Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 31;114(5):E717-E726.
doi: 10.1073/pnas.1620755114. Epub 2017 Jan 17.

Transcriptomic, Proteomic, and Metabolomic Landscape of Positional Memory in the Caudal Fin of Zebrafish

Affiliations
Free PMC article

Transcriptomic, Proteomic, and Metabolomic Landscape of Positional Memory in the Caudal Fin of Zebrafish

Jeremy S Rabinowitz et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.

Keywords: caudal fin; growth control; positional memory; regeneration; zebrafish.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transcriptomic and proteomic mapping of positional information in uninjured caudal fins. (A) Illustration of the three regions of the fin that were harvested for RNA-seq or LFQ proteomics (see also Fig. S1). (B) Heat maps of 566 transcripts or 238 proteins with proximally enriched (green) or distally enriched (blue) gradients. Each transcript was differentially expressed (FDR < 1%) between each region, and in the same direction across regions. The transcript values shown are the average RPM from all biological replicates, normalized to the middle region, and then log2 transformed. The average protein abundance from all technical replicates is shown as log2 abundance normalized to the middle region. All protein values for the heat map are derived from experiment 3 only (Fig. S1). Each protein was differentially expressed (FDR < 5%) between proximal and distal regions (see Dataset S21 description for more details). (C and D) PCA for RNA-seq (C) or proteomics (D) data. In C, the points represent biological replicates. In D, the points represent technical replicates from different experiments (indicated by numbers 1, 2, and 3). Experiments 2 and 2* are samples from the same group of fish analyzed on different mass spectrometers. (E and F) Volcano plots showing the relative abundances of transcripts (E) or proteins (F). Transcripts were considered differentially expressed at FDR < 1% and fold change > 3 between proximal and distal regions, whereas proteins were considered differentially expressed at FDR < 5%. Selected transcripts and proteins are highlighted with the gene or protein name.
Fig. S1.
Fig. S1.
Illustrations showing regions harvested for the proximodistal proteomics screens. Proximal (green) and distal (blue) regions were collected from three separate biological experiments. Experiment 1 regions were pooled from eight fish, whereas experiments 2 and 3 were pooled from 20 fish each. The middle (orange) region was only collected during experiment 3 from 20 pooled fish.
Fig. S2.
Fig. S2.
Middle-enriched and middle-depleted gradients were also identified by RNA-seq and LFQ proteomics. (A and B) Heat maps of 147 transcripts (A) or 45 proteins (B) with middle-depleted (red) or middle-enriched (brown) gradients. In A, transcript values shown are RPM normalized to middle and then log2 transformed. Each transcript was differentially expressed (FDR < 1%) between proximal and middle regions and also between middle and distal regions. In B, the average protein abundance from all technical replicates is shown as log2 abundance normalized to the middle region. All protein values for the heat map are derived from experiment 3 only (Fig. S1). Each protein was differentially expressed (FDR < 5%) between proximal and distal regions (see Dataset S21 description for more details).
Fig. S3.
Fig. S3.
Comparison between proximal and distal RNA-seq and LFQ proteomics reveals extensive posttranscriptional regulation. (A) RNA-seq identified and quantified 20,791 transcripts, whereas LFQ proteomics identified and quantified 3,061 proteins. There were 2,680 genes measured by both screens. (B and C) Comparing the proximal and distal differentially expressed transcripts and proteins, restricted to the 2,680 commonly measured by both screens, we found 24 genes were proximally enriched for both mRNA and protein (B) and 8 genes were distally enriched for both mRNA and protein (C). In both the proximal and distal regions, fewer than 50% of the differentially expressed transcripts were found to have similar differentially expressed protein.
Fig. 2.
Fig. 2.
Complex differential expression of RNAs identified along proximodistal axis of caudal fin. (A) Annotations for GO Biological Process terms were performed for RNA. Proximally enriched (green) and distally enriched (blue) RNAs (FDR < 1% and fold change > 3.0) were examined as separate groups by using g:Cocoa (under g:Profiler) with “Best per parent group” hierarchical filtering. Statistically significant GO terms had P < 0.05. (BG) Bar graphs show proximally enriched (green) and distally enriched (blue) transcripts identified by RNA-seq (FDR < 1% and fold change > 3) for transcription factors (B), membrane bound receptors (C), ion channels (D), RA signaling pathway (E), Wnt signaling pathway (F), and for Fgf genes (G). For BD, only the top 20 scored RNAs are shown (Datasets S7–S9).
Fig. 3.
Fig. 3.
Aldh1l1 and Ca2 abundance in WT and mutant fins. (A) Graphs of the relative abundance of aldh1l1 and ca2 transcripts in proximal compared with distal regions of uninjured fins determined by RT-qPCR. Data are normalized to the abundance of actb2 transcripts. Data are mean ± SEM of n = 6 experiments for aldh1l1 and n = 3 for ca2, with >8 fish per experiment. (B and C) Representative Western blots showing Aldh1l1 and Ca2 protein in the proximal (P), middle (M), and distal (D) regions of uninjured caudal fins (B) and uninjured pectoral and dorsal fins (C). Actb2 was used as a loading control. (D) Representative images of sof and lof uninjured caudal fins, with Western blots showing Aldh1l1 and Ca2 protein in the proximal (P), middle (M), and distal (D) regions of these mutant fins.
Fig. 4.
Fig. 4.
Metabolites are differentially expressed along the proximodistal axis of the fin. (A) Annotations for GO Biological Process terms were performed for protein. Proximally enriched (green) and distally enriched (blue) proteins (FDR < 5%) were examined as separate groups by using g:Cocoa (under g:Profiler) with Best per parent group hierarchical filtering. Statistically significant GO terms had P < 0.05. (B) PCA of metabolite abundance from proximal (green) and distal (blue) regions; points represent individual fish. (C) Heat maps of 42 metabolites with proximally enriched (green) or distally enriched (blue) gradients. Each metabolite was differentially abundant (FDR < 5%) between proximal and distal regions. The metabolite values shown are the abundance averages from all biological replicates, normalized to the middle region, and then log2 transformed.
Fig. 5.
Fig. 5.
Proteomic analyses of proximal and distal regions of regenerating caudal fins identifies proteins that maintain differential expression after injury. (A) Diagram showing the regions of fins collected before (0 dpa) or during (1 and 3 dpa) regeneration for analysis by LFQ proteomics. (B) PCA of samples from proximal (green) and distal (blue) regions at the indicated times. The points represent technical replicates of 20 fish per region and time point. (C and D) Graphs of differences in protein abundances between proximal and distal regions. Protein abundance is plotted as the absolute value of the difference between the log2 transformed protein abundance. The colors of the bars indicate proximally enriched (green), or distally enriched (blue) proteins. *FDR < 5%. The genes graphed in C are the ones found with proximally enriched protein expression at 0 and 1 dpa, and also have proximally enriched RNA expression in uninjured fins. The gene graphed in D is the only protein that was differentially expressed between proximal and distal regions at 0, 1, and 3 dpa.
Fig. S4.
Fig. S4.
Representative caudal fin showing regions harvested for dorsal, central, and ventral proteomics.
Fig. 6.
Fig. 6.
Molecular gradients identified in the fin. Summary of transcripts, proteins, and metabolites found in the illustrated patterns along the proximodistal axis of the caudal fin; ND, not detected.

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback