In mammals, pre-implantation embryo development (PED) encompasses the time from the fertilization to the placement of blastocyst in the uterus. This period of development goes through dynamic cellular and molecular changes essential for pregnancy. Key regulatory networks for human PED remain unclear. Advances in sequencing technologies allow us to detect molecular signatures of individual cells during PED. We have performed bioinformatic analyses on existing single-cell RNA-sequencing (scRNA-seq) datasets to understand the dynamic gene expression during PED. Analysis of expression profiling of 7500 most changed genes indicated that PRD-like homeobox proteins have a significant impact on the regulation of gene expression during PED. Out of nine PRD-like homeodomain encoding transcription factors (TFs), we have found significant and dynamic expression pattern of three PRD-like TFs (DUXA, DPRX, and ARGFX). Based on clustering analysis, we found significant upregulation of these three genes after 4-cell stage before reducing by the end of morula stage. Tissue-specific expression analysis revealed that our interested genes (DUXA, DPRX, and ARGFX) showed a higher level of expression in the reproductive tissue like testis.

We found 18.5% to 27.7% similarity in protein sequences of this three TFs indicating that PRD-class homeodomain has similar characteristics. Phylogenetic analysis of all eleven classes of homeodomain TFs indicated that DUXA, DPRX, and ARGFX located in the PRD-class. Using Ensembl Genome Browser and NCBI database for Entrez Gene we found 11 DUXA pseudogenes, 7 DPRX and 2 ARGFX pseudogenes in human. Chi-squared test with a p-value <.01 confirmed significant binding affinity of DUXA and DPRX motifs with genes having similar expression pattern.

These findings provide evidence that DUXA, DPRX, and ARGFX transcription factors may play an important role in regulating gene expression during human preimplantation embryo development.