Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids

Abstract

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

Keywords: Lake Malawi; Lake Tanganyika; RNA-seq; cichlids; functional conservation; snout flap.

Fig. 1.

Convergent cases of snout flap evolution. East African cichlid species used in this study. The area of the soft tissue that was dissected is depicted by dashed lines. O. nasuta, Ophthalmotilapia nasuta; O. ventralis, Ophthalmotilapia ventralis; L. trewavasae, Labeotropheus trewavasae; T. tropheops, Tropheops tropheops.

Fig. 2.

Differentially expressed genes in the snout regions. (A) Venn diagram of genes with differential expression between the snout regions (“snout” and “no snout”) for Lake Malawi and Lake Tanganyika and the overlap of 201 genes between the two comparisons of which 96 are downregulated and 75 are upregulated in the flapped snout of both comparisons. Dendrogram clusters of the overlapping annotated genes showing upregulation (B) and downregulation (C) in expression in the flapped snout tissue, as well as those showing differential expression in both comparisons but in opposing directions (including not annotated genes) (D). Orange and blue shadings indicate higher and lower relative expression, respectively. Lake Tanganyika: Ophthalmotilapia nasuta (O.n, dark blue), Ophthalmotilapia ventralis (O.v; light blue); Lake Malawi: Labeotropheus trewavasae (L.t; red), Tropheops tropheops (T.t; orange).

Fig. 3.

Functional analyses of the overlapping differentially expressed genes in the flapped snout. (A) Enrichment for gene ontologies of biological processes using the shared 201 differentially expressed genes. (B) Functional interactions between the differentially expressed genes predicted based on zebrafish databases in STRING v10 (http://string-db.org/).

Fig. 4.

qPCR expression analysis of a selected set of candidate genes. qPCR validation of expression differences for selected sets of genes showing upregulation (A) or downregulation (B) in snout tissues. (C) qPCR expression analysis of predicted transcription factors. The bars indicate mean and standard deviation of RQ expression values for five biological replicates per species. The asterisks above the bar represent significant expression differences (*P < 0.05; **P < 0.01; and ***P < 0.001). O.n, Ophthalmotilapia nasuta (dark blue); O.v, Ophthalmotilapia ventralis (light blue); L.t, Labeotropheus trewavasae (red); T.t, Tropheops tropheops (orange).