The zebrafish progranulin gene family and antisense transcripts

Abstract

Background: Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish.

Results: The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs.

Conclusion: In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.

Figure 1

Reversed-Phase HPLC purification of granulin-A and granulin-1 from extracts of two carp spleens. Panel A: HPLC fractions derived from an extract of two carp spleens and enriched in granulin-1 immunoreactivity were further purified by HPLC using 0.13% heptafluorobutyric acid as counterion. Two major components were identified, a peptide sharing 58% sequence conservation with human granulin-A (peak A) and granulin-1 (peak 1), each indicated by arrows. Panel B: Sequence comparison of carp granulin peptides with their respective candidate orthologues deduced from cloned zebrafish cDNA sequences (this study), and human granulin-A. Numbers correspond to amino acid position. Characteristic cysteines are shown in bold.

Figure 2

Comparison of the deduced translated sequences for zebrafish progranulin-1 and progranulin-2. Two zebrafish cDNAs sharing 92.3% identity (grn1 and grn2), each possessing a 441 nucleotide-long open reading frame (ORF), encode deduced precursors consisting of one full and one amino-terminal half granulin peptide, with a calculated mass of 14.5 kDa and 14.8 kDa, respectively. Both carry an identical signal peptide (italics), whose predicted cleavage site is indicated by an arrow. The predicted sequences for zebrafish granulins 1 and 2 are highlighted. Numbers correspond to amino acid position.

Figure 3

Genomic organization of zebrafish grn1, grn2 and their complementary antisense gene. Zebrafish grn1 and grn2 genes are found in tandem in a head-to-tail orientation and share an identical exonic organization (exons 1–5, orange and blue boxes, respectively), but differ in their respective intron lengths (A, C, D). The spliced and polyadenylated non-protein coding ASgrn1-2 is encoded on four exons (pink boxes) and shares exon/intron complementarity to both grn1 and grn2 (see text for details). Shown on top is a schematic representation of the chimeric progranulin transcript, suggesting trans-splicing as a mechanism for its generation. Bars (-) above or below exons indicate the relative position of primer pairs (grn1+2 forward and reverse; ASgrn1-2 forward and reverse) used for discriminating between ASgrn1-2 and combined grn1/grn2 expression using RT-PCR.

Figure 4

Sequence comparison of zebrafish progranulin-A and -B with human progranulin. Amino acid sequences were deduced from the cloned grna and grnb zebrafish cDNAs. Unlike human progranulin, which carries one-half and seven granulin peptide motifs, zebrafish progranulin-a and progranulin-b harbour 10 and 9 full copies of the granulin motif (boxed), and possess distinct putative signal peptides (italics). Sequences were aligned using the ClustalW method, and gaps were introduced as dashed lines for optimal alignment. Identical residues are indicated by an asterisk. Numbers on the right represent amino acid position. Human granulin motif nomenclature is listed (right).

Figure 5

Chromosomal assignment of zebrafish grn genes. Zebrafish grna is located close to genes (HoxB cluster, dlx8, pyy) that form an extensive bloc of conserved synteny with human chromosome 17 (Hsa17), indicating an orthologous relationship to human progranulin (grn). Zebrafish grnb, in contrast, maps to LG24 in a region devoid of syntenic correspondence to zebrafish LG3 or Hsa17 (data not shown). Grn1 and grn2 map to LG19, in a region that finds scattered synteny to two human chromosomes (Hsa 6 and Hsa 7). The presence of grn1 and grn2 on a zebrafish chromosome bearing the HoxA cluster, npy and dlx6 genes (i.e. paralogues of genes linked to zebrafish grna and human grn), suggests that grn1 or grn2 may have originated in concert with the mechanism leading to emergence of duplicated Hox clusters at the base of the vertebrate radiation. Map position on zebrafish chromosomes (LG) is presented in centiRays where 1 centiRay = 148 kilobases, the estimated average breakpoint frequency for the LN54 RH panel.

Figure 6

RT-PCR Analysis of zebrafish grns in adult tissues. Panel A: Zebrafish grna and grnb are ubiquitously expressed in various organs. Panel B: A comparison of the combined expression of grn1 and grn2 (grn1+2) relative to their antisense transcript. An increased number of cycles was used in the PCR to allow for the detection of ASgrn1-2 transcripts. Panel C: grn1, grn2 and hybrid grn are differentially regulated with the latter expressed only in the intestine. Results shown were generated using the forward and reverse1 primer pair (Additional File 10). Identical results were obtained using the forward and reverse2 primer pair (not shown). Hybrid grn was amplified using a grn1 forward and grn2 reverse primer combination. No product was obtained using a grn2 forward and grn1 reverse primer pair (not shown). Number of cycles for each reaction is indicated. Amplified PCR products were analyzed by electrophoresis next to a 100-bp DNA ladder. No template and amplification of actin mRNA were used as negative and positive controls, respectively. Similar results were obtained with two other experiments.

Figure 7

RT-PCR analysis of zebrafish grn expression during development. Panel A: grna and grnb transcripts are detected throughout all stages of development, whereas grn1 and grn2 expression is first detected by 48 hours post-fertilization. Maternal expression of grnb is more abundant than grna. Panel B: Combined expression of grn1 and grn2 relative to their antisense transcript. Ethidium bromide stain reveals the presence of sense transcription only (top). Detection of the antisense transcript is revealed by using a ³²P-labelled oligo as probe that recognizes both sense and antisense amplicons after Southern transfer. Note weak expression of grn1 and/or grn2 at earlier stages of development. Numbers of cycles used for the PCR are indicated. No template and actin were used as negative and positive controls, respectively. Developmental stages are as follows according to Kimmel et al. 1995: cleavage (16-cell); high (mid-blastula, 3 hpf); sphere-dome (late blastula, 4–4.3 hpf); shield (50% epiboly, 6 hpf); tailbud (10 hpf); 3–7 somites (11–12 hpf); 12–14 somites (14–16 hpf); early (24 hpf) and late (48 hpf) pharyngula; hatching (72 hpf); ealy larval period (96 and 120 hpf). Gene specific primers and amplicon sizes are listed in Additional File 10.

Figure 8

Developmental expression analysis of zebrafish grna and grnb mRNAs by whole mount in situ hybridization. An ontogeny of expression conducted for grna (A) and grnb (B) revealed similar expression patterns for these genes from fertilization to late segmentation stage (a–f), with grna being weaker than grnb. At the 4-cell stage (a) and 50% epiboly (b) ubiquitous expression is observed for grnb only. A lateral view of the 6-somite stage embryo (c) reveals discernable ubiquitous expression above background levels for grna, and increased grnb expression in the epithelium of the eye primordium and CNS, as well as the caudal region. In a dorsal view of the same animals (d), caudal expression in the axial mesoderm (arrow) is observed for both genes, whereas only grnb is detected in the paraxial mesoderm (arrowheads). Lateral (e) and frontal (f) views of the late somitogenesis stage embryo (18–20 hpf) show continued expression in the eye primordium, CNS and tailbud for both genes. In addition, grnb can be detected in the YSL (arrow) (e) and the adaxial cells (arrowheads) (f) flanking the axial mesoderm. At 24 hpf (g), grna expression is found in the tectum and eye retina, in a diffuse pattern in the anterior endoderm (arrowhead) and in a punctuate pattern within the ventral tail region of the ICM (arrow), whereas elevated expression persists for grnb in the forebrain, midbrain and ventral hindbrain region, the eyes, as well as in the YSL, concentrated at the tip of the yolk extension (arrow). In a lateral view at 48 hpf (h), grna expression in the ICM extends rostrally, is detected in the head vasculature, and is now apparent in the skin epithelium, whereas in a lateral view (i) both grna and grnb are transiently expressed in the AER of the pectoral fin buds (arrows). At 72 hpf (j), grna, but not grnb, is expressed in presumed dispersed leukocytes, while in a dorsolateral view (k), grnb can be detected in the swim bladder (arrow). AE, anterior endoderm; AER, apical ectodermal ridge of the pectoral fin buds; ICM, intermediate cell mass; LPM. lateral plate mesoderm; YSL, yolk syncytial layer.

Figure 9

Expression analysis of zebrafish grna and grnb mRNAs by whole mount in situ hybridisation at 5 dpf. At 120 hpf grna (A) exhibits widespread expression in the visceral region, including the pronephric kidneys (arrowheads in sections 1 and 3) and intestine (arrow in section 2), while grnb (B) remains expressed in the YSL and pancreas (arrows in section 1) and is strong in the proctodeum region of the intestine (arrow in section 2). At this stage, a hybridization signal for the sense riboprobe to grna, but not grnb, is detected in the brain, intestine and pronephric ducts (b). Numbered arrows denote the position of corresponding sections shown below (magnified 10×). PD, pronephric ducts; YSL, yolk syncytial layer.

Figure 10

Expression analysis of grn1, grn2, hybrid grn, and ASgrn1-2 in the hatching stage zebrafish embryo by whole mount in situ hybridization. Panel A: grn1 is expressed in the intestine and pharyngeal region (arrow), and at low levels in the pronephric ducts. Panel B: In contrast, grn2 mRNA is only weakly detected in the pharyngeal region (arrow) and the proctodeum (arrowhead), and is occasionally found in dispersed leukocytes (not shown). Panel C: The abundance of the trans-spliced product (hybrid grn) is stronger than grn2 in the proctodeum (arrowhead), but absent in the pharyngeal region. Panels D–F: The corresponding sense riboprobes to grn1 and grn2, but not to hybrid grn, detect ASgrn1-2 expression in the pharyngeal region (arrows). These expression patterns were reproduced in at least three independent experiments.

Figure 11

Expression analysis of grn1, grn2, hybrid grn, and ASgrn1-2 in the 5 day-old zebrafish larva by mRNA in situ hybridization. Panels A–C: grn1 is expressed in the intestine (arrow), swim bladder, and more abundantly in the head kidneys (arrowheads) than in the pronephric ducts. Panels D–F: grn2 is expressed similarly to grn1 in the head kidneys (arrowheads) and pronephric ducts, but is undetected in the intestine. In contrast, grn2 is strongly expressed in the brain and the branchial jaw region (compare A with D, and C with F), is distributed in a punctuate pattern along the ventral region of the animal in presumed myeloid progenitors (asterisks in D), and often found in randomly dispersed leukocytes (large cells in F). Panel G: Hybrid grn is found exclusively in the proctodeum. Panel H: The sense riboprobe to ASgrn1-2 (devoid of the tzf sequence) recapitulates the combined expression patterns for grn1 and grn2. Panels (I–N): Sense riboprobes to grn1 (I,J) or grn2 (K,L), but not hybrid grn (M), show that antisense transcription occurs in the jaw region (arrows in I and K), the swim bladder, and in the mid-region of the intestine, in a pattern identical to that observed for the antisense probe corresponding to ASgrn1-2 (N). B, E: dorsal views; C, F, I, K: ventral views. For each target mRNA, the use of corresponding sense and antisense (AS) riboprobes is indicated. OC, presumed ossification center; HK, head kidney; PD, pronephric duct; SB, swim bladder.

Figure 12

Diagrammatic representation of the structures and evolutionary origins of the granulin in multicelluar organisms. Evolutionary distances in millions of years derived from Hedges 2002 [77]. The estimated rounds of vertebrate genome duplication events are indicated (1R, 2R, 3R). The various progranulin structures were derived from various databases as outlined below. Land plant – (Arabidopsis thaliana) – Papain-like thiol protease bearing a carboxyl-terminal granulin domain (AAK71314); Slime mold – (Dictyostelium discoideum) – 1 copy progranulin from a single EST (AU267401) Trematode worm (Schistosoma japonicum) – 9 copy progranulin built from a combination of four ESTs (AY810079, BU790215, BU799560, BU771494); Nematode worm – (Caenorhabditis elegans) – 3 copy progranulin from single EST (NM_060580) – overall architecture confirmed by genome sequence (Z81595); Annelid worm (earthworm – Lumbricus rubellus) – 1 copy progranulin from a single EST (CO046860); Primitive chordate – (Sea squirt, Ciona intestinalis) – 7 copy progranulin predicted from draft genomic sequence (AABS01000126) and overall architecture confirmed by ESTs. Domains 2 to 7 nearly identical (BW368775, BW311239). Amphibian – (Frog, Xenopus laevis) co-orthologues progranulins A and B consisting of one-half domain followed by 12 full domains and one-half domain followed by nine full domains respectively [78]. These are structurally closely related and are a result of a recent tetraploidization event 30 mya [79]. Avian (chicken – Gallus gallus) – 4 copy progranulin built from three ESTs (BM440305, BU297352 and BX265765) – overall architecture confirmed by genome sequence (LOC426606); Human (Homo sapiens) – Progranulin composed of one half domain followed by 7 domains (UniProt entry: P28799); Teleost – Takifugu rubripes – 2, 3 and 11 copy progranulins predicted from draft genome sequences (M000077, S002118, S0001020) with overall structures confirmed by ESTs (CA846088, CA332411, AL842916, CA588603). Teleost – Danio rerio: from this study: co-orthologue progranulins A and B composed of 10 (NM_001001949) and 9 (AY289606) respectively; two smaller progranulins consisting of 1 and one half granulin repeats (AF273479, AF273480).