Expression of tollo mRNA is first detected in the cellular blastoderm, initially as prominent bands at both ends of the embryo. Very rapidly, tollo mRNA appears in dorsoventral bands repeated along the entire length of the embryo. As germband retraction begins, late in stage 12, the bands of tollo mRNA span the entire width of the germband, placing Tollo expression within ectodermal domains from which ventral nerve cord precursor cells differentiate and delaminate. By the time germband retraction is complete (stage 13), tollo mRNA expression disappears from the ventral ectoderm that underlies the delaminated, discrete nerve cord. Expression of the HRP epitope in the ventral nerve cord is first detected reproducibly at stage 14, shortly after tollo mRNA decreases. Thus, Tollo expression in ventral ectoderm coincides with a period of maximal contact with differentiating neurons and disappears once neurons segregate from the ectoderm to form a consolidated ventral nerve cord (Seppo, 2003).
In the lateral ectoderm, tollo mRNA is found in distinct, segmentally repeated domains at stage 13. Together, these repeated domains form continuous anteroposterior stripes of ectodermal expression. Within each domain, expression is not uniform. Cells at the segment boundaries express higher levels of tollo mRNA, forming ectodermal pockets that are partly lined with Tollo-expressing cells. By early stage 15, tollo mRNA is greatly reduced in the lateral ectoderm and expressing domains are attenuated to a few cells immediately adjacent to segment boundaries. Expression of the HRP epitope in the peripheral nervous system is first detected reproducibly at stage 15, shortly after tollo mRNA expression has decreased in the lateral ectoderm (Seppo, 2003).
Consistent with the determination that the TM3 chromosome has not lost the entire tollo gene, hybridization signal was detected in TM3/TM3 embryo. Thus, RNA that contains tollo sequence is produced in TM3 embryos despite being undetectable in blotted poly-A+ mRNA preparations. Hybridization to Brd15/Brd15 embryos was not detected at any stage, indicating that the anti-sense probe is specific for tollo and does not cross-hybridize to other embryonically expressed Toll-like receptors (Seppo, 2003).
The position of Tollo-expressing domains along the dorsoventral axis of the lateral ectoderm closely approximates the site of proneural cluster formation. Therefore, tollo mRNA expression was localized relative to the position of differentiating neurons in the peripheral nervous system. Within the lateral domains of Tollo expression found in each segment, maturing neurons occupy patches that display reduced or undetectable tollo mRNA. At stage 14, all but the earliest neurons to differentiate (which have actively begun to migrate away from their birthplace towards their final embryonic positions) are found in close association with ectodermal cells that express tollo mRNA. Thus, in the peripheral nervous system, as in the ventral nerve cord, Tollo expression coincides temporally with periods of neural differentiation that are characterized by maximal contact between the ectoderm and neural precursor cells (Seppo, 2003).
To determine whether tollo is sufficient to rescue expression of the HRP epitope in the Brd15 homozygote, a transformation construct was generated (pUASTtollo) that placed tollo-coding sequence under the control of UAS elements. A second transformation construct was prepared (ptolloGal4) that placed Gal4 expression under control of 2.5 kb of Drosophila genomic DNA found immediately upstream of the tollo initiation codon. Tollo-Gal4 transformant lines were crossed to a UAS-lacZ reporter line and embryo collections were stained for ß-galactosidase activity. Both in the germband extended embryo at stage 12 and in the lateral ectoderm of the stage 13 embryo, lacZ activity matched the distribution of tollo mRNA detected by in situ hybridization. Thus, the 2.5 kb of genomic DNA incorporated into the ptolloGal4 transformation vector contains control sequences sufficient to recapitulate normal Tollo expression (Seppo, 2003).
UAS-tollo and tollo-Gal4 transformant lines were separately prepared in the Brd15/TM3 background. HRP-epitope expression is absent from embryos collected from lines bearing either construct alone. However, when UAS-tollo and tollo-Gal4 lines are crossed to each other, HRP-epitope expression is rescued in embryos that lack (Brd15/TM3 and TM3/TM3 genotypes) and in embryos that possess (Brd15/Brd15) the head involution defect associated with the Brd15 deletion. Thus, the head involution defect is independent of HRP-epitope expression. Tollo-Gal4/UAS-tollo rescues HRP-epitope expression in the ventral nerve cord and in the peripheral nervous system (Seppo, 2003).
Other Gal4 driver lines were screened for their ability to rescue the HRP epitope in UAS-tollo transformants. Neither a pan-neural driver (ELAV-Gal4) nor a mesectodermal/midline glial driver (rhomboid-Gal4) rescued oligosaccharide expression when crossed to UAS-tollo, despite their ability to drive expression in cells that make extensive contact with neuronal surfaces. Therefore, simple juxtaposition of Tollo protein and a neuron is insufficient; induction of the neuronal HRP epitope requires Tollo expression in appropriate non-neural ectodermal cells (Seppo, 2003).
Heat-shock driven expression of Tollo in all cells (hsp70-Gal4/UAS-tollo) generates early embryonic lethality that precludes assessment of HRP-epitope rescue. However, in the course of these experiments, hsp70-Gal4/UAS-tollo embryos not subjected to heat shock were also collected and stained with anti-HRP antibody. Unexpectedly, unshocked embryos older than stage 15 express the HRP epitope in the salivary gland and in sensory neurons most proximal to the gland. Other neuronal populations were not stained, whether in the CNS or in more posterior segments of the PNS. Thus, leaky Gal4 expression in the salivary gland (verified by UAS-lacZ reporter) is sufficient to induce the HRP epitope in a tissue that does not normally express the glycan and is able to rescue the epitope in nearby sensory neurons (Seppo, 2003).
Antiserum against horseradish peroxidase (anti-HRP Ab) labels the surfaces of neurons in both Drosophila and grasshopper. The anti-HRP Ab immunoprecipitates at least 17 different membrane glycoproteins from the Drosophila embryo CNS (and a similar array from grasshopper). Anti-HRP Ab recognizes a neural-specific carbohydrate moiety expressed by most if not all of these proteins. Although the anti-HRP Ab stains all axon pathways, 2 of the anti-HRP glycoproteins, Fasciclin I and II, are expressed on specific subsets of axon pathways in the grasshopper embryo (Snow, 1987).
In embryos, the fasciclins are localized to axonal subsets, while the RPTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, a biochemical approach has been taken. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted 'HRP proteins') apparently bear the HRP carbohydrate epitope. Polyclonal anti-HRP antibodies have been used to purify these proteins from Drosophila embryos, and protein sequences have been obtained from seven HRP protein bands. These data define three major HRP proteins as Neurotactin, Fasciclin I, and an RPTP, Ptp69d. Fasciclin II, Neuroglian, Ptp10D, and Ptp99A are also HRP proteins (Desai, 1994).
Antibodies recognizing horse radish peroxidase (HRP) stain neurons in Drosophila and other insects. Western blots were used to analyze and characterize some of the anti-HRP reactive components from Drosophila melanogaster. Anti-HRP reactive components can be reproducibly detected during all developmental stages, although the pattern changes at different developmental times. In adults, there are at least 10 reproducibly stained components. Two of the bands, with molecular sizes of 42 and 80 kDa are likely to be the major contributors to neuronal anti-HRP staining in Drosophila. These components are enriched in adult fly heads. In contrast, many of the other anti-HRP reactive components in adults are enriched in abdomen and are present exclusively or at much higher levels in male flies. Two of the male specific components with molecular sizes of 62 and 48 kDa have been purified and characterized. Partial N-terminal amino acid sequencing revealed that the 62 kDa protein is identical to a part of D. melanogaster carboxylesterase, while the 48 kDa protein does not match any known sequences. Esterase-6 has previously been shown to be enriched in male accessory gland and consistent with this, anti-HRP antibodies also have been shown to stain this gland (Wang, 1994).
Characterizing the gene product(s) recognized by anti-HRP antibodies is of interest because it may be important for nervous system function and/or development. An anti-HRP-reactive Mr 42K glycoprotein has been identified and purified from adult Drosophila heads that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. These components have been collectively named Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system (Sun, 1995).
The nature of anti-HRP antigens was investigated in Drosophila and found to include a complex set of developmentally regulated proteins. Their common epitope appears to be a carbohydrate that shares features with the sugar moiety of pineapple stem bromelain, a plant glycoprotein whose carbohydrate structure has been determined. A mutation was identified that eliminates staining by the antibody in imaginal and adult neural tissue. Tissue specific glycoconjugates, although widespread in the animal kingdom, are little understood. This mutation provides a unique opportunity to address the consequences of altering a neural specific carbohydrate moiety in an otherwise intact and behaving animal. The mutation maps to 84F. A second mutation, contained on the third chromosome balancer, TM3, eliminates anti-HRP staining in embryos. These mutations appear to be separate genes (Katz, 1988).
Embryos homozygous for the TM3 balancer chromosome fail to express the HRP epitope in the ventral nerve cord and peripheral nervous system, although expression is maintained in three non-neural tissues. TM3 rearrangement breakpoints provide likely candidates for regions of the third chromosome that affect HRP-epitope expression. To assess the relevance of these breakpoints and to ensure that a gene located between TM3 breakpoints was not missed, overlapping deletion stocks that together cover approximately 80% of the third chromosome were screened for their ability to complement loss of the HRP epitope in a TM3 background. The smallest non-complementing deletion (breakpoints 71A1/2-71C1/2) is carried in a Bearded stock designated Brd15. The combination Brd15/TM3 or Brd15/TM3-lacZ produces viable and fertile adults (Seppo, 2003).
Of the relevant genotypes, only Brd15 homozygotes display gross morphologic aberrations. Brd15/Brd15 embryos develop normally until stage 14 when defects in the formation of anterior terminal structures become apparent. In particular, retraction of the clypeolabrum is stalled in Brd15 homozygotes, causing the supraesophageal ganglia (embryonic brain lobes) to appear exteriorized. The head involution defect provides an unambiguous, reliable diagnostic for the Brd15 homozygous genotype. Examination of neural tissue integrity by monoclonal antibody staining demonstrates that longitudinal and commissural bundles are present in the central nervous system (mAb BP102), appropriate cell numbers and approximate cellular relations are preserved in the peripheral nervous system (mAb 22C10), and efferent motor pathways develop normally (mAb 1D4) in Brd15 homozygotes. Thus, neural differentiation and axon extension, to the extent that they are revealed by these mAb markers, are unaffected by Brd15 (Seppo, 2003).
The proximal breakpoint of the Brd15 deletion (71C1/2) overlaps a TM3 rearrangement breakpoint at 71C. Therefore, P1 phage clones that map to the 71C1/2 interval were obtained and probed with 32P-end-labeled mRNA prepared from embryo collections from OreR or Brd15/TM3 stocks. The P1 phage clone designated DS06206 contains a 2.4 kb EcoRI fragment and a 1.1 kb BamHI/XbaI fragment that are both transcribed in OreR but not detected in Brd15/TM3 embryos. Subsequent sequence analysis placed the 1.1 kb fragment within the 2.4 kb fragment. Probe prepared from the 2.4 kb EcoRI fragment was used to probe Northern blots of poly-A+ RNA isolated from OreR or Brd15/TM3 embryos. A 6.5-7.0 kb band was identified in the OreR preparation that was not detected in Brd15/TM3 poly-A+ RNA. Genomic Southern analysis demonstrates multiple restriction fragment length polymorphisms in the Brd15/TM3 genotype. Probe prepared from the 2.4 kb EcoRI fragment hybridizes to a 1.5 kb HindIII fragment in OreR that is shifted to approximately 6 kb in Brd15/TM3. In turn, probe prepared from the 1.5 kb HindIII fragment identifies the same polymorphism as well as 876 bp HindIII/Xba1 and 280 bp PstI fragments that also differentiate the two genotypes. The sequenced 2.4 kb EcoRI genomic fragment yielded an open reading frame of 1250 bp which was extended to 4038 bp in length by further genomic sequencing (Seppo, 2003).
A total of 6735 nucleotides were sequenced, extending from 17 bp upstream of the ORF to 2677 bp beyond the first in-frame stop codon, and found to be co-linear with genomic sequence in GenBank Accession Number AE003531 and with Drosophila cDNA sequence LD33590. The sequence predicts that the HindIII, XbaI and PstI polymorphisms observed in Brd15/TM3 lie in the 3' UTR of the gene. To more precisely define the polymorphism, 3'-RACE was performed on poly-A+ RNA isolated from OreR and Brd15/TM3 embryos. The fragment amplified from Brd15/TM3 embryos yielded 969 nucleotides of sequence of which the first 237 matched previously sequenced genomic DNA. However, TM3 sequence diverged from wild-type at a position corresponding to nucleotide 5635, 1.6 kb downstream from the first in-frame stop codon of tollo (nucleotide 4039) and within the 3' UTR predicted by mRNA size. Sequence obtained for the first 732 bases of divergence matches a Drosophila transposable element designated '412', GenBank Accession Number X04132. It was not determined whether the divergent sequence reflects the insertion of an intact transposable element or identifies the site of the TM3 rearrangement breakpoint previously mapped to 71C (Seppo, 2003).
Reference names in red indicate recommended papers.
Desai, C. J., Popova, E. and Zinn, K. (1994). A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the `HRP' carbohydrate epitope. J. Neurosci. 14: 7272-7283. 7527841
Fabini, G., Freilinger, A., Altmann, F. and Wilson, I. B. (2001). Identification of core alpha 1,3-fucosylated glycans and cloning of the requisite fucosyltransferase cDNA from Drosophila melanogaster. Potential basis of the neural anti-horseradish peroxidase epitope. J. Biol. Chem. 276: 28058-28067. 11382750
Jan, L. Y. and Jan, Y. N. (1982). Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and grasshopper embryos. Proc. Natl. Acad. Sci. 79: 2700-2704. 6806816
Katz, F., Moats, W. and Jan, Y. N. (1988). A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate). EMBO J. 7: 3471-3477. 2463162
Phillis, R. W., Bramlage, A. T., Wotus, C., Whittaker, A., Gramates, L. S., Seppala, D., Farahanchi, F., Caruccio, P. and Murphey, R. K. (1993). Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster. Genetics 133: 581-592. 8454205
Seppo, A., Matani, P., Sharrow, M. and Tiemeyer, M. (2003). Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells. Development 130: 1439-1448. 12588858
Snow, P. M., Patel, N. H., Harrelson, A. L. and Goodman, C. S. (1987). Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embryos. J. Neurosci. 7: 4137-4144. 3320283
Sun, B. and Salvaterra, P. M. (1995). Characterization of nervana, a Drosophila melanogaster neuron-specific glycoprotein antigen recognized by anti-horseradish peroxidase antibodies. J. Neurochem. 65(1): 434-43. 7540667
Tauszig, S., Jouanguy, E., Hoffman, J. A. and Imler, J. L. (2000). Toll-related receptors and the control of antimicrobial peptide expression in Drosophila. Proc. Natl. Acad. Sci. 97: 10520-10525. 10973475
Wang, X., Sun, B., Yasuyama, K. and Salvaterra, P. M. (1994). Biochemical analysis of proteins recognized by anti-HRP antibodies in Drosophila melanogaster: identification and characterization of neuron specific and male specific glycoproteins. Insect Biochem. Mol. Biol. 24: 233-242. 8019574
Whitlock, K. E. (1993). Development of Drosophila wing sensory neurons in mutants with missing or modified cell surface molecules. Development 117: 1251-1260. 8404529
date revised: 23 May 2004
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