Transcriptomic and Epigenetic Landscapes of the Canine Zonary Placenta: Insights into Transcriptome Dynamics, Genomic Imprinting, and X Chromosome Inactivation

Abstract

The placenta is a pivotal evolutionary innovation that enables in utero fetal development. Although it serves conserved core functions, including nutrient transfer, waste removal, and fetal–maternal communication, placental structure and morphology vary substantially among mammalian lineages. In dogs, the placenta is classified as zonary and endotheliochorial, consisting of four major tissues: the allantochorion (AC), pigmented zone (PZ), transfer zone (TZ), and umbilical cord (UC). Despite their functional importance, the genetic and epigenetic characteristics of these tissues have not been comprehensively characterized.

The first part of this dissertation investigates transcriptomic landscapes and functional compartmentalization across the four tissues of the term canine placenta. Bulk RNA sequencing (RNA-seq) revealed distinct gene expression profiles indicative of tissue-specific functional specialization. A large number of inter-tissue differentially expressed genes (DEGs) and tissue-specific genes were identified. Genes that were highly expressed in AC were primarily enriched in immune-related functions, and AC also exhibited exceptionally high expression of the well-characterized imprinted genes IGF2 and H19. DEGs enriched in PZ were associated with neuronal processes, while numerous long non-coding RNA genes showed substantial expression uniquely in this tissue. Genes highly expressed in TZ were enriched in cellular transport and vascular regulation pathways, whereas UC-enriched genes were predominantly associated with muscle contraction and extracellular matrix organization.

Genomic imprinting refers to parent-of-origin-dependent monoallelic expression of certain genes. Imprinted genes are primarily involved in fetal and placental growth, developmental regulation, and neurological functions, with many showing particularly high expression in placental tissues. However, genomic imprinting has not been systematically characterized in the canine placenta. The second part of this dissertation presents a genome-wide survey of differential allelic expression using RNA-seq to identify candidate imprinted genes across placental tissues. Both tissue-specific and shared putative imprinted genes were identified, and differential allelic methylation at promoter regions was detected in several candidates.

To achieve dosage compensation of X-linked genes between XX females and XY males, one X chromosome in female therian mammals undergoes transcriptional silencing through a process known as X chromosome inactivation (XCI). Notably, placental XCI patterns differ among mammalian species. Placentas of marsupials, rodents, and cattle exhibit paternally imprinted XCI, whereas those of humans and equids display random XCI. The third part of this dissertation investigates whether canine placenta adopts imprinted or random XCI, with the results providing strong evidence against an imprinted XCI pattern.

Taken together, this dissertation provides a comprehensive characterization of the transcriptomic and epigenetic landscape of the canine placenta, offering new insights into placental biology, mammalian reproductive evolution, and mechanisms underlying developmental regulation in mammals.