Molecular conservation of marsupial and eutherian placentation and lactation

Abstract

Eutherians are often mistakenly termed 'placental mammals', but marsupials also have a placenta to mediate early embryonic development. Lactation is necessary for both infant and fetal development in eutherians and marsupials, although marsupials have a far more complex milk repertoire that facilitates morphogenesis of developmentally immature young. In this study, we demonstrate that the anatomically simple tammar placenta expresses a dynamic molecular program that is reminiscent of eutherian placentation, including both fetal and maternal signals. Further, we provide evidence that genes facilitating fetal development and nutrient transport display convergent co-option by placental and mammary gland cell types to optimize offspring success.

Keywords: developmental biology; evolutionary biology; genomics; lactation; marsupial; placenta; reproduction; stem cells; tammar wallaby; transcriptomics.

Figure 1.. Tammar placental structure.

Schematic of a day 24 tammar conceptus (right) highlighting placental cell types (middle). The tammar yolk sac placenta contains two cellular layers: the trophoblast (TL; blue) and the endoderm (EL; yellow). Further, the placental structure is divided into two halves. The ‘top’ non-vascularized tissue is termed the bilaminar omphalopleure (BOM) and the ‘bottom’ vascularized tissue is termed the trilaminar omphalopleure (TOM) which also contains a mesodermal layer (ML). H&E sections (left) demonstrate the cell types within the BOM and TOM components (Scale bars are 50 μm). DOI: http://dx.doi.org/10.7554/eLife.27450.003

Figure 2.. Tammar placental gene expression differs between tissues and over time.

(A) Transcripts differentially expressed between BOM and TOM (n = 445) with blue indicating high expression and red low expression (see Z-score insert upper right). (B) Transcripts differentially expressed between early (day 21–23) and late (day 25) placental time points (n = 1705). DOI: http://dx.doi.org/10.7554/eLife.27450.004

Figure 2—figure supplement 1.. Tammar placenta transcriptome exhibits distinct signatures in both tissue type and time.

(A) PCA plot depicting the 10 RNA-seq replicates colored by placental tissue type: BOM in red and TOM in blue, demonstrating tissue specific separation on principal component 1 (B) PCA re-plotted and colored by time point: early (day 21–23) in red, and late (day 25) in blue, demonstrating temporal separation on principal component 2. DOI: http://dx.doi.org/10.7554/eLife.27450.005

Figure 3.. Tammar placenta expresses eutherian placental markers.

Immunofluorescence of the eutherian placental markers: CDX2, CEBPB, GCM1, Pan- cytokeratins, and Vimentin, in day 24 tammar placenta. The left column shows protein expression. The middle is DAPI stain depicting the nucleus for the same section. The right column is the merge. No 1° Antibody is a negative control (bottom row). Scale bars are 50 μm. DOI: http://dx.doi.org/10.7554/eLife.27450.006

Figure 3—figure supplement 1.. Tammar placenta expresses eutherian placental markers.

(TOM) Immunofluorescence of key placenta development genes in day 24 wallaby TOM placenta tissue. The first column of panels depicts the expression of each gene, the second column of panels depicts a DAPI nuclear stain, and the third column of panels depicts a merge of both images. Genes depicted from top to bottom include: CDX2, CEBPB, GCM1, Pan-cytokeratins, Vimentin, and a negative control where no primary antibody was applied (Scale bars are 50 μm). DOI: http://dx.doi.org/10.7554/eLife.27450.007

Figure 4.. The tammar placenta is more similar to the midgestation mouse placenta.

(A) Comparison of mouse and tammar placental transcriptomes at different gestational ages using Spearman correlations (see values throughout graph). Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity. The mouse heart transcriptome used as a control (top). (B) Heat map depicting the expression of 410 transcripts that are specific for the mouse placenta of which 340 are expressed before and 70 are expressed after midgestation (e12.5). The tammar placenta expresses orthologs of 100 of these transcripts, which are highlighted in the yellow boxes on the heat map. 90 are classified as developmental, or expressed early in mouse, and 10 are classified as mature, or expressed late in mouse. Blue indicates high expression and red low expression (see Z-score on right). DOI: http://dx.doi.org/10.7554/eLife.27450.008

Figure 4—figure supplement 1.. Tammar placenta exhbits conservation of placenta-specific gene expression.

Heat map depicting a comparison of the mouse placental transcriptomes against tammar and adult mouse heart transcriptome to detect conservation organ-specific transcription. Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity. DOI: http://dx.doi.org/10.7554/eLife.27450.009

Figure 5.. Shuffling of reproduction genes between lactation and placentation.

(A) Venn-diagram (Left) comparing the genes expressed in the lactating tammar and mouse mammary glands with the tammar and eutherian placenta. Key ontology of genes in overlapping categories is highlighted. Genes listed for each category in Supplementary file 3. The table (Right) depicts key categories of genes from the venn-diagram and whether or not they are expressed in the placenta and mammary gland tissues of the tammar and eutherians. If the gene class is present in the tissue of a given lineage it is given a green ‘+”, if it is absent it is given a red “-“. (B) Immunofluorescence of GCM1 and GATA3. The first column depicts the expression of each protein, the second column is DAPI stain for nuclear DNA, and the third column is a merge of both images. The final row contains a negative control where no primary antibody was applied (Scale bars are 50 μm). DOI: http://dx.doi.org/10.7554/eLife.27450.010

Figure 5—figure supplement 1.. Tammar placenta and mammary gland have exceptionally distinct molecular functions.

Comparison of tammar and mouse organ transcriptomes including: placenta, mammary gland, liver and testis using Spearman correlations (see values throughout graph). Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity. DOI: http://dx.doi.org/10.7554/eLife.27450.011