Mouse Obox and Crxos modulate preimplantation transcriptional profiles revealing similarity between paralogous mouse and human homeobox genes

Abstract

Background: ETCHbox genes are eutherian-specific homeobox genes expressed during preimplantation development at a time when the first cell lineage decisions are being made. The mouse has an unusual repertoire of ETCHbox genes with several gene families lost in evolution and the remaining two, Crxos and Obox, greatly divergent in sequence and number. Each has undergone duplication to give a double homeodomain Crxos locus and a large cluster of over 60 Obox loci. The gene content differences between species raise important questions about how evolution can tolerate loss of genes implicated in key developmental events.

Results: We find that Crxos internal duplication occurred in the mouse lineage, while Obox duplication was stepwise, generating subgroups with distinct sequence and expression. Ectopic expression of three Obox genes and a Crxos transcript in primary mouse embryonic cells followed by transcriptome sequencing allowed investigation into their functional roles. We find distinct transcriptomic influences for different Obox subgroups and Crxos, including modulation of genes related to zygotic genome activation and preparation for blastocyst formation. Comparison with similar experiments performed using human homeobox genes reveals striking overlap between genes downstream of mouse Crxos and genes downstream of human ARGFX.

Conclusions: Mouse Crxos and human ARGFX homeobox genes are paralogous rather than orthologous, yet they have evolved to regulate a common set of genes. This suggests there was compensation of function alongside gene loss through co-option of a different locus. Functional compensation by non-orthologous genes with dissimilar sequences is unusual but may indicate underlying distributed robustness. Compensation may be driven by the strong evolutionary pressure for successful early embryo development.

Keywords: ARGFX; Blastocyst; Compensation; Gene duplication; Gene loss; Homeodomain; PRD class; Transcription factor.

Fig. 1

ETCHbox genes in human and mouse. The ETCHbox group of homeobox genes has six members: Leutx, Tprx1, Tprx2, Dprx, Argfx and Pargfx. In human, Pargfx has been lost in evolution. In mouse, only Tprx1 and Tprx2 orthologues remain and are referred to as Crxos and Obox, respectively; each has undergone sequence divergence and gene duplication

Fig. 2

ETCHbox organisation and expression in preimplantation development: a The Crxos locus has undergone a mouse-specific duplication and generates three transcripts: two with a single homeobox and a composite double-homeobox transcript. Crxos is the Tprx1 orthologue although originally named as a distinct gene. b Analysis of protein and nucleotide sequences of 66 Obox genes in mouse identified four groupings; sequences and expression profiles enable OboxA to be split into three subgroups. c The three Crxos transcripts have the same preimplantation temporal expression pattern, with the 3′ single-homeobox transcript generally having higher RNA levels

Fig. 3

Enriched profiles following Crxos or Obox ectopic expression. Following over-expression of Crxos, Oboxa1, Oboxa4 or Oboxa7 in cultured mouse embryonic cells, we identified genome-wide transcriptomic changes. Each set of up- or down-regulated genes was compared to sets of genes assigned to distinct temporal expression profiles to test for enrichment (Fisher’s exact test, corrected p values shown). a Genes up-regulated following Crxos ectopic expression are enriched for two profiles, 59 and 216, although we suggest profile 216 is an off-target effect. Genes down-regulated are enriched for profile 5. Pink shading indicates time of Crxos expression. b Genes in profile 101 are up-regulated by Oboxa1, Oboxa4 and Oboxa7. Blue, purple or green shading indicates time of expression for Oboxa1, Oboxa4 or Oboxa7, respectively. c Genes up-regulated by Oboxa4 ectopic expression are enriched for two profiles not affected by other Obox genes (226, 216) and one profile also affected by Oboxa1 (219). Genes down-regulated by Oboxa4 ectopic expression are enriched for six profiles not affected by other Obox genes (202, 84, 79, 124, 149, 129). Green curves relate to up-regulated genes; red curves relate to down-regulated genes. Oo = oocyte, Zy = zygote, 2C = two-cell embryo, 4C = four-cell embryo, 8C = eight-cell embryo, Mo = morula, Bl = blastocyst

Fig. 4

Overlap between genes downstream of Obox genes. Comparison of genes up- or down-regulated following ectopic expression of Obox genes; ectopic expression of the maternally expressed Oboxa4 gene affects expression of additional downstream genes not affected by Oboxa1 or Oboxa7 expression

Fig. 5

Similarity between genes downstream of human ARGFX and mouse Crxos. a The sets of genes up- or down-regulated following ectopic expression are compared between each mouse and human ETCHbox gene after filtering for one-to-one orthologues (human data from Ref. [7]); y-axis shows -log(p values) derived from pairwise Fisher’s exact test. Many comparisons are significant; the most striking similarities are between downstream targets of mouse Crxos and human ARGFX (up-regulated genes p = 3 × 10⁻⁵²; down-regulated genes p = 4.2 × 10⁻¹⁰²). Order of comparisons, from left to right, given in Additional file 6. b Proportional Venn diagrams showing extent of overlap between human and mouse one-to-one orthologues affected by Crxos ectopic expression in primary mouse embryonic fibroblasts and ARGFX in primary adult human fibroblasts

Fig. 6

Cellular and embryonic processes potentially regulated by mouse ETCHbox genes. Global transcriptomic changes elicited by ectopic expression, and the embryonic expression profiles of ETCHbox genes themselves, highlight possible developmental milestones regulated by ETCHbox genes. We suggest that Crxos is involved in preparing the embryo for the first cell fate decision prior to the early blastocyst stage. Obox genes likely regulate a range of biological processes in vivo; we suggest Oboxa4 is involved with early milestones including induction of zygotic gene expression, whereas Oboxa1 and Oboxa7 are involved in later events such as embryo compaction. Expression of the ETCHbox genes tested is represented by coloured lines (Crxos red, Oboxa1 blue, Oboxa4 purple, Oboxa7 green)

Fig. 7

Simplified scenario for the evolution of functional redundancy. a A group of genes contribute to several biological processes with partial overlap of functions. b Partial functional overlap permits gene loss to persist as a temporary state. c Redundancy can be restored through gene duplication and divergence. The temporal sequence of (b) and (c) can be reversed. Partial redundancy affords buffering against somatic failure of a gene or process