Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene

Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene

Gene Expression Patterns 8 (2008) 275–283 www.elsevier.com/locate/gep Embryonic expression patterns of Drosophila ACS family genes related to the hum...

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Gene Expression Patterns 8 (2008) 275–283 www.elsevier.com/locate/gep

Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene Bram Laridon a

a,b

, Patrick Callaerts

a,c

, Koen Norga

a,b,*

Laboratory of Developmental Genetics, V.I.B., Herestraat 49, Mailbox 602, B-3000 Leuven, Belgium b Department of Woman and Child, KULeuven, Belgium c Department of Human Genetics, KULeuven, Belgium

Received 1 December 2007; received in revised form 11 December 2007; accepted 12 December 2007 Available online 23 December 2007

Abstract The anion/cation symporter (ACS) family is a large subfamily of the major facilitator superfamily (MFS) of transporters. ACS family permeases are widely distributed in nature and transport either organic or inorganic anions in response to chemiosmotic cation gradients. The only protein in the ACS family to which a human disease has been linked, is sialin, the proton-driven lysosomal carrier for sialic acid. Genetic defects in sialin cause a lysosomal storage disease in humans. Here we have identified a group of conserved Drosophila ACS family genes related to sialin and studied their expression patterns throughout embryogenesis. Drosophila sialin-related genes are expressed in a wide variety of tissues. Expression is frequently observed throughout various parts of the intestinal tract, including Malpighian tubules and salivary glands. Additionally, some genes are expressed in vitellophages (yolk nuclei), nervous system, respiratory tract and a number of other embryonic tissues. These data will aid the establishment of a fruitfly model of human lysosomal storage disorders, the most common cause of neurodegeneration in childhood. Ó 2007 Elsevier B.V. All rights reserved. Keywords: ACS; Anion/cation symporter; Drosophila; Embryogenesis; In situ hybridization; Major facilitator superfamily; MFS; Sialic acid storage disease; Sialin

1. Results and discussion The anion/cation symporter (ACS) family is a large subfamily of the major facilitator superfamily (MFS) of transporters. MFS transporters are single polypeptides with 12–14 transmembrane (TM) domains capable of transporting a wide range of small solutes (including carbohydrates and amino acids) in response to chemiosmotic ion gradients (Pao et al., 1998). ACS family transporters recognize either inorganic (e.g. phosphate) or organic anions. Among the organic anions transported are glutamate, sialic acid, phosphate, glucarate, hexuronates, phthalate, allantoate and probably tartrate (Bellocchio et al., 2000; Crouzet * Corresponding author. Address: Laboratory of Developmental Genetics, V.I.B.-KULeuven, Herestraat 49, Mailbox 602, B-3000 Leuven, Belgium. Tel.: +32 16 343380; fax: +32 16 343842. E-mail address: [email protected] (K. Norga).

1567-133X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2007.12.003

and Otten, 1995; Havelaar et al., 1998; Magagnin et al., 1993; Rai et al., 1988). Proteins of the ACS family are widely distributed in nature. They are found in both gram-negative and gram-positive bacteria and in both the animal and fungal eukaryotic kingdoms (Pao et al., 1998). In humans, eight ACS carriers have been characterized: the Na+/phosphate co-transporters NPT1-4, sialin and the vesicular glutamate transporters VGLUT1-3 (Bai et al., 2001; Bellocchio et al., 2000; Chong et al., 1993; Gras et al., 2002; Magagnin et al., 1993; Ruddy et al., 1997; Verheijen et al., 1999). Sialin is the only protein in the ACS family to which a human disease has been linked. The protein is a predicted type-II transmembrane protein of 495 amino acids with 12 TM domains and N- and C-termini located in the cytoplasm. Sialin contains the characteristic ACS motif in the fourth TM-spanning region. The lysosomal sialic acid transporter is defective in sialic acid storage diseases (SASD, MIM 269920) (Morin et al., 2004;

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Verheijen et al., 1999; Wreden et al., 2005). These are autosomal recessive neurodegenerative disorders, which belong to the large group of lysosomal storage disorders. In SASD, free sialic acid accumulates in lysosomes, which leads to increased tissue concentrations, elevated urinary excretion and severe neurological and developmental problems (Lemyre et al., 1999; Tietze et al., 1989; Varho et al., 2002). Sialic acids occur mainly as terminal components of cell surface glycoproteins and glycolipids and comprise a large family of closely related derivatives of N-acetylneuraminic acid and N-glycolylneuraminic acid (Schauer, 2000; Tanner, 2005). The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes. In vertebrates, the regulated presence or absence of a polymer of sialic acid (PSA) on the neural cell adhesion molecule (NCAM) is required for the proper establishment and function of the nervous system (Bonfanti, 2006). Sialic acids are cleaved off from degraded sialoglycoconjugates by sialidase and are exported from lysosomes by the membrane transporter sialin. Sialic acids occur endogenously in Drosophila, but it has long been controversial whether Drosophila and insects in general have a sialic acid synthesis pathway (Roth et al., 1992). Recently however Drosophila homologs of sialic acid synthase, CMP-sialic synthase and sialyltransferase were identified and proven to be functional in expression systems (Kim et al., 2002; Koles et al., 2004; Viswanathan et al., 2006). A Drosophila ortholog of sialin has not been identified so far. As a first step towards this identification we examined the embryonic expression patterns of various conserved Drosophila ACS family genes related to sialin with in situ hybridizations. 1.1. Identification of ACS family genes related to sialin in the Drosophila genome We searched the Drosophila genome for homologs of sialin by means of a protein BLAST search with the BLASTP algorithm. This gave 66 hits of which we selected 21 genes with alignment scores higher than 200 (E-values ranging from 3e-102 to 7e-53) (Table 1). The closest homolog of sialin is CG4288 (E-value 3e-102). According to the cytological location of the genes there are 2 gene clusters. The first gene cluster is located at 55F3-F5 and consists of CG15094, CG15095 and CG15096. The other gene cluster is located at 59B2 and contains CG9826, CG9825, CG30265, CG12490 and CG30272 (Table 1). A phylogenetic analysis reveals that sialin clusters in a group with the Drosophila genes CG9864, CG10207, CG3036, CG4726, CG4288 and CG9887 (Fig. 1A). CG4288 is again the closest Drosophila relative. CG9887 is the Drosophila vesicular glutamate transporter (DVGLUT) (Daniels et al., 2004; Mahr and Aberle, 2006). A protein domain search with the Pfam database reveals that all 21 genes contain a MFS-1 domain (Pfam Accession: PF07690). The Drosophila genome contains 131

Table 1 List of Drosophila homologs of sialin with alignment scores higher than 200 Genea

Cytologya Alignment E-valueb cDNA Scoreb clonea

CG4288 CG4330 CG8098 (Picot)

92E14 11E1 53C15– 53D1 33B9 4D1

369 365 348

3e-102 6e-101 5e-96

GH23975 LD01958 LD22509

320 317

3e-87 1e-86

34C5 22E1

314 313

1e-85 2e-85

LD14545 Not available AT30085 RH74545

301 285

1e-81 5e-77

RE01809 GH07529

284 283

2e-76 3e-76

RE35348 GH05102

CG9864 CG15096 CG8791 CG10207 (Na+-dependent inorganic phosphate cotransporter) CG9826

21F1 55F3– 55F4 55F3 25B1– 25B2 56F11 55F5 43F2 51C5

280 280 273 256

2e-75 2e-75 2e-73 4e-68

LD41684 RH60267 LP07366 RE15779

59B2

233

2e-61

CG9825 CG30265 CG12490

59B2 59B2 59B2

224 223 214

1e-58 4e-58 1e-55

CG7881 CG30272

42A4 59B2

212 205

6e-55 7e-53

Not available RE70413 RE73545 Not available LP04804 Not available

CG5304 (lethal(2)01810) CG6978 CG9254 CG9887 (Vesicular glutamate transporter) CG4726 CGl5095 (lethal(2)08717) CG15094 CG3036

a

Gene names, cytology and cDNA clone IDs are from Flybase (http:// flybase.bio.indiana.edu/). b Alignment scores and E-values are from BLAST (http://130.14.29.110/ BLAST/).

MFS-1 containing proteins. All 21 genes also contain an ACS consensus sequence, although this consensus sequence is not always fully conserved (especially on the first glutamic acid) (Fig. 1B). In the 10 phylogenetically most distant genes the glutamic acid is replaced by a glutamine (Fig. 1A and B). The glutamic acid is also substituted for another amino acid in the clustering genes CG9864, CG10207, CG3036 and CG4726. 1.2. Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene CG4288 expression begins at stage 13 in the salivary glands (Fig. 2A). Stage 16 embryos show additional expression in the posterior spiracles (Fig. 2B and B’). At stage 17 expression is detected in the salivary glands and in the hindgut (Fig. 2C–C’’). We could not detect CG4330 expression during embryogenesis. Expression of CG8098 (Picot = putative inorganic phosphate cotransporter) begins at stage 13 where it is detected in the salivary glands (Fig. 3A).

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Fig. 1. Phylogenetic analysis. (A) Neighbor-Joining phylogenetic tree of sialin and its Drosophila homologs. Sialin clusters in a group with the genes CG9864, CG10207, CG3036, CG4726, CG4288 and CG9887. Numbers indicate bootstrap values for each node, obtained on 1000 replications. The branch length indicates the evolutionary distance between the genes. (B) Partial multiple alignment of the ACS consensus sequence, sialin and its Drosophila homologs. Amino acid identities with the ACS consensus sequence are indicated by black boxes. Similar amino acids are indicated with grey boxes.

Fig. 2. Embryonic expression pattern of CG4288. Nomenclature: sg, salivary glands; ps, posterior spiracles; hg, hindgut. The embryos are shown laterally except for panels (B) and (B’) which are ventral views.

Fig. 3. Embryonic expression pattern of CG8098. Nomenclature: sg, salivary glands; gl, glial cells; mt, Malpighian tubules; hg, hindgut. Panel (A) is a lateral view. Panels (B–C’’) are dorsal views.

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At stage 16 additional expression was detected in a subgroup of midline glial cells (Fig. 3B and B’). Stage 17 embryos still show expression in salivary glands and glial cells together with additional expression in the hindgut and Malpighian tubules (Fig. 3C–C’’). CG5304 expression is visible at stage 10 in the region where the Malpighian tubule primordia are situated (Fig. 4A and A’). At stage 17 CG5304 mRNA is found in the dorsal longitudinal tracheal trunks (Fig. 4B and B’), the posterior portion of the midgut (Fig. 4B and B’) and the somatic muscles (Fig. 4B and B’’). We could not detect any embryonic expression of CG9254. CG4726 is expressed from stage 11 on in the tracheal pits (Fig. 5A–B’). By stage 16 expression in the trachea has disappeared and is now observed in the dorsal median cells (Fig. 5C and C’), in the lateral cord surface glia (Fig. 5C’’) and in somatic muscles (Fig. 5C and C’’’). Transcription of CG15095 (lethal (2) 08717) is first visible at stage 5 at the posterior end of the embryo and also in a segmental pattern in the mid-ventral and mid-dorsal part of the embryo (Fig. 6A). During gastrulation (stages 6–7) the segmental expression in the mid-ventral part of the embryo increases and CG15095 is additionally activated at the amnioproctodeal invagination, at the stomodeal

invagination and in the vitellophages (yolk nuclei) (Fig. 6B and C). The expression at the amnioproctodeal and stomodeal invagination lasts until stage 9. From stage 9 till stage 13 expression is still detected in vitellophages (Fig. 6D–F). Stage 14 and stage 15 embryos show expression in the midgut and in the fat body (Fig. 6G–H). At stage 16 CG15095 expression is seen in the salivary glands and the midgut (Fig. 6I and I’). CG15094 is expressed from stage 10 till stage 13 in the amnioserosa (Fig. 7A–B’). From stage 15 on expression is visible in the salivary glands (Fig. 7C–D’). CG3036 mRNA is first detected during stage 4 at the anterior and posterior end of the embryo and also in the midventral area of the embryo (Fig. 8A). At stage 5 the expression in these areas becomes confined to the blastoderm cells. At this stage CG3036 is also expressed in vitellophages (Fig. 8B). At stage 6 there is still strong expression in the vitellophages. The expression in the anterior end and the midventral part of the embryo expands towards the centre (Fig. 8C). During stage 7 the expression at the anterior end and in the vitellophages is maintained. The midventral and posterior expression now extends to cover the whole germband and the amnioproctodeal invagination (Fig. 8D). Stage 9 embryos show strong expression

Fig. 4. Embryonic expression pattern of CG5304. Nomenclature: mg, midgut; sm, somatic muscles; dltt, dorsal longitudinal tracheal trunks. Panels (A) and (A’) are ventral views. Panels (B–B’’) are lateral views.

Fig. 5. Embryonic expression pattern of CG4726. Nomenclature: tp, tracheal pits; sm, somatic muscles; dmc, dorsal median cells; lcsg, lateral cord surface glia. Panels (A) and (A’) are lateral views. Panels (B–C’’’) are ventral views.

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Fig. 6. Embryonic expression pattern of CG15095. Nomenclature: v, vitellophages (yolk nuclei); sti, stomodeal invagination; api, amnioproctodeal invagination; mp, midgut primordium; fb, fat body; sg, salivary glands; mg, midgut. All embryos are shown laterally except for panels (G–I’) which are ventral views.

Fig. 7. Embryonic expression pattern of CG15094. Nomenclature: as, amnioserosa; sg, salivary glands. Panels (A–C) are lateral views. Panels (D) and (D’) are ventral views.

at the stomodeal invagination, in the ventral neurogenic region and at the amnioproctodeal invagination (Fig. 8E). At stage 10 and stage 11 mRNA of CG3036 is detected in the neuroblasts of the ventral nervous system, in the head region, where it corresponds to the procephalic neuroblasts, and at the amnioproctodeal invagination (Fig. 8F–H’). At stage 13 transcription of CG3036 was detected in the salivary glands, the oenocytes and in the proventriculus (Fig. 8 I and I’). Stage 16 embryos still show expression in the oenocytes and increased expression in the salivary glands (Fig. 8J and J’). CG9864 is only expressed maternally (Fig. 9A and B). CG15096 mRNA is first detected at stage 12 in the anterior and posterior midgut

rudiments (Fig. 10A and A’). At stage 16 expression is visible in the anterior and posterior portions of the midgut (Fig. 10B and B’). CG8791 expression in stage 10–stage 13 embryos is compatible with prohemocytes (Fig. 11A– B). From stage 14 on expression is detected in the salivary glands (Fig. 11C). Stage 16 embryos still show expression in the salivary glands (Fig. 11D and D’) together with expression in cells that coincide with the position of the true telson (Fig. 11D and D’’). CG10207 is expressed at stage 15 in the salivary glands and the Malpighian tubules (Fig. 12A–A’’). Stage 16 embryos only show expression in the salivary glands (Fig. 12B and B’). We could not detect any embryonic expression of CG9825. CG30265 is only

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Fig. 8. Embryonic expression pattern of CG3036. Nomenclature: v, vitellophages (yolk nuclei); api, amnioproctodeal invagination; sti, stomodeal invagination; nb, neuroblasts; pnb, procephalic neuroblasts; sg, salivary glands; pv, proventriculus; oen, oenocytes. The embryos are shown laterally except for panels (H) and (H’) which are dorsal views and panels (J) and (J’) which are ventral views.

Fig. 9. Embryonic expression pattern of CG9864.

expressed in isolated cells of stage 9 embryos, most likely corresponding to vitellophages (Fig. 13A and A’). CG7881 expression begins in the salivary glands at stage 13 (Fig. 14A). From stage 16 on expression is also visible

in the hindgut (Fig. 14B and B’). Stage 17 embryos show expression in two anteriorly situated groups of 6–8 cells (possibly sensory complexes) (Fig. 14C and C’), in the salivary glands (Fig. 14C and C’’) and in the hindgut (Fig. 14C and C’’’). In this study, we identified a group of conserved Drosophila ACS family genes homologous to sialin and studied their expression pattern throughout embryogenesis. Interestingly, while these genes are structurally related, their expression patterns do not overlap significantly. However, some overlap is present in the somatic muscles for CG5304 and CG4726. For CG5304 and CG15096 we find overlap in

Fig. 10. Embryonic expression pattern of CG15096. Nomenclature: amr, anterior midgut rudiment; pmr, posterior midgut rudiment; amg, anterior portion of the midgut; pmg, posterior portion of the midgut. Panels (A) and (A’) are ventral views. Panels (B) and (B’) are lateral views.

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Fig. 11. Embryonic expression pattern of CG8791. Nomenclature: h, prohemocytes; sg, salivary glands. Panels (A–B) are lateral views. Panels (C–D’’) are ventral views.

Fig. 12. Embryonic expression pattern of CG10207. Nomenclature: sg, salivary glands; mt, Malpighian tubules. Panels (A–A’’) are ventral views. Panels (B) and (B’) are lateral views.

Fig. 13. Embryonic expression pattern of CG30265. Panels (A) and (A’) are ventral views.

the midgut. CG3036 and CG15095 are both expressed in vitellophages. Both genes also have a similar expression pattern in stage 7 embryos. At this stage, both genes show a segmental expression in the germband, expression in vitellophages and strong expression at the amnioproctodeal invagination. CG15095 (lethal(2)08717) was previously identified in Kc cells as a putative target of Hairy (Bianchi-Frias et al., 2004). Our in situ hybridizations are compatible with a function of CG15095 as a target of Hairy as they show Hairy-like segmental expression of CG15095 in stage 5–7 embryos. Many genes also showed lumenal staining in the salivary glands at certain stages. We are not certain to what extent this staining is specific as we sometimes see similar lumenal staining with sense probes. However, salivary gland staining of embryos

hybridized with an antisense probe for CG15094 and CG3036 is most probably specific since in these cases staining is not only confined to the lumen. From the 21 ACS family genes related to sialin we identified, two of them were previously identified as essential genes: CG5304 (lethal(2)01810) and CG15095 (lethal(2)08717) (Spradling et al., 1999). So far functional studies have only been performed on CG9887, which was identified as the Drosophila vesicular glutamate transporter (Daniels et al., 2004). Our expression data are a first step towards the identification of the Drosophila ortholog of sialin and will aid the establishment of a fruitfly model of human lysosomal storage disorders. 2. Experimental procedures 2.1. Multiple-sequence alignment and phylogeny Protein alignments were carried out using Clustal X (Thompson et al., 1997) with no adjustment of the default parameters and were subsequently edited and manually improved in Genedoc Multiple Sequence Alignment Editor and Shading Utility, Version 2.6.000 (Nicholas and Nicholas, 1997). Neighbor-joining trees were constructed using the Jones–Taylor– Thornton (JTT) matrix (Jones et al., 1992) with MEGA4 (Tamura et al., 2007). Sites containing gaps were excluded from the analysis by

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Fig. 14. Embryonic expression pattern of CG7881. Nomenclature: sg, salivary glands; hg, hindgut. All embryos are shown laterally. using the pairwise-deletion option. The reliability of branching patterns was assessed by bootstrap analysis (1000 replications). The condensed tree (cut-off value of 50%) was manually reconstructed in Adobe Photoshop CS Version 8.0 using a cut-off value of 50%.

V.I.B., by a Research Programme of the Research Foundation-Flanders (FWO) (G.0397.06N) and by a Marie–Curie International Reintegration Grant (MIRG-CT-2004014819).

2.2. Whole-mount in situ hybridization

References Digoxigenin-labeled sense and antisense RNA probes were synthesized from cDNA clones from the Berkeley Drosophila Genome Project (see Table 1) by in vitro transcription with SP6, T7 and T3 polymerases using the DIG RNA labeling kit (Roche Applied Science). Probe length was reduced to approximately 200 bases by alkaline hydrolysis at 60° C in a solution containing 200 mM Na2CO3 and 200 mM Na2HCO3. Duration of the alkaline hydrolysis was estimated using the formula X = (Lo  Ld)/(0,11  Lo  Ld) with X = hydrolysis time in minutes; Lo = original length of the transcript in kb; Ld = desired length in kb = 0.2. Hydrolysis was stopped and the probe was precipitated by adding a solution of 300 ll ethanol, 5 ll 10% acetic acid, 11 ll 3 M NaAc pH 6.0 and 1.2 ll 1 M MgCl2 followed by incubation overnight at 20 °C and centrifugation (13,000 rpm, 4 °C, 15 min). Pellets were resuspended in 50 ll Ultra Pure Distilled Water (GIBCO) and the yield of DIG-labeled RNA was determined using DIG quantification and control teststrips (Roche Applied Science). Whole-mount in situ hybridization was performed using a variation of the protocol described by (Tautz and Pfeifle, 1989). Signal detection was carried out using anti-digoxigenin-AP Fab fragments (Roche Applied Science). Color development was performed in the dark using 0.5 mg/ml NBT (Roche Applied Science) and 0.25 mg/ml BCIP (Roche Applied Science). Zero to 17 h old yw embryos were used in this study. Embryos were staged according to Hartenstein (1993). Images were obtained using a light microscope (model BX61; Olympus) and Cell^D 2.6 imaging software. Panels labeled with (0 ) represent images of areas marked by frames, taken at higher magnification and in adjusted focal plane.

Acknowledgements We thank Volker Hartenstein for comments on the expression pattern analysis. We also thank Korneel Hens and Jason Clements for help with respectively phylogenetic analysis and Adobe Photoshop CS. B.L. is supported by the Research Foundation-Flanders (FWO) and KULeuven. K.N. is a Senior Clinical Investigator of the Research Foundation-Flanders (FWO). This work was supported by

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