Neuropeptidomic analysis of the embryonic Japanese quail diencephalon

Abstract

Background: Endogenous peptides such as neuropeptides are involved in numerous biological processes in the fully developed brain but very little is known about their role in brain development. Japanese quail is a commonly used bird model for studying sexual dimorphic brain development, especially adult male copulatory behavior in relation to manipulations of the embryonic endocrine system. This study uses a label-free liquid chromatography mass spectrometry approach to analyze the influence of age (embryonic days 12 vs 17), sex and embryonic day 3 ethinylestradiol exposure on the expression of multiple endogenous peptides in the developing diencephalon.

Results: We identified a total of 65 peptides whereof 38 were sufficiently present in all groups for statistical analysis. Age was the most defining variable in the data and sex had the least impact. Most identified peptides were more highly expressed in embryonic day 17. The top candidates for EE2 exposure and sex effects were neuropeptide K (downregulated by EE2 in males and females), gastrin-releasing peptide (more highly expressed in control and EE2 exposed males) and gonadotropin-inhibiting hormone related protein 2 (more highly expressed in control males and displaying interaction effects between age and sex). We also report a new potential secretogranin-2 derived neuropeptide and previously unknown phosphorylations in the C-terminal flanking protachykinin 1 neuropeptide.

Conclusions: This study is the first larger study on endogenous peptides in the developing brain and implies a previously unknown role for a number of neuropeptides in middle to late avian embryogenesis. It demonstrates the power of label-free liquid chromatography mass spectrometry to analyze the expression of multiple endogenous peptides and the potential to detect new putative peptide candidates in a developmental model.

Figure 1

Unsupervised hierarchical clustering and principal component analysis. (A) Visualization of group differences (ed12MC, ed12FC, ed12ME, ed12FE, ed17MC, ed17FC, ed17ME, ed17FE) for 204 peptides, using two-way clustering of the log2 ratio (Xclass-Xtot) between the group median value (X_class) and the total median value for every peptide (X_tot). (B) One-way clustering of 38 identified peptides. Positive values are colored red, negative are green. (C) Principal component analysis using two components (C1, C2). The last letter(s) in the group definitions ed12/17XX stand for M = male, F = female, C = controls, E = ethinylestradiol exposed. # designates the peptide numbers in the data set.

Figure 2

PLS/DA on age and EE₂ exposure using 204 quail peptides. (A) Three-dimensional score plot for all observations using the first three components (explaining ~75% of the data) in a model on age and EE₂ exposure effects. The PLS/DA model uses the dummy variables DA1 (ed12C, red color), DA2 (ed12E, green color), DA3 (ed17C, blue color), and DA4 (ed17E, black color). In (B), the second and third components are combined in a score scatter plot, separating controls (red and blue triangles) from EE₂ individuals (green and black boxes). The influence of identified peptides on the positioning of samples in the score scatter plot is seen in the loading scatter plot (C). The 38 identified peptides in the loading plot are listed in (D). Score/loading axes for DC2 and DC3 are designated t(2)/w*c(2) and t(3)/w*c(3) respectively. ed12/17C designates ed12/17 controls and ed12/17E designates ethinylestradiol exposed.

Figure 3

Virtual importance in the projection plot (VIP). The VIP shows the role of identified peptides in the separation of the four age and EE₂ exposure PLS/DA classes (ed12C, ed12E, ed17C, ed17E). The preprotachykinin-1 neuropeptide K (NPK) is the top candidate among identified peptides for being affected by both age and EE₂ exposure. Values above one are the most relevant for the separation of all classes. Unidentified peptides are not shown. Error bars designate confidence intervals derived by jack knifing.

Figure 4

Candidate diencephalon peptides sensitive to sex differences or EE₂. Visualization of the differences of candidate peptides (NPK, CRMP-2, GnIH-RP2, and GRP) between all eight groups (ed12MC, ed12FC, ed12ME, ed12FE, ed17MC, ed17FC, ed17ME, ed17FE) as calculated (Xclass-Xtot) for the hierarchical clustering in figure 1. The stars designate significance levels (*** p < 0.001, ** p < 0.01 and * p < 0.05) for the different factors in the model. ^a FDR adjusted p-value significance for F-test. Interaction effects were tested using Wald F-test. The last letters in the group definitions ed12/17XX stand for M = male, F = female, C = controls, E = ethinylestradiol exposed.

Figure 5

Japanese quail secretogranins. (A) Overview of avian secretogranin-2 (based on chicken sequence XP_422624) showing its granin protein domain and the position and multiple sequence alignment to other species for two identified peptides: TNEIVEEQYTPQSL (Secretoneurin peptide) and SGKLSFLEDE. Bt is Bos taurus, Ss is Sus scrofa, Hs is Homo sapiens, Mm is Mus musculus, Gg is Gallus gallus, Xl is Xenopus leavis, and Dr is Danio rerio. (B) MS/MS fragmentation graph for SGKLSFLEDE in GPM browser. RI stands for relative intensity. C, Visualization of expression differences between the groups as calculated (Xclass-Xtot) for the hierarchical clustering in figure 1. The stars designate significance levels (*** p < 0.001, ** p < 0.01 and * p < 0.05) for the different factors in the model. ^a FDR adjusted p-value significance for F-test. Interaction effects were tested using Wald F-test. The last letters in the group definitions ed12/17XX stand for M = male, F = female, C = controls, E = ethinylestradiol exposed.

Figure 6

The preprotachykinin-1 peptides. SLNSGSSERSIAQNYE is similar to the C-terminal flanking peptide (CTFP) in mammalian protachykinin 1. Two previously unknown serine phosphorylations at positions S6 or S7 and S10 were discovered. (A) FTICR derived MS/MS spectra for single phosphorylated form (Mw 1820) at S6 or S7 and (B) S10 (Mw 1900.3). Amino acid sequence alignment (C) between chicken (continuous sequence in black color) and human (red text) preprotachykinin 1 precursor shows the differences between birds and mammals in the detected tachykinins, especially neuropeptide K (NPK). (D) Visualization of differences between the groups as calculated (Xclass-Xtot) for the hierarchical clustering in figure 1. The stars designate significance levels (*** p < 0.001, ** p < 0.01 and * p < 0.05) for the different factors in the model. ^a FDR adjusted p-value significance for F-test. Interaction effects were tested using Wald F-test. The last letters in the group definitions ed12/17XX stand for M = male, F = female, C = controls, E = ethinylestradiol exposed.