QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00347

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Seppo, A. Articles by Tiemeyer, M. Search for Related Content PubMed PubMed Citation Articles by Seppo, A. Articles by Tiemeyer, M.

Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells

Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA

View larger version (117K): [in a new window]   Fig. 1. A third chromosome deletion uncovers an aberration on the TM3 balancer that is responsible for loss of the HRP epitope. (A) In balanced wild-type embryos (+/TM3-lacZ), anti-HRP antibodies stain the central nervous system (vnc, ventral nerve cord) and the peripheral nervous system (out of the plane of focus). Three non-neural tissues also express the HRP epitope: the garland gland (gg), anal pads (ap) and the posterior hindgut (phg). Blue staining at the anterior end of the embryo (towards the left in all panels) indicates the relative dose of the TM3-lacZ chromosome. (B) In TM3 homozygotes, the HRP epitope is absent from the nervous system but still detected in non-neural tissues. (C) Brd15 fails to complement the TM3 defect (Brd15/TM3-lacZ) and the epitope is still detected in non-neural tissues. (D-G) Whole-mount embryos were double stained: first for lacZ activity to confirm genotype; and then with mAb BP102 to provide landmarks for comparison of embryonic gross morphology. Dorsal views of stage 15 and 16 embryo pairs are shown for the genotypes (D) +/+, (E) TM3-lacZ/TM3-lacZ, (F) Brd15/TM3-lacZ and (G) Brd15/Brd15. Brd15 homozygotes display a characteristic defect in head involution (arrowhead in G). Scale bar: 350 µm in A-C; 500 µm in D-G.

View larger version (14K): [in a new window]   Fig. 2. Deletion non-complementation maps loss of the HRP epitope to a TM3 breakpoint at 71C. Deletions covering the third chromosome were screened for loss of the HRP epitope in combination with the TM3 balancer. Breakpoints are indicated for complementing deletions (white) and non-complementing deletions (black). The gray zone labeled HRP- indicates the smallest region that contains a gene responsible for HRP-epitope expression. The distal end of the interval is defined by the TM3 breakpoint at 71C. The proximal breakpoint is limited to 71C1/2 by D5rv14 and Brd15.

View larger version (80K): [in a new window]   Fig. 3. A differentially expressed transcript and genomic polymorphisms distinguish Brd15/TM3 from OreR embryos. (A-C) Reverse-northern analysis. DNA from P1 phage DS06206 was digested with BamHI (lane 1), XbaI (lane 2), BamHI/XbaI (lane 3) or EcoRI (lane 4) then electrophoresed (m indicates marker lane, 1 kb ladder). (A) Ethidium bromide staining of agarose gel. Blots prepared from the gel were probed with 32P-end-labeled mRNA isolated from OreR (B) or from Brd15/TM3 embryos (C). A differentially expressed 2.4 kb EcoRI band was subcloned. (D) Northern analysis. Poly-A+ RNA isolated from OreR (lane 1) or Brd15/TM3 embryos (lane 2) was probed with the 2.4 kb EcoRI fragment. (E-G) Southern analysis. Genomic DNA prepared from OreR (lane 1) or from Brd15/TM3 (lane 2) adults was digested with (E) HindIII, (F) HindIII/XbaI or (G) PstI and probed with a 1.5 kb HindIII fragment (see Fig. 4 for restriction map and probe locations). Digestion with PstI produces the smallest polymorphic fragment, a 280 bp fragment in OreR that is shifted to 7.5 kb in Brd15/TM3.

View larger version (20K): [in a new window]   Fig. 4. The disrupted gene in Brd15/TM3 embryos encodes a Toll-like receptor. The wild-type tollo gene predicts a protein of 1346 amino acids. (Top) Conserved sequence domains identify the gene as a Toll-like Receptor (black rectangles, leucine-rich repeats, LRR; discontinuous black ovals, cysteine-rich domains, CR; gray oval, Toll homology signaling domain, TH; putative transmembrane domain, TM). (Bottom) Genomic sequence (nucleotides 17001 to 30000 from AE003531) contains a 4038 nucleotide ORF (gray box) within a single exon. Nucleotide 1 in the tollo ORF corresponds to nucleotide 18564 in AE003531. The boxed region (6735 nucleotides) was sequenced, either from genomic DNA derived from P1 phage (gray and white, nucleotides -17 to 5717) or by overlapping 3'-RACE (hatched, nucleotides 5398 to 6718). Arrow at the 5' end indicates a predicted promoter. Four consensus polyadenylation signals (A) are found within 5 kb of the first inframe stop codon. Sequence divergence in the TM3 chromosome is indicated by a broken line transposable element (TE). A polymorphic PstI genomic fragment confirms the site of divergence and is indicated in the aligned restriction map for the region (P, PstI; X, XbaI; H, HindIII). Probe 1 (EcoRI fragment) used for in situ hybridization and northern blots. Probe 2 (HindIII fragment) used for genomic Southern analysis.

View larger version (89K): [in a new window]   Fig. 5. Tollo mRNA is dynamically expressed throughout embryogenesis. The distribution of tollo mRNA was visualized by in situ hybridization with digoxigenin-labeled probes in OreR (A-F), TM3/TM3 (G) and Brd15/Brd15 (H) embryos. Message is first detected in distinct anterior and posterior bands at the cellular blastoderm stage (A) but rapidly spreads to include bands along the entire length of the embryo (B). At stage 12 (C,D), the extended germ-band demonstrates segmental expression. By stage 13 (E) ectodermal expression is restricted to domains within each segment. At higher magnification (F), stage 13 expression of tollo mRNA is not uniform within lateral ectodermal domains. TM3 homozygotes (G, stage 13) exhibit weak hybridization signal, while Brd15 homozygotes (H, stage 14) completely lack message. Scale bar: 450 µm in A-C,E,G,H; 125 µm in D,F.

View larger version (111K): [in a new window]   Fig. 6. Tollo mRNA is expressed in ectodermal cells that surround differentiating neurons. After in situ hybridization (purple), embryos were stained with mAb 22C10 (brown) to visualize the relationship between differentiating neurons and Tollo-expressing ectodermal cells. (A) Segments of the peripheral ectoderm are shown (T2-A4, anterior towards the left, dorsal towards the top of the panel) from an early stage 14 embryo. Neurons differentiate within domains of tollo mRNA expression. (B) Higher magnification of T2 and T3 from A shows the intimate relationship between Tollo-expressing ectodermal cells and differentiating neurons. Scale bar: 60 µm in A; 20 µm in B.

View larger version (119K): [in a new window]   Fig. 7. A tollo-Gal4 transgene drives lacZ expression (from UAS-lacZ) in the same cells that express Tollo mRNA. (A,C) lacZ activity visualized in tollo-Gal4; UAS-lacZ embryos. (B,D) Tollo mRNA detected in OreR embryos by in situ hybridization. In stage 12, germ band-extended embryos (A,B), lacZ activity (A) is detected in segmentally repeated bands of ectodermal cells that mirror Tollo mRNA (B). In the peripheral ectoderm at stage 13 (C,D) lacZ activity (C) is detected in domains of ectodermal cells. Segments T3 — A2 are shown in C,D, anterior towards the left and dorsal towards the top of the panel. Scale bar: 350 µm in A,B; 60 µm in C,D.

View larger version (120K): [in a new window]   Fig. 8. Transgenic expression of Tollo in Brd15 homozygotes rescues expression of the HRP epitope in the ventral nerve cord and in the peripheral nervous system. All panels show embryos probed with anti-HRP antibody. Whole-mount (A,D,G), ventral nerve cord (B,E,H) and three segments (C,F,I) of the peripheral nervous system are shown in OreR (A-C, stage 15), Brd15/Brd15 (D-F, stage 16) and tollo-Gal4; UAS-tollo; Brd15/Brd15 (G-I, stage 16) embryos. Transgenic Tollo rescues expression of the HRP epitope in the ventral nerve cord (G,H) but does not correct the head involution defect associated with the Brd15 deletion (compare arrowheads in D and G). HRP-epitope expression in elements of the peripheral nervous system first becomes apparent at stage 15 in OreR (short arrow in C). Loss of the HRP epitope in the peripheral nervous system of Brd15 homozygotes (F) is rescued by Tollo (short arrow in I). Tollo expression under control of hsp70-Gal4 without heat shock (J,K) induces HRP-epitope expression in the salivary gland (sg, paired structures at top and bottom of J,K) and rescues epitope expression in nearby sensory neurons (short arrows in K). The anterior end of dissected hsp70-Gal4; UAS-tollo; Brd15/TM3-lacZ embryos at stage 14 (J) and at stage 16 (K) are shown, anterior towards the left, after sequential lacZ activity staining (blue) and anti-HRP antibody staining. At dissection, the anterodorsal ectoderm of the head was sliced at the dorsal midline and flattened laterally. Subsequent removal of the embryonic brain lobes reveals the rescued sensory neurons (gg, garland gland; vnc, ventral nerve cord). Scale bar: 450 µm in A,D,G; 125 µm in B,E,H; 63 µm in C,F,I; 115 µm in J,K.