, prolonged/extended expression in neurons resulted in embryonic lethality and ventral cord axon fascicle patterning defects. Whole-mount immunostains of late stage 14 and older elav-Gal4/UAS-nerfin-1 embryos identified multiple defects in axon scaffolding throughout the CNS. Prolonged expression in neurons interfered with the development of Fas2-positive longitudinal connective fascicles. These experiments also revealed that Nerfin-1 misexpression had a differential effect on the organization of Fas2 positive axon fascicles, with the intermediate and lateral fascicles exhibiting a greater degree of defasciculation than the medial fascicle. Immunostains with antibodies specific for the different Robo family members also revealed that misexpression of Nerfin-1 in neurons affected the distribution of Robo3. Robo3 exhibited a wider more diffuse ventral cord distribution in elav-Gal4/UAS-nerfin-1 embryos, and it was found in axons extending across the midline, suggesting either that the subcellular distribution of Robo3 had been altered or that axons within the Robo3-positive axons had defasciculated from the connectives and crossed the midline. Interestingly, no significant effect on the other two Robos, Robo and Robo2, were observed in elav-Gal4/UAS-nerfin-1 embryos. In addition, no significant changes in the expression of other axon guidance genes or cell-fate determinants were detected in elav-Gal4/UAS-nerfin-1 embryos. Prolonged misexpression did not significantly alter the patterning of motoneuron axon tracks that exit the CNS nor did it adversely affect axon patterning in the PNS (Kuzin, 2005).
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 contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).
RNAi of several genes affected the number of class I neurons as well as morphogenesis of class I dendrites; RNAi of seven genes caused supernumerary cells and RNAi of four genes caused high penetrance cell loss in addition to dendrite defects. For example, RNAi of the zinc finger TF nerfin-1 caused an increase in neurons labeled by Gal4221 with as many as eight neurons visible in some segments. Unlike wild-type class I neurons, neurons from nerfin-1(RNAi)-treated embryos extended mostly unbranched dendrites. In many cases, the routing pattern of the dendrites appeared abnormal, but the cell number defects make it difficult to resolve the projection pattern of individual dendrites or conclusively determine whether each neuron projects the same number of primary dendrites. RNAi of six other genes, including jumeau, a winged-helix TF known to regulate neuroblast cell fate and the number of PNS neurons, similarly caused an increase in neuronal number as well as defects in dendrite morphogenesis (Parrish, 2006).
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date revised: 30 May 2008
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