Delineating the molecular basis of adaptive morphologies is essential to understand their evolution and as well as their potential contribution to speciation. In this study, we investigated the soft-tissue craniofacial trait of the hypertrophic lip in G. permaxillaris, in a comparative transcriptomic framework with G. bellcrossi, which are both from the Limnochromini tribe belonging to the cichlid adaptive radiation of Lake Tanganyika. Gnathochromis permaxillaris is a deepwater cichlid living over mud bottom as far as oxygenated water reaches down and has a unique mode of foraging. The species slowly swims directly over the mud surface with its protruding snout hovering at the surface and at the same time opening the protruding mouth underneath, while ingesting mud that is then filtered through the gill arches. Its common name Vacuum cleaner cichlid is an adequate description of this mode of feeding. Thereby, the protruding upper lip with its small hypertrophy at its tip seems to be adaptive by boosting the efficiency of filtration (unpublished behavioural observations).
Using differential gene expression analysis in the hypertrophic lip region as input for gene ontology analysis, we found multiple biological processes to be enriched. These processes involve cell motility, adhesion and developmental growth, as well as regulation of GTPase mediated signals, particularly, the RAS signalling pathway. A recent integrated genomic and transcriptomic study in humans has revealed that cell adhesion, cell junction and extracellular structure organizations are among the major biological processes involving lip and cleft development and morphogenesis . Furthermore, as studies on human and mouse have shown, biological processes involving developmental growth and cell proliferation are known to be the key mechanisms in upper lip development and morphogenesis . On the other hand, RAS/MAPK signalling is among the well-known molecular pathways involved in development and morphogenesis of craniofacial structures in vertebrates [4, 27]. Activation of Ras signalling promotes cell proliferation, growth and survival in various tissues [28, 29], and because of these roles, several components of the Ras pathway are considered as therapeutic targets in different types of cancer . In mammals, Ras signaling plays a pivotal role in skin development, dermal thickenning and skin carcinogenesis . Defective activity of the Ras pathway can cause a wide range of skin anomalies, such as thickened palms and soles, redundant skin, papilloma formation, excessive proliferation of keratinocytes and increased skin folds . Therefore, the hypertophic lip region in the anterior upper lip of G. permaxillaris can be a result of increased activity of Ras signaling in this region. However, further functional studies (such as a protein manipulation method recently used in cichlids to investigate regional activity of a growth signal ) are required to find out the molecular reason for the anotomically limited activation of this signal only in the anterior part of the upper lip in G. permaxillaris.
We found that many of the genes with increased expression in the hypertrophic region of dorsal lip were already demonstrated to play a role in lip morphogensis and pathobiology in other vertebrates, including alx1 , alx3 , angptl2 , arid3a , crip2 , ddr2 , dpysl2 , itga5 , lmna , mgp , rgs5 , rhob [42, 43], rxfp2 , sostdc1 [42, 45], thbs3 , vcan [47, 48], and vtn . Among the transcriptionally repressed genes, we also found candidates with functions which were previously implicated in defective lip morphogenesis in other vertebarates such as cd96 , ep300 , fgfrl1 , frzb , hectd1 [55, 56], mmp13 , slc16a6 , and syne1 . Taken together, these results suggest that a similar set of genes might be involved in lip morphogenesis, not just across teleosts, but across vertebrates, and further functional studies are required to investigate their specific role in morphological divergence of soft tissues in fish.
Among the genes with reduced expression in the hypertophic lip regions, we validated four genes with qPCR, cyp1a, gimap8, lama5 and rasal3, and found all of them showing a similar expression reduction pattern in all of the hypertophic lip regions of both species. Cytochrome P450 (CYP) 1 alpha, cyp1a, encodes an enzyme with an important role in the cytochrome p450 xenobiotic metabolism and the synthesis of steroids and other lipids. Cyp1a is a downstream target for AHR, RAS and Wnt/β-Catenin signaling pathways [59, 60], and all of these signals are demonstrated to play important roles in morphogenesis and adaptive radiations of craniofacial elements in teleost fish . Differential regulation of cyp1a is implicated in craniofcial morphological divergence in fish . Differential regulation of AHR, RAS and Wnt/β-Catenin signals, as well as cyp1a, is also involved in the defective formation of the lip and palate in vertebrates [62–66]. We found reduced expression of cyp1a in the hypertophic lip regions in both cichlid species, which can be explained by its inhibitory role on cell proliferation [67, 68].
The second gene, GTPase immunity-associated protein family member 8 or gimap8, encodes a nucleotide-binding protein that plays a role in the maintenance and survival of lymphocytes in mammals . Consistent with our result, the same gene was found to be repressed in lip tissue of thick-lipped Midas cichlid ; however, its exact function during development and morphogenesis of soft tissues in vertebrates remained unclear. In humans, increased expression of gimap8 orthologue has been reported during adipocyte differentiation, indicating its potential role in cell differentiation . The third gene, lama5, encodes one of the vertebrate laminin alpha chain proteins, a family of extracellular matrix glycoproteins, which are the major noncollagenous constituent of basement membranes. In humans, lama5 has been implicated in pathologenesis of lip inflammation and carcinogenesis . In mouse, loss of lama5 causes hyper-proliferation of basal keratinocytes, an increase in the number of immune cells and thickening of epidermis; thus, reduced expression of lama5 in the hypertophic lip regions might result in an increased number of keratinocytes and subsequently thicker epidermis in these regions . The last gene, rasal3, RAS protein activator like-3, encodes a negative regulator of Ras signalling pathway and its duplication and deletion are both linked to defective lip morphogenesis in humans [73, 74]. The reduced expression of rasal3 in the hypertophic lip regions suggests higher activity of RAS signaling in this region, which is consitent with the enriched RAS related gene ontology for the differentially expressed genes.
Two of the genes with increased expression in hypertrophic lip regions, apoda and fhl2, were particularly interesting, since both genes have been already reported as potential molecular players in the formation of thick-lipped phenotype of central American cichlids . However, in contrast to our results, the expression of both genes have been shown to be repressed in hypertophic lips of Midas cichlid. Four-and-a-half LIM domains 2, fhl2, encodes a transcriptional modulator of cell proliferation and [75, 76], while apolipoprotein Da, apoda, encodes a multi-ligand transporter involved in neural cell survival . The molecular reason for this discrepancy is unclear, but it is likely that these genes have dual and opposite modulatory functions under different cellular conditions. Future functional investigations are required to unravel this discrepancy. It should be noted that fhl2 is also reported as an important molecular player in formation of egg-spot in cichlids indicating its functional diversity in the adaptive morphological divergence of cichlid fishes .
We predicted binding sites for Forkhead Box (FOX) transcription factors and the basic helix-loop-helix (bHLH) for transcription factor 12 (tcf12/heb) to be enriched on upstream regulatory sequences of many of the differentially expressed genes by RNA-sEq. Among the differentially expressed genes, we only found foxf1 as potential candidate that could bind to the enriched FOX binding site. Interestingly, micro-deletion in mammalian orthologue of foxf1 have been shown to be associated with lip and palate deformities in mouse and human [79, 80]. However, the qPCR analysis revealed that, although foxf1 expression was increased in the hypertophic lip region of G. permaxillaris, its expression was not increased in the hypertophic lip regions of G. bellcrossi. This could indicate that another member of the FOX family might be involved, whose expression difference was not detected by RNA-sEq. In addition to the core FOX binding site, we also found a binding site for foxp1 (another member of FOX family) to be enriched multipe times on the regulatory sequences. The qPCR analysis revealed that foxp1 expression has a significant expression reduction in all the hypertophic lip regions of both species. The reduced expression of foxp1 suggests a potential repressive effect on transcription of the genes induced in the hypertophic regions. Interestingly, foxp1 has been already demonstrated to have repressive effects on transcription of many of its downstream targets . Moreover, a mutation affecting foxp1 function has been associated with hypertophy of vermilion borders (side edges) of upper lip in humans . The role of foxp1 in lip hypertophy might be linked to its function in inhibition of cell proliferation by repressing the transcription of growth/cell cycle stimulating factors [83–85]. The second predicted TF, tcf12, has been recently shown to be involved in hypertophy of frontal head soft tissues (nuchal hump) in another East African cichlid species , however, we did not find its consistent expression difference between the lip regions by neither the RNA-seq nor the qPCR method in this study.