A gene expression study of ornamental fin shape in Neolamprologus brichardi, an African cichlid species

Abstract

The diversity of fin morphology within and across fish taxa offers great, but still largely unexplored, opportunities to investigate the proximate mechanisms underlying fin shape variation. Relying on available genetic knowledge brought forth mainly by the comprehensive study of the zebrafish caudal fin, we explored candidate molecular mechanisms for the maintenance and formation of the conspicuously elongated filaments adorning the unpaired fins of the East African “princess cichlid” Neolamprologus brichardi. Via qPCR assays, we detected expression differences of candidate genes between elongated and short regions of intact and regenerating fins. The identified genes include skeletogenic and growth factors (igf2b, fgf3, bmp2 and bmp4), components of the WNT pathway (lef1, wnt5b and wnt10) and a regulatory network determining fin ray segment size and junction (cx43, esco2 and sema3d), as well as other genes with different roles (mmp9, msxb and pea3). Interestingly, some of these genes showed fin specific expression differences which are often neglected in studies of model fish that focus on the caudal fin. Moreover, while the observed expression patterns were generally consistent with zebrafish results, we also detected deviating expression correlations and gene functions.

Towards a gene regulatory network shaping the fins of the Princess cichlid

“…Extensive research has been launched to identify genes underlying fin growth and regeneration with a strong focus on the caudal fin of the zebrafish model Danio rerio 2 , 8 – 10 . It is only in recent years that the molecular basis of the morphological diversity of fins within and across species has attracted some attention 11 – 15 . Studies capitalizing on the natural variation in fin morphology addressed, for instance, the ventral elongation of the caudal fin in swordtail fish 11 , interspecific divergence in pectoral fin morphology in cichlids from Lake Malawi 13 and the twin-tail phenotype of goldfish 14 .…”

“…In a previous study, we hypothesized that positional differences in gene expression levels underlie the extreme elongation of filaments, and tested a series of candidate genes involved in fin formation and regeneration for differential expression between elongated (L) and regular (i.e. short, S) fin regions 15 . Comparing gene expression levels between L and S tissue sampled from both intact and regenerating fins, we detected several genes with either higher L-expression or higher S-expression 15 .…”

“…short, S) fin regions 15 . Comparing gene expression levels between L and S tissue sampled from both intact and regenerating fins, we detected several genes with either higher L-expression or higher S-expression 15 . Particularly strong and consistent signals were obtained for cx43/ Gja1 and mmp9 , both showing elevated L-expression, and sema3d with elevated S-expression.…”

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“…Furthermore, we chose eight candidate genes which are frequently used as reference genes in qPCR studies of different tissues in East African cichlids (Yang et al, 2013;Ahi & Sefc, 2017a,b;Ahi, Richter & Sefc, 2017). To design primers, we used conserved coding sequence regions based on the transcriptomes of several East African haplochromine species (Pundamilia nyererei, Simochromis diagramma, Gnathochromis pfefferi, Metriaclima zebra, and Astatotilapia burtoni) and two other cichlid species belonging to distant tribes (Oreochromis niloticus and Neolamprologus brichardi) (Brawand et al, 2014;Singh et al, 2017).…”

“…In order to precisely measure the expression of the appetite-regulating genes in the brains, identification of stably expressed reference gene(s) with minimum expression variation among the samples is considered as first crucial step in qPCR (Kubista et al, 2006). The eight candidate reference genes were among the frequently used reference genes in qPCR studies of different tissues in East African cichlids (Yang et al, 2013;Ahi & Sefc, 2017a,b;Ahi, Richter & Sefc, 2017). The expression levels of candidate reference genes were variable; from the lowest expression level (highest Cq value) of tbp to the highest expression level (lowest Cq value) of actb1 ( Figure 1B).…”