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. 2014 Dec 6;15:1074.
doi: 10.1186/1471-2164-15-1074.

Transcriptomic Analysis of the Lesser Spotted Catshark (Scyliorhinus Canicula) Pancreas, Liver and Brain Reveals Molecular Level Conservation of Vertebrate Pancreas Function

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Free PMC article

Transcriptomic Analysis of the Lesser Spotted Catshark (Scyliorhinus Canicula) Pancreas, Liver and Brain Reveals Molecular Level Conservation of Vertebrate Pancreas Function

John F Mulley et al. BMC Genomics. .
Free PMC article

Abstract

Background: Understanding the evolution of the vertebrate pancreas is key to understanding its functions. The chondrichthyes (cartilaginous fish such as sharks and rays) have often been suggested to possess the most ancient example of a distinct pancreas with both hormonal (endocrine) and digestive (exocrine) roles. The lack of genetic, genomic and transcriptomic data for cartilaginous fish has hindered a more thorough understanding of the molecular-level functions of the chondrichthyan pancreas, particularly with respect to their "unusual" energy metabolism (where ketone bodies and amino acids are the main oxidative fuel source) and their paradoxical ability to both maintain stable blood glucose levels and tolerate extensive periods of hypoglycemia. In order to shed light on some of these processes, we carried out the first large-scale comparative transcriptomic survey of multiple cartilaginous fish tissues: the pancreas, brain and liver of the lesser spotted catshark, Scyliorhinus canicula.

Results: We generated a mutli-tissue assembly comprising 86,006 contigs, of which 44,794 were assigned to a particular tissue or combination of tissues based on mapping of sequencing reads. We have characterised transcripts encoding genes involved in insulin regulation, glucose sensing, transcriptional regulation, signaling and digestion, as well as many peptide hormone precursors and their receptors for the first time. Comparisons to mammalian pancreas transcriptomes reveals that mechanisms of glucose sensing and insulin regulation used to establish and maintain a stable internal environment are conserved across jawed vertebrates and likely pre-date the vertebrate radiation. Conservation of pancreatic hormones and genes encoding digestive proteins support the single, early evolution of a distinct pancreatic gland with endocrine and exocrine functions in jawed vertebrates. In addition, we demonstrate that chondrichthyes lack pancreatic polypeptide (PP) and that reports of PP in the literature are likely due cross-reaction with PYY and/or NPY in the pancreas. A three hormone islet organ is therefore the ancestral jawed vertebrate condition, later elaborated upon only in the tetrapod lineage.

Conclusions: The cartilaginous fish are a great untapped resource for the reconstruction of patterns and processes of vertebrate evolution and new approaches such as those described in this paper will greatly facilitate their incorporation into the rank of "model organism".

Figures

Figure 1
Figure 1
Phylogenetic tree of the major extant vertebrate groups. The relationships of the most common chondrichthyan (cartilaginous fish) model species (Elephant shark, Callorhinchus milii; Little skate, Leucoraja erinacea; Lesser spotted catshark, Scyliorhinus canicula; Spiny dogfish, Squalus acanthias) are shown, as are representative lineages from the ray-finned (actinopterygian) and lobe-finned (sarcopterygian) fish. The origin of the combined endocrine and exocrine pancreatic gland at the base of the jawed vertebrates is indicated.
Figure 2
Figure 2
Tissue distribution of transcripts, as determined by mapping the sequencing reads derived from each tissue to a combined, all-tissue assembly. Contig values of ≥1 FPKM (Fragments Per Kilobase of exon per Million fragments mapped) were taken as evidence for expression.
Figure 3
Figure 3
Length and tissue distribution of open reading frames (ORFs) derived from assembled contigs.
Figure 4
Figure 4
Proportion of transcripts assigned to each of the top 25 gene ontology (GO) slim ‘Biological Process’ terms for catshark pancreas, brain and liver tissue-specific transcripts.
Figure 5
Figure 5
Proportion of transcripts assigned to each of the top 25 gene ontology (GO) slim ‘Molecular Function’ terms for catshark pancreas, brain and liver tissue-specific transcripts.
Figure 6
Figure 6
Annotation of the precursor peptides of catshark preproinsulin (A) and preproglucagon (B). Signal peptides (amino acids 1–24 and 1–20 respectively) are underlined and basic amino acid cleavage sites are lowercase. Glucagon-like peptides (GLP) 1a and 1b are annotated based on similarity to the duplicated GLP1 peptides in the unpublished Squalus acanthias and Hydrolagus colliei proglucagon sequences available on Genbank (accession numbers AAS57653 and AAS57654). An oxyntomodulin-like peptide has been purified from H. colliei and corresponds to amino acids 47–82 in the catshark preproglucagon.
Figure 7
Figure 7
Amino acid alignment of vertebrate Peptide YY (PYY), Neuropeptide Y (NPY) and Pancreatic polypeptide (PP) sequences. Genbank accession numbers are given in square brackets. Sca, Scyliorhinus canicula (lesser spotted catshark); Sac, Squalus acanthias (spiny dogfish); Ler, Leucoraja erinacea (little skate); Cmi, Callorhinchus milii (elephant shark); Hsa, Homo sapiens (human); Lfl, Lampetra planeri (brook lamprey); Loc, Leucoraja ocellata (winter skate).
Figure 8
Figure 8
Immunolocalization of pancreatic hormones and pancreatic polypeptide and PYY in catshark pancreas. (A) The distribution of the pancreatic hormones insulin (blue), glucagon (green) and somatostatin (Red) in uniquely shaped islet structures. (B) Pancreatic polypeptide (red) specific antisera fail to stain a specific subset of endocrine cells in the pancreas, while insulin (blue) and glucagon show a normal distribution. (C-D) PYY shows colocalization with most of the insulin immunoreactive cells but not glucagon or somatostatin (E-F). All images are 250x magnification.
Figure 9
Figure 9
Protein domains in vertebrate PDX1 and PDX2. Transactivation domains A-E[119], PCIF1-interaction domains [118], homeodomains, DNA-binding domains (i) and nuclear localisation signals (ii) are highlighted. Domain E contains the PBX-interacting hexapeptide motif [117]. There is very little conservation of amino acid sequence between the paralogous PDX1 and PDX2 suggesting that they carry out distinct functions within the pancreas, although clearly both are localised to the nucleus, bind DNA and interact with PBX proteins. Hsa, human (Homo sapiens); Mmu, mouse (Mus musculus); Rno, rat (Rattus norvegicus); Gga, chicken (Gallus gallus); Acar, Anole lizard (Anolis carolinensis); Xla, Xenopus laevis; Xtr, Xenopus tropicalis; Lme, Indonesian coelacanth (Latimeria menadoensis); Lch, African coelacanth (Latimeria chalumnae); Acal, Bowfin (Amia calva); Loc, Spotted gar (Lepisosteus oculatus); Dre, zebrafish (Danio rerio); Tru, fugu (Takifugu rubripes); Ola, medaka (Oryzias latipes); Gac, stickleback; Tni, Green spotted puffer (Tetraodon nigroviridis); Sca, lesser spotted catshark (Scyliorhinus canicula); Ler, little skate (Leucoraja erinacea); Cmi, elephant shark (Callorhinchus milii); Bfl, amphioxus (Branchiostoma floridae).
Figure 10
Figure 10
Stages of vertebrate pancreas evolution. The earliest vertebrates likely possessed diffuse islet organs associated with the bile duct as found in extant jawless fish lineages such as hagfish and lampreys. The development of a distinct pancreatic gland with endocrine (insulin, glucagon and somatostatin) and exocrine (digestive) functions is a jawed vertebrate innovation and only coelacanths and tetrapods possess a four hormone (insulin, glucagon, somatostatin and pancreatic polypeptide (PP)) organ. Many of the key genes in jawed vertebrate pancreas development and function are members of multi-gene families, produced during the two rounds of whole genome duplication that took place early in vertebrate evolution.

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