The top 50 adult (but not larval) tubule enriched probesets represent 16 known, 16 and 16 novel genes with and without GO terms respectively; including 2 unannotated probesets (Table 4-2A). The known genes include Hsp70Aa, Hsp70Bbb, alpha-Est6, Tsp42Eq, p38c, JhI-26, PhKgamma, Hsp70Bc, Kua, Cyp6a2, mthl14, l(2)08717, Oscillin, fusl, Fmo-1, and comm3.
The novel GO: Transport genes include CG14694, CG10226, CG17664 and
CG7720. The CG14694 has an OMIM entry: SLC19A3, a member of micronutrient transporter family, that transport reduced folate and has been implicated in biotin-responsive basal ganglia disease. It was first diagnosed in patients with consanguineous parents with origins from Saudi, Syria and Yemen. The CG10226 belongs to an ABC transporter, ABCA4 in humans, with an OMIM entry: age related macular degeneration.
The CG17664 has an OMIM entry: AQP3, a water channel implicated in blood group GIL. The CG7720 encodes a putative SLC family transporter, a sodium- iodide symporter in humans, with an OMIM entry: SLC5A5, implicated in congenital hypothyroidism. The human counterpart plays a key role in the
plasma membranes of the lactating breast and other tissues in I- uptake, the first step in the biosynthesis of iodine-containing thyroid hormones (Dohan et al., 2007).
The top 50 larval (but not adult) Malpighian tubule enriched probesets represent 19 known, 16 and 12 novel genes with and without GO terms respectively;
including 2 unannotated probesets (Table 4-2B). The known genes include Btd, Jhe, Jhedup, rdgc, TwdlG, SelR, bw, csul, Or35a, cad, ome, Pvf1, AttD,
l(3)82Fd, E23, BG642312, sprt, pyd and shn.
The novel GO: transport genes include CG10505, CG8850, CG7888, CG11897 and CG6293. The CG10505 has an OMIM entry: PMP70, an ABC transporter associated peroxisomal disorder with Zellweger spectrum. The CG6293 is included in the family that code permeases that may transport xanthine, uracil and vitamin C according to Pfam (Finn et al., 2010).
The top 50 commonly enriched probesets in both adult and larval tubules represent 9 known, 23 and 14 novel genes with and without GO terms respectively; including an additional unannotated probeset (Table 4-2C).
Interestingly, scarlet which is highly enriched and only found in tubules, at both larval and adult stages, encodes a protein that participates in the eye pigment biosynthetic process indicating tubule’s key role in this function.
Gp150-like is highly enriched and has a human ortholog: aspirin (ASPN) with OMIM entry associated with lumbar disc degeneration/osteoarthritis. ASPN
belongs to a family of leucine-rich repeat proteins, and is an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of the disorder (Kizawa et al., 2005). Why this is important in a simple invertebrate like a fly? The functionally distinct initial segment of the fly anterior tubule (Sozen et al., 1997) is a place for mineralised concretions (Wessing et al., 1992). Gp150-like found to be highly enriched in the initial segment of the tubule in our previous microarray study (unpublished). This broadens the horizon of an invertebrate tissue as a place to study a gene,
important in human pathology, given its genetic and physiological amenability. The NaPi-T is highly enriched in both stages and found to be highly specific to tubules. Other genes enriched include the Oatp58Da and Oatp58Da which have no human orthologs; irk3 and Sr-CIV, encoding a protein product with a STAT binding consensi (Kwon et al., 2008), involved in defence response.
Table 4-2 The top 50 genes enriched in Malpighian tubules.
(A) Adult Enrichment (B) Larval Enrichment (C) Common Enrichment
Gene Symbol FCA Gene Symbol FCA Gene Symbol FCA(at vs
awf) FCA( lft vs wlf) CG32024 59 CG13312 41 Sr-CIV 49 29 CG32843 57 Btd 34 CG33282 91 43 CG13313 49 Jhe 34 CG15408 68 27 CG17636 46 Jhedup 33 CG8837 85 25 CG32023 42 CG6475 30 CG3285 63 22 CG6602 42 CG3264 25 CG15406 32 20 CG14694 39 CG10505 20 CG18095 85 54 CG4484 34 CG8850 19 CG15279 35 22 CG7144 34 rdgC 18 Irk3 50 35 CG33012 29 CG14958 18 CG34043 53 40 CG9444 28 CG13516 18 Oatp58Da 57 47 CG10226 27 CG13836 18 Oatp58Db/gb 61 30 CG7992 27 CG32626 16 CG3270 45 21 CG10170 25 rdgC 15 NaPi-T 78 27 CG7881 23 CG17646 14 CG13905 75 35 CG13309 23 CG14949 13 CG32195 43 22 Hsp70Aa/b 23 CG14963 13 st 98 24 CG13656 22 CG3303 12 CG10006 45 30 CG17664 22 TwdlG 11 CG14606 62 40 CG9629 22 CG7888 11 CG3014 42 41 CG5431 22 CG15771 10 CG5361 58 25 Hsp70Bbb 22 SelR 10 CG6465 52 22 1638611_at 21 bw 10 Ugt86Dd 29 21 CG33258 20 1631349_s_at 10 CG14857 22 21 alpha-Est6 20 csul 10 CG17751 84 36 Tsp42Eq 20 Or35a 10 CG16727 94 21 p38c 20 cad 10 CG11659 150 45 CG5849 20 CG9062 9 CG5697 40 27 CG1315 20 CG14856 9 CG6733 26 20 JhI-26 19 CG6225 9 CG17110 61 30 CG8079 19 1638280_at 9 CG31106 48 25 PhKgamma 18 ome 8 CG10553 24 20 Hsp70Bc 18 CG34198 8 CG31097 46 32 CG7720 18 Pvf1 8 CG31380 48 23 CG15706 18 AttD 8 CG42235 62 33 Kua 17 CG11897 8 CG42235 74 61 Cyp6a2 17 CG32234 8 CG42235 69 52 CG30411 17 CG4586 8 CG42235 60 23 CG31562 17 l(3)82Fd 8 CG42235 66 23 CG13604 17 E23 7 CG2187 33 28 mthl14 17 BG642312 7 CG11889 26 33 l(2)08717 17 CG6364 7 CG3690 62 33 CG6891 17 CG6293 7 CG2680 29 26 CG13827 17 sprt 7 CG15221 39 40 1639729_s_at 16 CG30375 7 CG8028 78 46 Oscillin 16 pyd 7 CG14195 31 21 CG42329 16 CG10301 7 CG18814 19 31 fusl 16 CG7431 7 1631526_s_at 19 23 Fmo-1 16 shn 7 Ugt35b 20 37 comm3 16 CG9328 7 Cyp6a8 31 20
Sr-CIV has been shown to be upregulated in Nurf mutants (that show
inflammatory syndrome) in which NURF was implicated as a regulator of a large set of JAK/STAT target genes (Kwon et al., 2008). The tubule senses the bacteria and may constitute a cell-autonomous system of immunity (McGettigan et al., 2005). Sr-CIV has a putative concanavalin A-like lectin/glucanase domain.
The lectins and glucanases are found in all orders of life and show the common property of reversibly binding to specific complex carbohydrates. Some catalyse beta-glucans found in microorganisms (Hahn et al., 1995). This observation reinforces the idea that tubules may act as an independent system in
potentiating innate immune responses to the pathogenic bacteria within their milieu.
Other genes that did not show up in the top list, but did in the 10-fold or over upregulated lists include the famous xanthine dehydrogenase (or rosy), white, urate oxidase (uro). In that, rosy and white are well characterised classical mutants. The localisation of rosy and uro have been established to be
peroxisomal (Beard and Holtzman, 1987; Wallrath et al., 1990). Mutations in rosy and white cause eye colour phenotypes, thus are important in the associated pigment biosynthesis and transport processes (Beyenbach et al., 2010b). The rosy gene encodes a bifunctional oxidoreductase that can act as a dehydrogenase or an oxidase depending on the substrate and acceptor
availability (Parks and Granger, 1986; Stirpe and Della Corte, 1969). It catalyses the conversion of hypoxanthine to xanthine, and further to uric acid (Beard and Holtzman, 1987; Reaume et al., 1989). Most interestingly, the Drosophila rosy mutants recapitulate the human inborn error of metabolism, xanthinuria type I, caused by mutations in the human homolog of rosy (Beyenbach et al., 2010b). In conjunction, the studies using sedimentation gradient centrifugation,
established that rosy-localised peroxisomal size, shape, and centrifugal
behaviour are similar to peroxisomes of vertebrates (Beard and Holtzman, 1987; Parks and Granger, 1986).
Uro encodes a functional urate oxidase that converts uric acid to 5-
hydroxyisourate which eventually is converted to allantoin. In humans and many primates uric acid is the end product of the catabolism of purines because they have a non-functional gene (Wu et al., 1989).
The peroxisomal localisation of rosy is interesting which could regulate the downstream effectors of the peroxisomal resident enzymes in the purine degradation pathway.
For example, by the end of embryogenesis, distal tubule cells transport the organic solutes of urate (urates) into the lumen, where they precipitate as uric acid crystals but not until they hatch they are able to clear it by fluid transport process (Beyenbach et al., 2010a). As insects go through successive
developmental stages from larvae to adult, the excretory load increases on the organism. For this task, Malpighian tubules have to rapidly adapt, as they are largely established during embryogenesis unlike other epithelial tissues
(Beyenbach et al., 2010a; Skaer, 1993). Nevertheless, peroxisomes in the tubules play essential roles in the transport and excretory mechanisms of tubules, at both adult and larval stages.