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First published online August 17, 2006
Journal of Experimental Biology 209, 3383-3404 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02328

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Comparative sequence analysis and tissue localization of members of the SLC6 family of transporters in adult Drosophila melanogaster

Matthew S. Thimgan^1,*, Jonathan S. Berg² and Ann E. Stuart¹

¹ Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza Houston, TX 77030, USA

View larger version (223K): [in a new window]   Fig. 1. Twenty-one sequences in the Drosophila genome are homologous to SLC6 transporters. (A) Schematic illustration of the structure SLC6 family transporters (based on the crystal structure of Yamashita et al., 2005). Transmembrane domains are represented by grey rectangles and are numbered according to Yamashita et al., while intracellular and extracellular loops are represented by a thick dark line. Many metazoan SLC6 transporters have large N-terminal and C-terminal extensions, represented by a broken thick line, and most metazoan SLC6 transporters have a 10-50 amino acid extracellular loop between TM3 and TM4, indicated by a thick broken line. Conserved intracellular domains are indicated by white rectangles, and conserved extracellular domains are indicated by black rectangles. The arrows indicate the position of the beta sheet conformation predicted by Yamashita et al. Members of the orphan neurotransmitter subfamily have divergent extracellular loops between TM7 and TM8, and between TM11 and TM12 (dotted lines). (B) Multiple sequence alignment generated in ClustalX using 21 putative Drosophila SLC6 transporters and the leucine transporter from Aquifex aeolicus, manually adjusted to maximize comparison to the Aquifex aeolicus transporter. Protein domains are annotated at the top of each sequence block, indicating transmembrane (TM) regions, intracellular linker regions (IL), extracellular linker regions (EL) alpha helical structure (), and beta-sheets (arrows); designation of the protein domains is based on the published alignment of Yamashita et al., with minor changes. The names of each protein sequence are on the left (see Table S1 in supplementary material for accession numbers), asterisks are used to mark 10 amino acid intervals, and the residue numbers are indicated at far right. Amino acid residues are shaded according to the degree of conservation (black=100%, dark grey=80%, light grey=60%; amino acids with similar chemical properties are considered equivalent for the purposes of determining conserved residues). Gaps are represented by dashes and residues removed from the alignment for space reasons are indicated by numbers in parentheses. Selected residues that were considered important for transporter function by Yamashita et al. are indicated by symbols at the bottom of each sequence block (, charged residues at extracellular and cytoplasmic entrances; ,, residues important for coordinating sodium ions 1 and 2, respectively) and residues considered to be strictly conserved by Yamashita et al. are indicated as `Invariant' on the bottom line of each sequence block to facilitate comparison between alignments.

View larger version (60K): [in a new window]   Fig. 2. Phylogenetic analysis assigns many Drosophila SLC6 proteins to previously identified subfamilies and a novel IAAT subfamily. An unrooted phylogenetic tree displaying Drosophila candidate amino acid (AA) sequences with cloned and predicted SLC6 transporter amino acid sequences from multiple organisms. Candidate Drosophila SLC6 transporters are labelled in bold and underlined. Prominently represented among the insect sequences are putative SLC6s from Anopheles gambiae (Ag), a mosquito known for carrying the malaria parasite and whose genome sequence was recently reported (Holt et al., 2002). We also have included known transporter sequences from insects such as the cabbage looper, Trichoplusia ni (Tn), the tobacco caterpillar Manduca sexta (Ms) and from mammals. See Table S1 in supplementary material for a list of the sequence names, abbreviations and accession numbers. Subfamilies (monoamine transporters, GABA transporters, orphan neurotransmitter transporters, insect amino acid transporters) are indicated. As expected, a number of sequences do not group with the previously identified subfamilies. Many of the C. elegans transporters are quite divergent and group with each other; likewise, the prokaryote transporters are most similar to each other but do not segregate into a recognized subfamily with eukaryote transporters. Several of the candidate Drosophila transporters form a small subgroup that does not convincingly group with any other transporters. Nodes that are identical in >90% of bootstrap trials are denoted by filled circles and nodes that are identical in 75-90% of bootstrap trials are denoted by empty circles. The complete multiple sequence alignment from which this phylogenetic tree was generated is available as Fig. S1 in supplementary material.

View larger version (65K): [in a new window]   Fig. 3. The segmental mRNA expression profile of the SLC6 homologs in the head, thorax and abdomen of both male and female flies determined by RT-PCR. The head (H), thorax (T) and abdomen (A) were isolated from either male or female flies and RT-PCR was performed to determine presence or absence of transcript in a given body segment. Candidates are arranged from top to bottom as: (i) those expressed differentially in the three segments; (ii) those showing gender specificity; (iii) those expressed equally in the segments; (iv) control genes HDC and rp49. HDC is known to have enriched expression in the head and thorax and rp49 controlled for equivalent starting template in the PCR reactions. The column labelled `No RT' showed controls confirming that the amplicons were dependent on the presence of cDNA. Band size is measured in base pairs (bp).

View larger version (135K): [in a new window]   Fig. 4. Localization of transcripts involved in monoaminergic neurotransmission using in situ hybridization. Representative frontal sections showing the labelling in the head (A,C,E,G-J) or the thorax (B,D,E). In (A,C,E) the ellipsoid body is circled with a dotted line to illustrate that sections are at approximately same depth in the head. (A,B) The SerT anti-sense riboprobe labels discrete cells in the head and in the thorax. Anatomical positions of the labelled cells in (A) are consistent with the SP1 (arrows), LP1 (notched arrow), and SE2 (barbed arrow) clusters of serotonergic neurons. Cells in (B) are present in the posterior portion of the ventral ganglion (arrows). (C,D) The DAT anti-sense riboprobe labels discrete cells in the head (C) and thorax (D). The anatomical position of the labelled cells suggests these cells are part of the DM (arrows), DL1 (white arrowheads), and DL2 (carrot) clusters of neurons. The cells in (D) are present in the posterior portion of the ventral ganglion, and are likely to be part of the dorsal lateral cluster (barbed arrow) and the medial cluster (arrows). (E,F) The riboprobe for CG33528 labels many cells (markers have remained consistent with A and C) throughout the head (E) and thorax (F) that are in similar anatomical locations as cells that label for the transporters, SerT and DAT. Gray open arrows denote labelling of the medullary layer (MC cells). (G) Wider view of CG33528 labelling showing the cells of the brain that are labelled (arrows, barbed arrows and white arrowheads) and that a layer of cells at the base of the photoreceptors is also labelled (black arrowheads). Box denotes area of higher magnification in (H,I). (H,I) Serially sectioned preparation illustrating the region boxed in G but in a different preparation. (H) Section labelled with CG33528 shows label right at the base of the photoreceptors (PR). Block arrows denote examples where the CG33528 label has penetrated the photoreceptor layer. The dotted line represents the outline of the lamina neuropil (LaN). (I, top) Fluorescence image showing NrxIV antibody labelling glial septate junctions at the base of the photoreceptors in the same anatomical position as the CG33528 label in (H). (I, bottom) For clarity, the brightfield image of the fluorescence image has been presented so that the morphological features (i.e. the characteristic striations of both the photoreceptor layer and the lamina neuropil) that define the different layers in optic lobes can be distinguished. The broken line outlines the lamina neuropil and the solid line denotes the base of the photoreceptors. (J) The riboprobe for the histamine synthesizing enzyme, histidine decarboxylase (HDC), labels cells in the central brain region (arrows) as well as the photoreceptor cell bodies (barbed arrows). In all panels dorsal is up. Scale bars, 50 µm.

View larger version (120K): [in a new window]   Fig. 5. CG1732 and CG15088 are expressed in a glial-like pattern. The various glial subtypes are shown in (B) and the corresponding area in a sequential section is shown in (A). Examples of perineural (arrow) and subperineural (carrot) glia are marked [nomenclature according to Eule et al. (Eule et al., 1995)]. Inner chiasmatic glia are marked by the barbed arrow. Neuropil are labelled Medulla/Lobula (Me/Lo), suboesophageal ganglion (SEG), central brain (CB), and photoreceptors (PR). (A) Frontal section of the fly head showing the brain and optic lobes. Cells labelled with the anti-sense riboprobe for CG1732 surround the neuropils of both structures. The label occurs in regularly spaced intervals; the neuropils have a purple hue suggesting that the mRNA may be present in penetrating processes. (B) Sequential section to A labelled with the glial marker, Repo. Cells that express CG1732 are likely to be glia because they occupy the same anatomical position and approximate spacing as cells that label with the nuclear glia marker Repo. Boxed areas in A and B denote areas shown at higher magnification in C and D, respectively. (C) Higher magnification of the optic lobes in A. In both C and D, arrowheads indicate labelled cells in either outer chiasm giant glia or marginal glia at the proximal margin of the lamina neuropil. Medulla neuropil (MeN), Medulla cell bodies (MeCB), and lamina neuropil (LaN) are denoted. Arrows show the location of glia at the distal margin of the lamina; open arrows show position occupied by epithelial glia. Notched arrows mark positions of medulla neuropil glia. Glia distal to the lamina neuropil do not label with CG1732 and epithelial glia at the distal margin may faintly label for CG1732. (D) Higher magnification of B showing Repo label in the large glial nuclei of the outer chiasm glia (arrowheads) at the proximal margin of the lamina. Arrows show glia at the distal border of the lamina and open arrows show the epithelial glia; both label for Repo. Notched arrows mark the nuclei of medulla neuropil glia. (E) Pattern of label of CG15088 probe. (F) the Repo antibody labels nuclei in similar sections from different preparations. Asterisks denote comparable neuropils. Like Repo, CG15088 appears to label glia throughout the cortex. All sections are frontal sections with dorsal up; scale bars, 200 µm (A,B,E,F), 50 µm (C,D).

View larger version (143K): [in a new window]   Fig. 6. CG5226, CG5549, and CG10804 label cells broadly throughout the cell body layer in the adult head. (A) Representative horizontal section illustrating that the riboprobe for CG5226 labels cells throughout the CNS cell body layer of the head (A) and representative frontal section showing labelling in the thorax (B). We did not detect glial labelling in the optic chiasm giant glia in addition to the cortical cell bodies. (C,D) Representative frontal sections showing the probe designed against CG5549 labels the cellular cortex and the photoreceptors in the head (C) and CNS cells in the thoracic ganglion (#) and cardia (*) in (D). (E,F) The riboprobe for CG10804 labels cells throughout the adult Drosophila head, including inner chiasm giant glia (arrow) (E) and CNS cells in the thorax (F). All panels are frontal sections with dorsal up; scale bars, 50 µm.

View larger version (125K): [in a new window]   Fig. 7. Eight candidates label cells exclusively outside of the CNS. Panels show representative sections of a variety of tissues labelled using in situ hybridization, and are organized vertically by type of tissue labelled: (A-D) alimentary canal, (E-G) male reproductive system, (H-J) female reproductive system, and (K,L) other tissues. Frontal sections through the gut demonstrate that the riboprobe for CG3252 (A) and CG15279 (B) both label cells in the cardia (car; *), a structure at the transition between the foregut and the midgut. Ventral nerve cord is marked (vnc). (C,D) The anti-sense riboprobe for CG8850 (C) and CG1698 (D) both label ventricular cells (vc; filled arrows) that line the alimentary canal (ac) that runs from the thorax (th) into the abdomen. CG8850 labels cells in a restricted domain of ventricular cells in the abdomen; neighbouring ventricular cells along the alimentary canal are not labelled. Sagittal sections reveal that CG7075 (E), CG1698 (F) and CG8291 (G) label male reproductive tissue, likely the testis (tes) in the male abdomen (arrows). In addition, CG8291 (G) labels the rectal bulb (rb; open arrows), a hindgut structure at the end of the alimentary canal. Insets show boxed areas at higher magnification. Horizontal (H) and sagittal (I,J) sections show that CG4476 (H), CG1698 (I), and CG8291 (J) label structures in the female reproductive system. CG4476 (H) and CG8291 (I) label nurse cells (nc; arrows) and developing oocytes (oo; *). CG1698 (I) labels a structure called the calyx (cal; arrows). (K) CG15279 (mt; arrows) also labels two closely apposed Malpighian tubules. (L) Midgut precursor cells (mgp; arrowhead) and the Garland cells (gc; arrows) were labelled by CG13795 in a stage 14 embryo. At later stages, only the Garland cells are labelled. Anterior of the embryo is in the upper left corner and this is a dorsal view of the embryo. Scale bars, 50 µm (A,B), 100 µm (C,F,I-K) and 200 µm (D,E,G,H). A, anterior; D, dorsal.