Views of the NIDA, NINDS Or the National Summed Across the Three Auditory Forebrain Lobule Sec- Institutes of Health
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Xie et al. BMC Biology 2010, 8:28 http://www.biomedcentral.com/1741-7007/8/28 RESEARCH ARTICLE Open Access The zebra finch neuropeptidome: prediction, detection and expression Fang Xie1, Sarah E London2,6, Bruce R Southey1,3, Suresh P Annangudi1,6, Andinet Amare1, Sandra L Rodriguez-Zas2,3,5, David F Clayton2,4,5,6, Jonathan V Sweedler1,2,5,6* Abstract Background: Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions. Results: Complementary bioinformatic resources were integrated to survey the zebra finch genome, identifying 70 putative prohormones. Ninety peptides derived from 24 predicted prohormones were characterized using several MS platforms; tandem MS confirmed a majority of the sequences. Most of the peptides described here were not known in the zebra finch or other avian species, although homologous prohormones exist in the chicken genome. Among the zebra finch peptides discovered were several unique vasoactive intestinal and adenylate cyclase activating polypeptide 1 peptides created by cleavage at sites previously unreported in mammalian prohormones. MS-based profiling of brain areas required for singing detected 13 peptides within one brain nucleus, HVC; in situ hybridization detected 13 of the 15 prohormone genes examined within at least one major song control nucleus. Expression mapping also identified prohormone messenger RNAs in areas associated with spatial learning and social behaviours. Based on the whole-genome analysis, 40 prohormone probes were found on a commonly used zebra finch brain microarray. Analysis of these newly annotated transcripts revealed that six prohormone probes showed altered expression after birds heard song playbacks in a paradigm of song recognition learning; we partially verify this result experimentally. Conclusions: The zebra finch peptidome and prohormone complement is now characterized. Based on previous microarray results on zebra finch vocal learning and synaptic plasticity, a number of these prohormones show significant changes during learning. Interestingly, most mammalian prohormones have counterparts in the zebra finch, demonstrating that this songbird uses similar biochemical pathways for neurotransmission and hormonal regulation. These findings enhance investigation into neuropeptide-mediated mechanisms of brain function, learning and behaviour in this model. Background interest in songbird neurobiology is the set of telence- Songbirds, including zebra finches (Taeniopygia guttata), phalic nuclei, referred to collectively as the song control are well-established model organisms for a variety of system. This brain circuit is required for vocal learning biological functions and are notable for their complex and song production in male zebra finches and in other natural behaviours such as vocal communication, learn- songbirds and is also connected to the auditory fore- ing and social living structures [1-3]. Of particular brain lobule that provides the system with auditory information [4,5]. * Correspondence: [email protected] Neuropeptides, a complex group of cell-to-cell signalling 1 Department of Chemistry, University of Illinois at Urbana-Champaign, molecules, can act as neurotransmitters, neuromodulators, Urbana, IL, 61801 USA © 2010 Xie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Xie et al. BMC Biology 2010, 8:28 Page 2 of 17 http://www.biomedcentral.com/1741-7007/8/28 or peptide hormones [6,7]. A few neuropeptides have been sequences for some matches. The GenBank zebra finch previously examined in songbirds [8-15]; those studies expressed sequence tag (EST) database was used to con- demonstrated that neuropeptides could act within brain firm the identification and recover sequences. For regions relevant to song and other behaviours. Given the instance, somastatin (SST) was identified using the EST potential for these signalling molecules to impact a wide [GenBank:CK234915] because of insufficient genome cov- range of behaviorally-relevant neural functions, the present erage and sequencing errors. In other cases, the lack of study aimed to identify a large number of neuropeptides. genome and EST sequences prevented complete recovery Neuropeptide research is complicated by several of the prohormone. As an example, only a 22 amino acid different factors. Typically, the biosynthesis of neuro- prediction was obtained for appetite-regulating hormone peptides starts with the production of a large protein (ghrelin/obestatin prepropeptide, GHRL) compared to the prohormone, which undergoes a variety of processing 116 amino acid chicken GHRL protein sequence. events before the final products-bioactive peptides-are Slight differences in assembly releases resulted in the generated. The gene that codes for a neuropeptide may incomplete presence of nociceptin (PNOC) and pancrea- also contain sequences encoding several other peptides. tic polypeptide (PPY) prohormones. An EST [Genbank: Peptides can be predicted from prohormone sequences CK234392] was matched to the chicken PNOC and based on common proteolytic cleavage sites [16-19] and translation of the EST recovered the first 77 amino directly measured in their bioactive forms from brain acids. This EST was not present in the genomic data as samples [20]. The processing of a single prohormone only 35 bases were matched in the trace archives. can vary depending on the tissues and/or the develop- A match for PPY was identified in the pre-release mental stages and, therefore, neuropeptide localization assembly but not in the release assembly. However, is not always consistent with transcript localization. there was no supporting EST data. The complete Consequently, the comprehensive identification, mea- chicken PPY prohormone was reported in UniProt but surement and localization of neuropeptides in any spe- was not present in the available chicken genome. Pep- cies require a multi-faceted approach. tide sequences have also been reported in UniProt for Taking advantage of the newly-released zebra finch the gull, turkey and ostrich, implying that the zebra genome sequence [21], we predict, measure and localize finch version may be present. the expression of a large complement of neuropeptides Using the EST database and chicken data, possible in the zebra finch brain using a variety of techniques. A alternative splicing was detected for six genes. survey of the zebra finch prohormone complement was Three prohormones - pituitary adenylate cyclase-acti- undertaken using bioinformatic tools. These results vating polypeptide (ADCYAP1), glucagon (GLUC) and were then used to annotate prohormone probes on a vasoactive intestinal peptide (VIP) - have been reported widely used zebra finch microarray platform [22]. Neu- with alternative isoforms in chicken. Tachykinin 1 ropeptidomic analyses using previously described mass (TAC1) has multiple mammalian isoforms and two spectrometry (MS) approaches [20,23-26] were indepen- zebra finch isoforms were identified and subsequently dently conducted to identify the signalling peptides cre- confirmed by ESTs. However, although no chicken ated from these genes in the zebra finch brain and TAC1 isoforms have been reported, four TAC1 chicken pituitary. In situ hybridization (ISH) was performed for isoforms are predicted in the corresponding National a subset of prohormone genes. Both ISH and MS profil- Center for Biotechnology Information gene entry. Two ing were employed to localize the potential for neuro- prohormones, augurin or chromosome 2 open reading peptide function in individual song control nuclei. The frame 40 (C2orf40) and urotensin 2 domain containing integration of these different methodologies results in a (UTS2D), had a single isoform that was supported by more comprehensive suite of neuropeptide data that will EST data. In both cases, an alternative sequence was accelerate investigation into their function in songbirds. predicted using the chicken sequence with Wise2 [27]. The zebra finch prohormone complement is similar to Results and discussion the chicken and to mammals, with evidence for 68 pro- Genomic annotation of neuropeptide prohormone genes hormone homologues in either or both the avian and There were 70 matches to known chicken and mammalian mammalian genomes. This included six prohormones neuropeptide prohormone genes in the zebra finch gen- that matched the chicken genome reported by Delfino et ome resources, resulting in the identification of 51 prohor- al. [28]. Urocortin 1 (UCN), identified in the zebra finch mones with complete sequences. Table 1 provides the by EST [GenBank:DV950835], was not identified in the predicted zebra finch prohormones and homologous chicken genome. However, UCN still may be