Spatial and temporal expression of the 23 murine Prolactin/Placental Lactogen-related genes is not associated with their position in the locus

Abstract

Background: The Prolactin (PRL) hormone gene family shows considerable variation among placental mammals. Whereas there is a single PRL gene in humans that is expressed by the pituitary, there are an additional 22 genes in mice including the placental lactogens (PL) and Prolactin-related proteins (PLPs) whose expression is limited to the placenta. To understand the regulation and potential functions of these genes, we conducted a detailed temporal and spatial expression study in the placenta between embryonic days 7.5 and E18.5 in three genetic strains.

Results: Of the 22 PRL/PL genes examined, only minor differences were observed among strains of mice. We found that not one family member has the same expression pattern as another when both temporal and spatial data were examined. There was also no correlation in expression between genes that were most closely related or between adjacent genes in the PRL/PL locus. Bioinformatic analysis of upstream regulatory regions identified conserved combinations (modules) of putative transcription factor binding sites shared by genes expressed in the same trophoblast subtype, supporting the notion that local regulatory elements, rather than locus control regions, specify subtype-specific expression. Further diversification in expression was also detected as splice variants for several genes.

Conclusion: In the present study, a detailed temporal and spatial placental expression map was generated for all murine PRL/PL family members from E7.5 to E18.5 of gestation in three genetic strains. This detailed analysis uncovered several new markers for some trophoblast cell types that will be useful for future analysis of placental structure in mutant mice with placental phenotypes. More importantly, several main conclusions about regulation of the locus are apparent. First, no two family members have the same expression pattern when both temporal and spatial data are examined. Second, most genes are expressed in multiple trophoblast cell subtypes though none were detected in the chorion, where trophoblast stem cells reside, or in syncytiotrophoblast of the labyrinth layer. Third, bioinformatic comparisons of upstream regulatory regions identified predicted transcription factor binding site modules that are shared by genes expressed in the same trophoblast subtype. Fourth, further diversification of gene products from the PRL/PL locus occurs through alternative splice isoforms for several genes.

Figure 1

Placental anatomy and trophoblast subtypes at early and mid-gestation. A, B. Embryonic day 8.5 implantation site. A – The collapse of the ectoplacental cone cavity (~E8.0) brings the base of the ectoplacental cone into contact with the distal surface of the chorion, while the allantois makes contact with, and adheres to, the basal surface of the chorion by E8.5. B – Large parietal TGC cells line the implantationsite. C-H. Embryonic day 14.5 placenta. C – Spiral artery lined by spiral artery TGCs. D – parietal TGC with glycogen trophoblast cells located beneath within the spongiotrophoblast layer as well as glycogen trophoblast cells which have invaded above the parietal TGC layer into the decidua. E – Clusters of glycogen trophoblast cells within the spongiotrophoblastlayer. F – Large central canal lined by canal TGCs. G – The maternal blood sinuses and fetal blood vessels (lined by endothelial cells) of the labyrinth are separated by a trilaminar trophoblast layer; sinusoidal TGCs that line the maternal sinusoids and two layers of syncytiotrophoblast. H – Clusters of glycogen trophoblast cells invade into the decidua beginning on E12.5. Ch – chorion, C-TGC – canal trophoblast giant cell, Dec – decidua, EPC – ectoplacental cone, Endo – endothelial cell, GlyT – glycogen trophoblast cell, P-TGC – parietal trophoblast giant cell, S-TGC – sinusoidal trophoblast giant cell, SpA-TGC – spiral-associated trophoblast giant cell, SpT – spongiotrophoblast, SynT – syncytiotrophoblast cell (I – layer I, II- layer II). Black bar represents 0.1 mm.

Figure 2

Temporal and spatial expression profiles of PRL/PL family members compared with phylogenetic tree analysis. A – Scale diagram of the PRL/PL gene locus on chromosome 13. Rectangles indicate the location of PRL family member genes and ovals represent the location of pseudogenes within the locus. Red arrows indicate the orientation of each gene within the cluster. Note the large inversion from Prl2b1 to Prl7c1. In addition, three Prl2c genes are located approximately 14 Mb upstream of the PRL/PL locus on chromosome 13. *The Prl2c gene located within the main prolactin family cluster (27–28 Mb) does not correspond to any of the 4 Prl2c genes previously annotated from cDNA sequences (Prl2c2-5) and is therefore referred to as Plf, the original gene symbol. B – Phylogenetic tree depicting the evolutionary relationship between the members of the PRL family coupled with temporal (northern blot analysis) and spatial (in situ hybridization summary) expression data for each gene. Blue boxes indicate some positive expression within the trophoblast subtype population while white boxes represent an absence of gene expression. **As the differences between the Prl2c genes (Prl2c2-5) cannot be discerned by northern blot analysis, the results in the current analysis reflect the combined expression of all these genes, and is therefore labeled simply as Prl2c.

Figure 3

Expression of numerous PRL/PL family members within the ectoplacental cone. In situ hybridization of PRL/PL family members in E8.5 implantations sites. Black boxes represent the area magnified in the image below. Dec – decidua, EPC – ectoplacental cone. Black bar represents 1.5 mm. Red bar represents 100 μm.

Figure 4

Spongiotrophoblast and glycogen trophoblast expression of PRL/PL family members. A – Double in situ hybridization of Pcdh12 (Blue) and Prl7a2 (Brown) in E14.5 placenta. B – Double in situ hybridization of Prl6a1 (Blue) and Prl7a2 (Brown) in E14.5 placenta. C – Summary of spongiotrophoblast and glycogen trophoblast-specific expression patterns as well as genes expressed in both populations.

Figure 5

Common modules of transcription factor binding sites within the promoters of commonly expressed PRL/PL family members. A – 3000 bases upstream of the coding sequence plus 200 bases within the coding sequence of 5 genes that are expressed within the glycogen trophoblast population. B – 3000 bases upstream of the coding sequence plus 200 bases within the coding sequence of 3 genes that are expressed within the sinusoidal TGC population within the labyrinth. Examples of 2 different modules are shown.

Figure 6

Several PRL family members have multiple splice variants. The splice patterns of both the originally reported transcripts and their predicted novel isoforms are depicted on the left. Arrows indicate the location and direction of PCR primers used to verify expression of each isoform. Arrow heads (>) indicate the direction of transcription. * indicates predicted transcript. Gel pictures showing PCR amplicons for all the isoforms with the corresponding fragment sizes are shown on the right.