Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development

Abstract

The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various Prnp transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the Prnp-knockout-Sprn-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various Prnp and Sprn genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in Eutherians and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

“…It is of interest to recall that an early interpretation of the impact of PrP knockout on cerebellar degeneration 30 had to be revisited subsequent to a demonstration of an artifactual effect of certain Prnp null alleles on the adjacent Prnd transcription unit 31 , 32 . However, Mtg 1 transcript levels are reported as being unaltered by shRNA vectors in a follow-up study, 33 tipping the balance to a consideration of off-axis effects of shRNAs against irrelevant genes. Further validation of shRNA knockdown strategies could involve (1) confirming successful knockdown of the Sho protein and (2) genetic rescue of the knockdown effect on protein levels and embryonic development by co-administration of a second vector with the shRNA vector: this second vector would encode a wt Sho protein open reading frame upstream of a heterologous 3'UTR (i.e., a 3' UTR not targeted by the current anti- Sprn shRNAs).…”

The role of prion protein in stem cell regulation

“…However, the inactivation of PrP C in zebrafish induced embryonic lethality (Malaga-Trillo & Sempou 2009), highlighting the lack of a clear model that could connect the observations. Recently, it has been reported that this lethal consequence is due to the function of the prion protein family in controlling trophoblastic cell lineage maintenance and differentiation (Passet et al 2012). Several approaches have been employed in this search, from newly designed KO models to cell culture, where PrP C appeared to be involved in oxidative stress , neuroprotection (Li & Harris 2005), copper metabolism (Brown et al 1997), synaptic and nervous transmission (Bremer et al 2010), and cellular adhesion ).…”

The prion protein family

“…Origin of this lethality was recently found to result from a developmental failure of the trophectoderm-derived compartment 20 . RNA interference is known to potentially induce knockdown of off-targets and thus false phenotypes.…”

“…However, since it was observed with two independent ShRNAs targeting the Shadoo mRNA, it seems unlikely that this is at the origin of the observed lethality. Furthermore, an attractive alternative hypothesis, that the ShRNA were also targeting the Mtg1 transcript that overlaps the 3′-end of the Shadoo transcript, 16 , 21 could be dismissed by (1) the locations of the ShRNA targeted sequences outside the overlapping region 19 and (2) the absence of differential expression of the Mtg1 transcript in the Shadoo knockdown embryos 20 . Instead, recent data from the literature appear to be in frame with the induced embryonic lethal phenotype and to indirectly comfort this observation.…”