Pronounced expression of extracellular matrix proteoglycans regulated by Wnt pathway underlies the parallel evolution of lip hypertrophy in East African cichlids

Abstract

Cichlid fishes inhabiting the East African Great Lakes, Victoria, Malawi, and Tanganyika, are textbook examples of parallel evolution, as they have acquired similar traits independently in each of the three lakes during the process of adaptive radiation. In particular, 'hypertrophied lip' has been highlighted as a prominent example of parallel evolution. However, the underlying molecular mechanisms remain poorly understood. In this study, we conducted an integrated comparative analysis between the hypertrophied and normal lips of cichlids across three lakes based on histology, proteomics, and transcriptomics. Histological and proteomic analyses revealed that the hypertrophied lips were characterized by enlargement of the proteoglycan-rich layer, in which versican and periostin proteins were abundant. Transcriptome analysis revealed that the expression of extracellular matrix-related genes, including collagens, glycoproteins, and proteoglycans, was higher in hypertrophied lips, regardless of their phylogenetic relationships. In addition, the genes in Wnt signaling pathway, which is involved in promoting proteoglycan expression, was highly expressed in both the juvenile and adult stages of hypertrophied lips. Our comprehensive analyses showed that hypertrophied lips of the three different phylogenetic origins can be explained by similar proteomic and transcriptomic profiles, which may provide important clues into the molecular mechanisms underlying phenotypic parallelisms in East African cichlids.

Keywords: cichlid; evolutionary biology; hypertrophied lip; multi-omics; parallel evolution.

Figure 1.. Histological comparisons of hypertrophied and normal lips of cichlids among lakes, focusing on proteoglycan-rich loose connective tissue.

Examples of typical cichlids with hypertrophied lips (A, Haplochromis chilotes) and normal lips (B, H. sauvagei). The lower jaw and lip are highlighted by black squares. (C) Phylogenetic relationships among cichlids from Lake Victoria, Lake Malawi, and Lake Tanganyika. The midline of the lips, based on the lower dentary bone of the lower jaw (marked by asterisks), in cichlids from Lake Victoria (D, E, H. chilotes with hypertrophied lips; F, G, H. sauvagei with normal lips), Lake Malawi (H, I, Placidochromis milomo with hypertrophied lips; J, K, Maylandia lombardoi with normal lips), and Lake Tanganyika (L, M, Lobochilotes labiatus with hypertrophied lips; N, O, Tropheus moorii with normal lips) were sectioned in sagittal, and stained with AB: Alcian blue (D, H, L, F, J, and N), or VG: Van Gieson (E, I, M, F, J, and N). Each section is shown with the dorsal side up and the anterior side to the right. The area surrounded by dotted lines shows proteoglycan-rich tissue (stained dense blue by AB) or collagen-rich tissue (stained dense red by VG). Each species showed mutually exclusive staining by AB and VG, and AB-positive (proteoglycan-rich) tissues were commonly enlarged in hypertrophied lips among the cichlids from all three lakes with statistical significance. Scale bars are 1 mm.

Figure 1—figure supplement 1.. The midlines of the lips were defined by the shape of the lower dentary bone.

Hematoxylin–eosin stain (HE stain) was used to examine serial sections of the lower lips and lower jaws of cichlids from Lake Victoria (A, H. chilotes with hypertrophied lips; B, H. sauvagei with normal lips), Lake Malawi (C, Placidochromis milomo with hypertrophied lips; D, Maylandia lombardoi with normal lips), and Lake Tanganyika (E, Lobochilotes labiatus with hypertrophied lips; F, Tropheus moorii with normal lips). Each section is shown with the dorsal side up and the anterior side to the left. The lower dentary bone became smaller as it approached the midline, and just at the midline, the bone was composed of cartilage. Scale bars are 1 mm.

Figure 1—figure supplement 2.. The relative area of the proteoglycan-rich layer was significantly enlarged in the hypertrophied lips of Victoria cichlids.

The stained area percentage of Alcian blue stain (AB stain; A) and Van Gieson stain (VG stain; B) relative to the whole-lip area was calculated using ImageJ software. H. chilotes with hypertrophied lips (yellow box) and H. sauvagei with normal lips (blue box) were used in the experiment. Each plot is an average value calculated by three sections near the midline of the lips. Mann–Whitney U-test was used to examine the statistical significance (*p-value <0.05, **p-value <0.01).

Figure 2.. Chondroitin sulfate proteoglycans accumulate in hypertrophied lips.

(A) Volcano plot of proteomics comparison between the tips of hypertrophied and normal lips. Statistical significance was examined using Welch’s t-test. Yellow plots highlight highly accumulated proteins in hypertrophied lips, and blue plots highlight highly accumulated proteins in normal lips with the criteria of p-value <0.05 and log2 fold change >1. The top 20 proteins highly accumulated in normal (B) and hypertrophied lips (C) are shown along with log2 fold change relative to normal lips. (D) The relative amount of each proteoglycan-related protein. MS2 intensity indicates the intensity of peptides obtained from LC–MS/MS and DIA/SWATH analysis using DIA-NN software by comparing three parts of the lip: the base of hypertrophied lips (HLip), the tip of HLip, and normal lips (NLip). Welch’s t-test was used to examine statistical significance (*p-value <0.05). Digestion of chondroitin sulfate by treatment with (upper panels) and without (lower panels) chondroitinase in hypertrophied lips of cichlids from Lake Victoria (E), Lake Malawi (F), and Lake Tanganyika (G). Each section is shown with the dorsal side up and the anterior side to the right. Scale bars are 1 mm.

Figure 2—figure supplement 1.. Volcano plot of proteomics comparison between the tip of hypertrophied lips and the base of hypertrophied lips (A) and the base of hypertrophied and normal lips (B).

Statistical significance was examined by Welch’s t-test. Differentially accumulated proteins in each comparison are highlighted in the black plot with the following criteria: p-value <0.05, log2 fold change >1.

Figure 3.. Hypertrophied lips of cichlids are characterized by higher expression of extracellular matrix (ECM)-related genes.

Transcriptomic analysis using three species of Lake Victoria cichlids (H. chilotes (Hch) with hypertrophied lips; H. sauvagei (Hsa) and H. pyrrhocephalus (Hpy) with normal lips), three species of Lake Malawi cichlids (Placidochromis milomo (Pmi) with hypertrophied lips; Maylandia zebra (Mze) and Labidochromis caeruleus (Lca) with normal lips), and two species of Lake Tanganyika cichlids (Lobochilotes labiatus (Lla) with hypertrophied lips; Tropheus moorii (Tmo) with normal lips). Principal component analysis (PCA) plots of all expressed genes showed differences depending on the lakes (A), while those of ECM-related genes showed differences depending on lip morphology: hypertrophied or normal (B). (C) Heatmap of ECM-related gene expression between hypertrophied and normal lips. The expression rate (TPM) normalized by Z score of each gene is compared for Lake Victoria (yellow; Hch, Has, and Hpy), Lake Malawi (blue; Pmi, Lca, and Mze), and Lake Tanganyika (red; Lla and Tmo). The intensity of the relative expression rate is shown in the bottom right of the figure. (D) Cumulative fold change of the ECM-related genes comparing hypertrophied and normal lips. The gene symbol corresponds to (C). The color represents the lake information. The gray dotted line represents the value at which higher expression was observed in the hypertrophied lips.

Figure 4.. Comparison of juvenile and adult cichlids to examine gene expression changes during the development of hypertrophied lips.

(A) The lips of the cichlids used in this analysis are shown with the dorsal side up and the anterior side to the right: the juvenile and adult stages of Haplochromis chilotes (Hch, representative of hypertrophied lips: HLip) and H. sauvagei (Hsa, representative of normal lips: NLip) from Lake Victoria. Hypertrophied lips at juvenile stage are not notably enlarged. Scale bars are 0.5 mm. (B) Spearman correlation matrix of the extracellular matrix (ECM)-related gene expression rate (TPM) normalized by Z score. The raw correlation values are indicated on the right side and the average correlation values within each stage and species are indicated on the left side. The orange frame highlights the correlations between the hypertrophied and normal lips at the adult stage, and the blue frame highlights those at the juvenile stage. The black frame highlights the correlations between juvenile and adult samples of each species. (C) Cluster heatmap of the ECM-related gene expression rate (TPM) normalized by Z score in each gene. Each gene is clustered by similarity of expression rates. The intensity of the relative expression rate is shown on the bottom left of the figure. (D) The log10 cumulative fold change of the ECM-related genes comparing hypertrophied and normal lips. The gene symbol corresponds to (C). The positive values indicate high expression in hypertrophied lips, whereas the negative values indicate low expression in hypertrophied lips. The colors of the bars indicate the stage of the samples.

Figure 5.. Wnt pathway-related genes are highly expressed in hypertrophied lips of juvenile and adult cichlids.

(A, B) Enrichment analysis of highly expressed genes in the hypertrophied lips. Labels are according to the top annotation in DAVID annotation clusters. Asterisks indicate statistically significant annotation clusters (FDR <0.05). The Wnt-related annotations are highlighted in yellow. (C–N) Expression of the specific Wnt pathway-related genes. (C, J) The size of the dots represents the log fold change, and the color of the dots represents the p-value. Blue dots represent genes that are highly expressed in hypertrophied lips (HLip), and red dots represent genes that are highly expressed in normal lips (NLip). (D–I, K–N) Box plots show the expression rate (TPM) of Wnt pathway-related genes. Yellow boxes represent genes that are highly expressed in hypertrophied lips and blue boxes represent genes that are highly expressed in normal lips, respectively. Mann–Whitney U-test was used to examine the statistical significance (*p-value <0.05). (C–I) The canonical Wnt pathway-related genes and (J–N) the noncanonical Wnt pathway-related genes.

Figure 6.. The trend of higher expression of Wnt-related genes in hypertrophied lips.

(A) Box plots of expression rate (TPM) of Wnt-related genes of hypertrophied lips (HLip: yellow) and normal lips (NLip: blue). Mann–Whitney U-test was used to examine the statistical significance (*p-value <0.05, **p-value <0.01). (B) Illustration of the canonical Wnt pathway. Differentially expressed genes (DEGs) are marked in the bold.

Author response image 1.