Periostin as a biomarker of the amniotic membrane

Abstract

Tracing the precise developmental origin of amnion and amnion-derived stem cells is still challenging and depends chiefly on analyzing powerful genetic model amniotes like mouse. Profound understanding of the fundamental differences in amnion development in both the disc-shaped primate and human embryo and the cup-shaped mouse embryo is pivotal in particular when sampling amniotic membrane from nonprimate species for isolating candidate amniotic stem cells. The availability of molecular marker genes that are specifically expressed in the amniotic membrane and not in other extraembryonic membranes would be instrumental to validate unequivocally the starting material under investigation. So far such amniotic markers have not been reported. We postulated that bone morphogenetic protein (BMP) target genes are putative amniotic membrane markers mainly because deficiency in one of several components of the BMP signaling cascade in mice has been documented to result in defective development of the early amnion. Comparative gene expression analysis of acknowledged target genes for BMP in different extraembryonic tissues, combined with in situ hybridization, identified Periostin (Postn) mRNA enrichment in amnion throughout gestation. In addition, we identify and propose a combination of markers as transcriptional signature for the different extraembryonic tissues in mouse.

Figure 1

(a)–(c) Schematic representation of a mouse embryo illustrating the position of the extraembryonic tissues before and after axial rotation. During the process of axial rotation the embryo becomes enwrapped in its extraembryonic membranes. Extraembryonic mesoderm is shown in red; yellow represents amniotic ectoderm and embryonic ectoderm (embryonic mesoderm is not depicted); green represents trophectoderm-derived extraembryonic lineages; blue shows extraembryonic endoderm. For more detailed description of the extraembryonic membranes, see [2]. (d)–(h) Sagittal sections through BRE: LacZ mouse embryos in the time range between amnion closure (E7.0) and head fold stages (E8.0). X-gal staining (blue) for β-galactosidase detection in BRE : LacZ heterozygous embryos [5] reports dynamic SMAD1/5/8 mediated BMP signaling in the developing amnion. Abbreviations: E: embryonic day; LSEB: late streak-early bud; NP: neural plate; NPLB: neural plate-late bud; EHF: early head fold; LHF: late head fold.

Figure 2

RNA profiling of putative amnion markers in mouse extraembryonic tissues by RT-qPCR. (a)–(b) Validation of microdissected amniotic membrane tissues collected at different stages of development by relative RNA expression analysis of markers for (a) nonneural ectoderm (Ap-2) and allantois (Tbx4) and (b) primitive red blood cells (ζ-globin) and yolk sac endoderm (Afp). (c)–(e) Expression analysis of acknowledged target genes of SMAD-mediated BMP signaling (Tbx2, Tbx5, Hand1, Hand2, Msx1, Msx2) and (f) Postn, in extraembryonic tissues. Relative RNA levels were obtained by setting the sample with lowest expression for each target to 1. The expression of different targets cannot be directly compared. Abbreviations: Am: amnion; Al: allantois; YS: yolk sac.

Figure 3

In situ hybridization on sections of mouse embryos. (a) RNA localization (blue) of Postn in E8.5 mouse embryos. Boxed areas are shown at a higher magnification. Postn appears localized in the amniotic mesoderm. (b) RNA localization (blue) of Postn in E9.5 mouse embryos. Abbreviations: Am: amnion; Al: allantois; AmM: amniotic mesoderm; AmEC: amniotic ectoderm; Ch: chorionic plate; Ht: heart; Hd: head; Pl: placenta; YS: yolk sac.

Figure 4

(a) RNA profiling of POSTN in human extraembryonic tissues of three individual embryos by RT-qPCR. Gestational age is represented by weeks (w) and days (d). (b) Relative RNA expression of Postn/POSTN in mouse and human amnion samples from different developmental stages. In human amnion POSTN is expressed at very high levels during the first trimester, followed by a significant drop in expression, whilst Postn expression in mouse amnion is stable during gestation. Mouse and human developmental stages are positioned along an arbitrary interval scale. Mouse and human gestation approximates 19.5 days and 38 weeks respectively. Mouse and human developmental stages do not match pairwise. Abbreviations: Am: amnion; Ch: chorion, E: embryonic day; Pl: placenta, UC: umbilical cord, 8–38 w: weeks of gestation.

Figure 5

Relative RNA expression of Postn, AP-2, ζ-globin, Afp, and Tbx4 in extraembryonic tissues of (a) E8.5 and (b) E9.5 mouse embryos. Here, the relative RNA expression values were multiplied or divided by a scale factor, in order to represent values for different transcripts with different expression levels in a single graph. Expression levels of the different genes cannot be compared. Amnion is enriched in Postn and AP-2, while visceral yolk sac (Afp), allantois (Tbx4), and primitive red blood cell (ζ-globin) markers are neglectable. In contrast, yolk sac and allantois express low levels of Postn and AP-2. Abbreviations: Am: amnion; Al: allantois; YS: yolk sac.