H/358 mRNA Is Preferentially Expressed in the Labyrinth Region of the Mature Chorioallantoic Placenta
As the chorioallantoic placenta matured between Days 10 through 14, the labyrinth zone became highly vascularized and contained a large number of Hp58-positive fetal erythroid cells. In addition, at Day 14 of pregnancy Hp58 was detected in the syncytiotrophoblast of the labyrinthine placenta (data not shown). We hypothesized that if Hp58 was synthesized in cells preferentially located in the labyrinthine placenta, then the amount of Hp58 mRNA would be higher in that region compared to the junctional zone. To test whether Hp58 mRNA was expressed in the mature chorioallantoic placenta (Day 18) and if there were differences in the amount of transcripts expressed between the labyrinth and junctional regions, we measured Hp58 mRNA. Total placental RNA (10 ^g) from the labyrinth and junctional zones of the placenta at Day 18 of pregnancy were combined with single-stranded riboprobes and analyzed by RNase protection. A representative RNase protection assay is shown in Figure 7A.
The full-length, singlestranded transcript from the Hp58 template migrated at the predicted size (~350 bases) in the absence of placental RNA and RNase digestion (lane 1). In the absence of target RNA and RNase digestion (lane 2) the full-length G3PDH transcript migrated at the expected size (~400 bases). No bands were evident in reactions digested with RNase that contained both riboprobes and lacked placental RNA (lane 3, control). When placental RNA from the labyrinth (lane 4) and junctional (lane 5) regions of the placenta were hybridized with the Hp58 riboprobe, the smaller protected fragment corresponding to placental Hp58 was evident. The protected fragment for placental rat G3PDH was the expected size of 316 base pairs (lanes 4 and 5). Those protected fragments were absent from reactions lacking placental RNA (lanes 1-3). The amount of Hp58 mRNA in the two different regions of the placenta was measured by densitometric scanning of an autoradiograph from samples analyzed in triplicate and adjusted to G3PDH as described by us previously. There was a significant (P < 0.05, 3.6-fold) difference in Hp58 mRNA in the labyrinth compared with the junctional zone of the placenta (Fig. 7B).
FIG. 7. Expression of Hp58 mRNA in the labyrinth and junctional regions of the rat chorioallantoic placenta at Day 1 8 of pregnancy. A) Representative RNase protection assay of H^58 mRNA from the labyrinth and junctional zones of the placenta. Reactions (except lanes 1 and 2) were digested with RNase as described in the text. Lane 1, the full-length H^58 transcript synthesized using T3 RNA polymerase; lane 2, the full-length G3PDH transcript synthesized using SP6 polymerase; lane 3, H^58 and G3PDH riboprobes without placental RNA; lane 4, placental RNA (10 l^g) from the labyrinth region plus H^58 and G3PDH riboprobes; lane 5, placental RNA (10 ^g) isolated from the junctional region plus H^58 and G3PDH riboprobes. Arrowheads on the left indicate the position of RNA size markers at 100, 200, 300, 400, and 500 bases, respectively. B) Increased expression of H^58 mRNA in the labyrinth compared to the junctional region. The amount of H^58 mRNA in the labyrinth and junctional regions of the placenta at Day 18 of pregnancy was measured by densi-tometric scanning. There was a significant (P < 0.05, 3.6-fold) increase in Hp58 transcripts in the labyrinth zone. Data are the mean ± SEM of triplicate samples analyzed on the same autoradiograph. Results are representative of three independent RNase protection assays.