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In developmental and cell biology it is crucial to evaluate the dynamic profiles of metabolites. An emerging frog model system using Xenopus tropicalis, whose genome sequence and inbred strains are available, is now ready for metabolomics investigation in amphibians. In this study we applied matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) analysis to identify and visualize metabolomic molecular markers in tadpoles of Xenopus tropicalis We detected tissue-specific peaks and visualized their distribution in tissues, and distinguished 19 tissues and their specific peaks. We identified, for the first time, some of their molecular localizations via tandem mass spectrometric analysis: hydrocortisone in artery, L-DOPA in rhombencephalon, taurine in eye, corticosterone in gill, heme in heart, inosine monophosphate and carnosine in muscle, dopamine in nerves, and phosphatidylethanolamine (16:0/20:4) in pharynx. This is the first MALDI-MSI study of X. tropicalis tadpoles, as in small tadpoles it is hard to distinguish and dissect the various organs. Furthermore, until now there has been no data about the metabolomic profile of each organ. Our results suggest that MALDI-MSI is potentially a powerful tool for examining the dynamics of metabolomics in metamorphosis as well as conformational changes due to metabolic changes.
Fig. 1. Characteristics of tadpole samples. Left panel shows an upper view of the head of a tadpole. The dotted lines (a, b and c) show the position of each cross section cut. Hematoxylin and eosin−stained cross sections at positions a, b and c are shown in the right panel. Scale bar: 2 mm.
Fig. 2. Mass spectra of extracts from 10 different tissues in negative ion mode. Detected peaks are summarized in Table 1.
Fig. 3. Scheme of mass spectrometry imaging. Consecutive sections were cut using a cryostat and mounted onto ITO-coated glass slides for MSI. Slides were uniformly sprayed with a matrix solution. Two-dimensional laser scanning was performed to get mass spectra from each spot. Image reconstruction from each peak was done by specialized imaging software.
Fig. 4. Mass spectrometry imaging for each tissue-specific peak. In section 1 molecular ions specifically distributed in the retina, olfactory bulb, lens, choanal canal, internal gills, and epidermis. In the same manner, inner ear, rhombencephalon, nerves, internal gills, pharynx, heart, and artery were separated by tissue-specific peaks in section 2, and molecular markers of muscle, notochord, gonad, liver, stomach, spleen, gall bladder, and intestine were imaged in section 3. Table 2 lists the detected peaks. We tested 3 individual animals and confirmed the same localization (N=3).
Fig. 5. Identification of molecular marker for each tissue. Eight distinct tissue-specific molecular markers were identified by tandem mass spectrometric analyses on tissue sections. The result of each analysis and tissue localizations are shown. PE, phosphatidylethanolamine.
Xenopus tropicalis tadpole NF stage 56, head and pharyngeal region in cross section: Left panel shows an upper view of the head of a NF stage 56 tadpole. The dotted lines (a, b and c) show the position of each cross section cut. Hematoxylin and eosinâ stained cross sections at positions a, b and c are shown in the right panel. Scale bar: 2 mm.
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