Novel Approach Combining Transcriptional and Evolutionary Signatures to Identify New Multiciliation Genes

Abstract

Multiciliogenesis is a complex process that allows the generation of hundreds of motile cilia on the surface of specialized cells, to create fluid flow across epithelial surfaces. Dysfunction of human multiciliated cells is associated with diseases of the brain, airway and reproductive tracts. Despite recent efforts to characterize the transcriptional events responsible for the differentiation of multiciliated cells, a lot of actors remain to be identified. In this work, we capitalize on the ever-growing quantity of high-throughput data to search for new candidate genes involved in multiciliation. After performing a large-scale screening using 10 transcriptomics datasets dedicated to multiciliation, we established a specific evolutionary signature involving Otomorpha fish to use as a criterion to select the most likely targets. Combining both approaches highlighted a list of 114 potential multiciliated candidates. We characterized these genes first by generating protein interaction networks, which showed various clusters of ciliated and multiciliated genes, and then by computing phylogenetic profiles. In the end, we selected 11 poorly characterized genes that seem like particularly promising multiciliated candidates. By combining functional and comparative genomics methods, we developed a novel type of approach to study biological processes and identify new promising candidates linked to that process.

Keywords: comparative genomics; evolution; functional genomics; multi-omics data integration; multiciliation; rare diseases.

Figure 1

Distribution of MCCs in different metazoan clades. A green circle indicates the presence of MCCs, a red circle indicates their absence. A yellow circle indicates the existence of both multiciliated species and strictly monociliated species in the same branch. Gray circles indicate a lack of information regarding multiciliation. Xen.: xenacoelomorpha; Non-Bilat: non-bilaterian. Phylogeny based on the results of Giribet [12].

Figure 2

Schematic representation of the main stages of multiciliogenesis, with major genes. Flat-ended arrows depict the repression of a gene, regular arrows depict the activation of a gene.

Figure 3

Schematic representation of the clustering of the results from the transcriptomics datasets. Circle size is proportional to the percentage of the cluster’s genes found in each dataset. ¹GSE76342; ²GSE116690; ³GSE32452; ⁴GSE75715; ⁵GSE59309; ⁶GSE73331; ⁷GSE60365; ⁸GSE89271. ‘Early’ genes (STK11, p73, MCIDAS and E2F4) are responsible for the overall regulation of multiciliation. ‘Late’ genes (Myb, Foxn4 and Foxj1) activate the generation of motile cilia.

Figure 4

Phylogenetic distribution of multiciliated genes in Metazoa. Only a selected pertinent subset of the studied species is represented. A black square indicates the presence of an ortholog in the concerned species, a blank square indicates the absence of an ortholog. Black rectangles indicate the presence of an ancestral gene before its split in two paralogs. Gray squares indicate the presence of a homologue with an abormaly divergent sequence.

Figure 5

Overview of CCNO multiple sequence alignment. Framed areas show parts of the alignment with the most divergence in Otomorpha fish when compared to other species. Oto.: Otomorpha; Actino.: Actinopterygii.

Figure 6

Multiciliated locus in selected vertebrate species. The locus contains MCIDAS, CCNO and CDC20B, three genes essential for multiciliation. This synteny block is conserved in most vertebrate species except in Otomorpha fish (Danio rerio, Astyanax mexicanus).

Figure 7

Interaction network computed from the list of 114 potential multiciliated targets as well as genes presenting a functional interaction with at least two genes of that list. These clusters contain 57 of our genes (circled in bold) and a total of 919 genes.

Figure 8

Evolutionary clustering of 984 human genes based on their phylogenetic distribution in 711 eukaryotic species. Rows represent genes and columns represent species, with each colored square indicating the presence of an ortholog of the concerned gene in the chosen species. The three lines above the profiles indicate the main clades in Eukaryota, Metazoa and Chordata respectively.