XB-ART-51076BMC Biol August 1, 2015; 13 56.
Cdx ParaHox genes acquired distinct developmental roles after gene duplication in vertebrate evolution.
The functional consequences of whole genome duplications in vertebrate evolution are not fully understood. It remains unclear, for instance, why paralogues were retained in some gene families but extensively lost in others. Cdx homeobox genes encode conserved transcription factors controlling posterior development across diverse bilaterians. These genes are part of the ParaHox gene cluster. Multiple Cdx copies were retained after genome duplication, raising questions about how functional divergence, overlap, and redundancy respectively contributed to their retention and evolutionary fate. We examined the degree of regulatory and functional overlap between the three vertebrate Cdx genes using single and triple morpholino knock-down in Xenopus tropicalis followed by RNA-seq. We found that one paralogue, Cdx4, has a much stronger effect on gene expression than the others, including a strong regulatory effect on FGF and Wnt genes. Functional annotation revealed distinct and overlapping roles and subtly different temporal windows of action for each gene. The data also reveal a colinear-like effect of Cdx genes on Hox genes, with repression of Hox paralogy groups 1 and 2, and activation increasing from Hox group 5 to 11. We also highlight cases in which duplicated genes regulate distinct paralogous targets revealing pathway elaboration after whole genome duplication. Despite shared core pathways, Cdx paralogues have acquired distinct regulatory roles during development. This implies that the degree of functional overlap between paralogues is relatively low and that gene expression pattern alone should be used with caution when investigating the functional evolution of duplicated genes. We therefore suggest that developmental programmes were extensively rewired after whole genome duplication in the early evolution of vertebrates.
PubMed ID: 26231746
PMC ID: PMC4522105
Article link: BMC Biol
Genes referenced: cdx1 cdx2 cdx4
Morpholinos: cdx1 MO2 cdx2 MO1 cdx4 MO1
Article Images: [+] show captions
|Transcriptomic assessment of Cdx paralogue function during Xenopus tropicalis development. a Duplication of the ancestral ParaHox cluster and subsequent gene loss in vertebrates resulting in three Cdx genes. b Experimental procedure: injection of eggs with morpholino oligonucleotides (MO) targeting specific Cdx copy, collection of stage 14 (early neurula) embryos for RNA extraction, and transcriptomic characterisation by RNA-seq. c Gene set overlap of the three Cdx MO targets inferred by differential expression analysis of RNA-seq data (see Methods). Venn diagram categories are specified for all genes (black), upregulated genes (red), and down-regulated genes (blue). d Gene set overlap for three Cdx MOs and co-injection of all three MOs (triple). e Gene expression and fold-change intensities across differential expression for all three Cdx MOs. Above scale fold-change values are noted as triangles. This representation reveals a strong quantitative extent of Cdx4 MO effect compared with Cdx1 and Cdx2 MOs|
|Fig 3. K-mean clustering of multiple Cdx MOs effects. a Heatmap representation of gene expression fold-changes triggered by the three Cdx MOs and the co-injection triple MOs. Genes are arranged according to k-mean clustering and 12 clusters (left) are delineated to capture the diversity of responses to treatments. b Detail of gene expression response to Cdx MOs in the 12 selected clusters. The average response is plotted as a bold line while response of each member gene of the cluster is plotted in a thin grey line|
|Fig. 4. Gene set enrichment analysis of Cdx MO effects. Functional annotation derived from Panther pathways and the GO biological process version of Panther database were employed for term enrichment analysis using distinct tests accounting for direction of expression change: distinct directional (DD), mixed directional (MD), non-directional (ND), as well as UP or DOWN regulation. The scheme yielding the best enrichment score was retained as the one providing the best description of the enrichment for the term (bubble fill colour). Displayed terms were retained as showing an enrichment >5 in at least one condition|