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Human Molecular Genetics Advance Access originally published online on October 6, 2006
Human Molecular Genetics 2006 15(22):3293-3305; doi:10.1093/hmg/ddl405
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Analysis of Nsdhl-deficient embryos reveals a role for Hedgehog signaling in early placental development

Fenglei Jiang and Gail E. Herman*

Center for Molecular and Human Genetics, Columbus Children's Research Institute, Department of Pediatrics, The Ohio State University, 700 Children's Dr., Room W403, Columbus, OH 43205, USA

* To whom correspondence should be addressed. Tel: +1 6147222849(direct)/2848(secretary); Fax: +1 6147222817; Email: hermang{at}ccri.net

Received August 28, 2006; Accepted September 27, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The X-linked Nsdhl gene encodes a sterol dehydrogenase involved in cholesterol biosynthesis. Mutations in this gene cause the male lethal phenotypes in human CHILD syndrome and bare patches (Bpa) mice. Affected male embryos for several mutant Nsdhl alleles die in mid-gestation with a thin and poorly vascularized placental labyrinth. The timing and specific abnormalities noted suggest a defect in one or more developmental signaling pathways as a possible mechanism. Here, we examined the possible involvement of the hedgehog signaling pathway in the placental pathology of Nsdhl mutants using a transgenic mouse line (Ptch1tm1Mps) that contains a lacZ reporter under the control of the promoter for Ptch1, the gene that encodes the major hedgehog receptor. We demonstrate expression of Ptch1 in allantoic mesoderm of the placenta from wild-type mid-gestation embryos. The evidence suggests that the signaling is induced by Indian hedgehog that is produced by distal (ectoplacental) visceral endoderm cells that migrate into the allantoic mesoderm before embryonic day 10.0. Using a ubiquitously expressed, X-linked lacZ transgene that undergoes normal X-inactivation, we demonstrate that the placental defects in Nsdhl/+ female embryos are non-cell autonomous. Further, affected placentas from mutant NsdhlBpa-8H male embryos demonstrate markedly decreased or no Ptch1-lacZ staining and no migration of Ihh expressing cells into the developing placenta. These data strongly implicate the hedgehog signaling pathway in the pathogenesis of the placental defects in NSDHL deficiency and provide evidence for a role for the hedgehog pathway in the development of a functional mammalian placenta.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nsdhl (for NADH sterol dehydrogenase-like) encodes a sterol dehydrogenase involved in the removal of C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in this gene are associated with the X-linked, male lethal, mouse mutants bare patches (Bpa) and striated (Str). Heterozygous Bpa females have a skeletal dysplasia and are dwarfed compared with normal littermates. They develop a hyperkeratotic skin eruption on post-natal day 5–7 that resolves, producing a striping of the adult coat consistent with random X-inactivation [reviewed in (1)]. Milder Bpa alleles, originally thought to be a distinct locus called striated (Str), cannot be distinguished from normal littermates until day 12–14 when the striped coat is first apparent. We have detected mutations in Nsdhl in seven Bpa/Str alleles, including the nonsense mutation K103X in the original Bpa1H mutant mouse that produces a truncated protein that is predicted to be a null (2,3). All of the known Nsdhl mutations are lethal prenatally in hemizygous males. Heterozygous mutations in the human NSDHL gene are found in the majority of cases of CHILD syndrome, a rare X-linked, male lethal malformation syndrome characterized by unilateral ichthyosiform skin lesions and limb reduction defects [reviewed in (4)].

Recently, we have demonstrated that affected male embryos for mild (Str) and moderate (Bpa8H) Nsdhl alleles die in mid-gestation, between embryonic day (E) 10.5 and 13.5 (5). Although no consistent primary anomalies were identified in affected male embryos themselves, the labyrinth layer of the fetal placenta was always thinner, with fewer fetal vessels and decreased proliferation of labyrinth trophoblast cells as measured by 5'-bromo-3'-deoxyuridine (BrdU) incorporation. In several affected placentas, embryonic blood vessels in the mutant placenta remained restricted to the chorioallantoic plate region, and there was a complete absence of fetal blood vessels in close apposition with maternal blood sinuses. At E10.5, minimal to no labyrinth layer could be detected in the most severely affected embryos. Fetal and maternal erythrocytes were present, suggesting that the placental failure was not due to a severe defect in hematopoiesis. The numbers and appearance of fetal placental giant cells and spongiotrophoblasts were similar in affected versus normal embryos.

We were initially surprised by our finding of a primary placental problem in these embryos, as X-inactivation is not random in most extraembryonic lineages in the mouse (6,7). Rather, the paternal X chromosome is preferentially inactivated in fetal trophoblast and extraembryonic endodermal lineages in female embryos. A primary defect in one of these lineages would be expected to produce identical effects in mutant male and female embryos. However, since allantoic mesoderm is derived from the epiblast (8), it exhibits random X-inactivation (7), and a defect in this lineage would be expected to preferentially affect mutant males. Following fusion of the allantois with the placental chorion, these mesodermally derived cells differentiate into endothelial cells lining the fetal vessels of the placental labyrinth and contribute to the mesenchymal cell population. Thus, we hypothesized that the primary defect in Nsdhl-deficient embryos must occur in this lineage.

The mechanism whereby a mutation in a gene encoding a cholesterol biosynthetic enzyme would produce placental defects was not readily apparent. It cannot be attributed to a lack of cholesterol or total sterols since comparable sterol levels were present in both normal and affected embryos, almost certainly as the result of maternal transport (5). Comparable sterol levels were also found in normal and affected placentas from Str1H dams at the time of death, whereas placentas associated with normal embryos for Bpa8H dams actually had lower sterol levels than those from affected embryos. We also do not believe that a generalized defect in membrane function causes the male lethality, since it would be likely to occur earlier in gestation, perhaps, even prior to implantation. Rather, we hypothesized that the specific effects on proliferation and vascularization of the placental labyrinth would be more compatible with a defect in one or more signaling pathways.

As described below, we initially focussed our investigations on abnormalities in the hedgehog pathway. There are three mammalian hedgehog proteins—sonic hedgehog (SHH), Indian hedgehog (IHH) and desert hedgehog (DHH), which is closest in amino acid sequence to the original Drosophila hedgehog protein (9,10). Hedgehog proteins act as dose-dependent morphogens affecting patterning in Drosophila larvae, while in mammals these proteins function throughout embryogenesis to affect cell fate determination, as well as cellular proliferation and programmed cell death. Hedgehog proteins undergo autocatalytic processing, with the covalent attachment of a cholesterol moiety to the C-terminus of the active N-terminal peptide, prior to their secretion. Palmitoylation of the N-terminal cysteine of the active ligand also occurs (10,11).

Secretion of hedgehog proteins requires the protein dispatched (DISP) and is not well understood. The major receptor for hedgehog proteins on target cells is the integral membrane protein patched (PTCH). There are two mammalian patched genes, with the major receptor encoded by Ptch1. In the absence of ligand, the PTCH1 protein represses a second membrane protein called smoothened (SMO), by an unknown, indirect mechanism. In Drosophila, stoichiometric binding of hedgehog to its receptor releases this repression, enabling SMO to undergo a conformational change that, in conjunction with a complex of additional proteins, prevents proteolytic cleavage of the transcription factor cubitus interruptus (Ci), and enables the activation of transcription of target genes. In mammals, there are three homologs of Ci, GLI1–GLI3, that divide the repressor and activator functions of Ci. The transcriptional targets of the hedgehog signaling cascade include Ptch1 itself, as well as one or more Gli genes [reviewed in (12,13)].

Aberrant hedgehog signaling has been associated with a variety of human cancers, as well as congenital malformations, including holoprosencephaly (13,14). Secondary abnormalities in hedgehog signaling have been speculated to play a role in the birth defects associated with inherited human developmental disorders of cholesterol synthesis [reviewed in (4,15)]. Further, a defective response to hedgehog signaling, likely at the level of SMO, has been demonstrated in cultured mouse embryonic fibroblasts (MEFs) generated from mice with targeted mutations in the 7-dehydrocholesterol reductase (Dhcr7) or lathosterol-5-desaturase (Sc5d) genes (16). These mouse mutants serve as models for the human cholesterol biosynthetic disorders Smith–Lemli–Opitz syndrome (SLOS) and lathosterolosis, respectively.

Expression of a hedgehog protein or hedgehog signaling has not been demonstrated in the murine fetal placenta, although Indian hedgehog (Ihh) is expressed in maternal uterine tissue at the time of implantation (17,18). Ihh is also expressed in the visceral endoderm of the murine yolk sac by E6.5, where it has been studied extensively (19–22). Ptch1 is expressed in adjacent yolk sac mesothelial and, possibly, some smooth muscle cells, but not in endothelial cells themselves. Studies of embryoid bodies and yolk sacs derived from Ihh–/– and Smo–/– ES cell lines or embryos, respectively, demonstrate that hedgehog signaling is necessary for the proper development of the yolk sac vasculature (21).

Here, we demonstrate the expression of Ihh and its receptor Ptch1 in the early murine fetal placenta. Further, this expression is severely impaired in Nsdhl-deficient placentas, providing the first in vivo demonstration of a defect in hedgehog signaling in a mammalian disorder of cholesterol biosynthesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NSDHL acts in a non-cell autonomous manner in the early mammalian placenta. To begin examining the molecular mechanisms resulting in the labyrinth defects in NSDHL-deficient placentas, we employed mice carrying a ubiquitously expressed X-linked lacZ transgene that undergoes normal X-inactivation (23). To minimize skewing of X-inactivation and effects from genetic background, we bred the transgenic mice onto C57BL/6J for more than 10 generations prior to these experiments (5). Bpa8H/+ females were crossed to lacZ transgenic males, and female embryos and placentas examined at E9.5 and E10.5. These female embryos should express the paternal X chromosome containing the lacZ reporter and a wild-type Nsdhl allele in approximately one-half of the cells derived from allantoic mesoderm that contribute to fetal vessels in the placental labyrinth (see schematic Fig. 1A).


Figure 4051
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  		Figure 1. X-gal staining of cells of the allantoic mesodermal lineage in E10.5 placentas. (A) Schematic representation of the mouse cross. A heterozygous Bpa8H/+ female was mated to a male carrying a ubiquitously expressed, X-linked lacZ transgene that undergoes normal X-inactivation (5,23). The affected X chromosome of the Bpa8H female is shown in pink and the normal X in green. The single X of the transgenic male that expresses lacZ and a wild-type Nsdhl allele is shown in blue. Male embryos from this cross would carry a normal X (green) or an affected X (pink) that is expressed in all cells. The latter are lost by E12.5 (5). Resultant normal female embryos would express a normal X (green) inherited from the mother or the X carrying the transgene (blue) inherited from the father. Expression of the X chromosomes would be random in all embryonic lineages (denoted as blue–green striping) and in extraembryonic lineages derived from mesoderm (blue dots in otherwise green placenta). In the remaining placental lineages, the maternal X (green, lacZ negative) would be expressed in all cells. Similarly, an affected heterozygous Bpa8H female would have random expression of the affected X (pink) or lacZ expressing X chromosome (blue) in the embryo (pink-blue striping) and in approximately one-half of the mesodermally derived cells in the placenta (blue dots in otherwise pink placenta). (B) A representative X-gal stained placental section is shown from a female embryo that is heterozygous affected (XBpa8H/XlacZ) or wild-type (X/XlacZ) at the Nsdhl locus. (C) More severely affected Bpa8H/+ female from a different litter with labyrinth development limited to the chorionic plate. The space between the chorionic plate and spongiotrophoblast/giant cell region (SG) is an artifact that occurred during processing. Sections are counterstained with nuclear fast red. A, allantois; L, labyrinth including region of the chorionic plate adjacent to the allantoic mesoderm; SG, giant cells and spongiotrophoblasts; M, maternal decidua. The boxed regions in the panels on the left in (B) and (C) are enlarged in the panels on the right. Arrows indicate lacZ negative endothelial cells due to random X-inactivation. Endothelial cells and fetal vessels in the affected placentas are restricted primarily (B) or completely (C) to the chorionic plate region of the fetal labyrinth (L).

 
There was a lack of invasion of blue-staining mesodermally derived cells in affected female placentas (XBpa8H/XlacZ) compared with those from normal female littermates (X/XlacZ) at both E9.5 and E10.5. In particular, at E10.5, in affected females, clusters of LacZ+ cells expressing a wild-type Nsdhl allele were found primarily or completely restricted to the chorionic plate, whereas in placentas from normal females, a well-formed labyrinth was dotted with blue-staining cells (Fig. 1B and C). Further, in the affected placentas, for those mesodermally derived cells that had migrated, most remained in small clusters and did not line well-formed vascular structures containing a lumen and fetal erythrocytes (compare Fig. 1B, top and bottom). Examination of LacZ staining in tissues of the embryo proper revealed a random pattern (not shown), excluding generalized skewing of X-inactivation as a cause of the placental abnormalities. In placentas from both mutant and wild-type female embryos, some of the endothelial cells lining the fetal vessels did not express lacZ, demonstrating that cells expressing the mutant Nsdhl allele were not completely excluded from this lineage (see arrows, Fig. 1B and C). These results suggest that NSDHL deficiency acts in a non-cell autonomous manner, at least with respect to these placental defects.

Hedgehog signaling in the early murine fetal placenta. To examine whether evidence of hedgehog signaling could be detected in the early fetal placenta, we next established timed matings between heterozygous Bpa8H/+ or normal B6CBA females and males carrying a lacZ reporter under the control of the Ptch1 promoter [(24); designated as Ptch-lacZ/+]. Upregulation of Ptch1, as detected by X-gal staining, serves as a sensitive and easily visualized indicator of in vivo hedgehog signaling. As shown in Figure 2A, the pattern of X-gal staining in wild-type embryos reflected previously reported expression domains of Ptch1, including ventral regions of the developing brain, ventral somites, developing gut and posterior aspects of the limb buds [zone of polarizing activity (ZPA), yellow arrow]. The placental surfaces shown face the embryo with the site of allantoic attachment at the center. At E9.5, a faint ring of blue, X-gal positive cells was detected in some, but not all, of the normal placentas (Fig. 2B), whereas at E10.5, a circumferential ring of blue staining indicating Ptch1 gene expression was found in all of the placentas examined (Fig. 2C). This ring coincided with the site of yolk sac attachment. By E10.5, there were also ‘spokes’ of Ptch1 expression that radiated inward and outward from this ring and increased in length and number, at least until E15.5, the latest time-point examined (Fig. 2D–F). In sections of X-gal stained wild-type placentas, we found Ptch1 expression in mesenchymal cells of the allantoic mesoderm (Fig. 2G). At higher magnification, the X-gal positive cells were found throughout the allantoic mesenchyme, but did not contribute to the endothelial cell population lining the fetal vessels (Fig. 2G). This finding was confirmed using double staining for the expression of Ptch1 and the endothelial marker PECAM-1, where, as in the yolk sac, we found that the former was not expressed in placental endothelial cells themselves (Fig. 2H).


Figure 4052
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  		Figure 2. Expression of Ptch1 in a normal embryo and post-fusion fetal placentas. A Bpa8H/+ female was mated to a Ptch1-lacZ/+ male and embryos and placentas dissected as described (Methods). (A) X-gal staining of E10.5 normal embryo. The yellow arrow denotes appropriate expression in the ZPA of the forelimb bud. (B–F) Ptch1 expression on coronal surfaces of normal fetal placentas. Note the faint circumferential ring of X-gal staining at E9.5 (B) that coincides with the site of yolk sac attachment to the placenta. By E10.5 (C), faint ‘spokes’ extend out from the ring. These increase at later times in gestation, E11.5 (D), E12.5 (E) and E15.5 (F). Remnants of yolk sacs that remained attached to the placentas in B, D and F are outlined by dotted black lines. X-gal staining of the embryo (A) was performed for 24 h, whereas all placental samples were stained for 48 h. (G) Five micrometer thin section of E12.5 placenta. Note blue X-gal staining indicative of Ptch1 gene expression in mesenchymal cells of the allantoic mesoderm but not in endothelial cells lining the fetal vessels shown. (H) Non-overlapping expression of Ptch1 (X-gal, blue) in mesenchymal cells and PECAM-1 (immunohistochemistry, brown) in endothelial cells of the allantoic mesoderm. The boxed regions in the left panels in (G) and (H) are shown at higher magnification on the right. The fetal surface is at the bottom of all of the images. A, allantoic mesoderm; L, labyrinth.

 
We next examined which hedgehog ligand was responsible for the upregulation of Ptch1. As shown in Figure 3A, at E10.5, only Ihh was expressed in the fetal placenta, whereas expression of all three hedgehog genes was detected in samples prepared from whole embryos. Similar results were observed using samples obtained from E9.5 or E11.5 embryos and placentas (not shown). Since Ihh is also expressed in the yolk sac visceral endoderm (20,21), we could not exclude the possibility of contamination of our placental samples. For this reason and to localize the sites of Ihh expression in the fetal placenta, we performed in situ hybridization on sections from E8.5 to E15.5 normal placentas, using an Ihh-specific antisense probe. As shown in Figure 3B, we noted the expected pattern of Ihh expression in the yolk sac. However, following chorioallantoic fusion, by E9.5, there were small clusters or ‘rests’ of Ihh-expressing cells within the allantoic mesoderm that had the morphological characteristics of visceral endoderm cells (Fig. 3B and C). The number of rests increased at later stages (compare dark-field images at E9.5 and E11.5 in Fig. 3B). Within the allantoic mesoderm, they were surrounded by the areas of Ptch expression in mesenchymal cells (see Figs 2G and 3C). At E15.5, Ihh expression was noted primarily in epithelial cells of the intraplacental yolk sac (IPY) that faced the fetus and lined the sinus of Duval (SD) (Fig. 3B, E15.5). Its expression in the fetal placenta continued throughout gestation (not shown).


Figure 4053
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  		Figure 3. Ihh expression in the fetal placenta. (A) RT-PCR for Ptch1 and the three hedgehog genes in E10.5 fetal placentas (P) dissected away from maternal decidua or in whole embryos (E). RT-PCR for Gapdh served as a control for genomic DNA contamination and RNA quality. The samples P- and E- contained no reverse transcriptase and produced no detectable PCR product. Controls containing no RNA also produced no signal (not shown). (B) 35S in situ hybridization with an Ihh antisense probe in normal placentas. Bright-field (left) and dark-field (right) images showing Ihh expression in extraembryonic tissues. The day of gestation is shown at the left. The fetal surface of the placenta is always at the bottom of each image. Note the strong expression of Ihh in visceral endoderm of the yolk sac at E8.5–E11.5. At E8.5, there is no detectable Ihh expression in the placenta itself, whereas at later stages, increasing numbers of small clusters of Ihh positive cells are noted within the allantoic mesoderm (yellow arrows in dark-field images). By E11.5, there are clusters of Ihh-expressing cells throughout the allantoic mesoderm. At E15.5, there is a well-defined IPY surrounding the SD. Ihh is expressed in the fetal-facing (lower), but not maternal-facing (upper) surface epithelium of the IPY. A sense probe for Ihh produced no signal (not shown). (C) Higher magnification bright-field (left) of Ihh in situ hybridization and hematoxylin/eosin (right) staining of boxed region in the E11.5 bright-field section in (B). Note the expression of Ihh in small clusters of epithelial-appearing cells (black arrows) with identical morphology to the columnar epithelium of the visceral endoderm. AL, allantois; CP, chorionic plate; VYS, visceral yolk sac including mesoderm and endoderm.

 
The IPY is unique to the rodent placenta and was first described by Duval in 1891 (25). It is located at the junction of the chorioallantoic plate and the labyrinth. It is first visible as a distinct, well-formed structure at ~E12.5 in the mouse and is believed to function in calcium transport needed for bone growth late in gestation (26,27). Thus, studies of the IPY have generally examined expression of selected genes at times late in gestation (>15.5), and other possible functions of the IPY have not been well studied.

There is also virtually nothing known about the formation of the IPY beyond the detailed morphological descriptions provided by Duval (25). He suggested that the IPY forms by invagination of distal visceral endoderm (dve) or, as he called it, ‘ectoplacental endoderm’ (25) as follows: Formation of the placental chorion at ~E7.25 creates two cavities, the more distal and transient ectoplacental cavity and the more proximal exocoelomic cavity, adjacent to the amnion and embryo proper (6,28). The dve is defined as extraembryonic endodermal cells that surround the ectoplacental cavity and overlie chorionic ectoderm. In contrast, proximal visceral endoderm (pve) lies adjacent to extraembryonic mesoderm and forms the yolk sac visceral endoderm that surrounds the exocoelomic cavity.

The mature, E15.5 IPY consists of a columnar epithelium that faces the fetal surface of the placenta and a flatter, cuboidal or squamous-appearing epithelium that faces the maternal decidua [see Fig. 132 in (25) and Fig. 2 in (26)]. These epithelial surfaces are separated by a potential cavity, the SD. Duval (25) and later Jollie [(29), see Fig. 5, for example] demonstrated in camera lucida drawings or photomicrographs, respectively, that the maternal-facing epithelial surface of the IPY is contiguous with parietal endoderm, whereas the fetal-facing epithelium is contiguous with dve.

To further define the origin of the placental ‘rests’ that expressed Ihh, we next performed additional in situ hybridizations using probes for genes known to be expressed in the pve and/or dve. The pve of the yolk sac is a secretory and absorptive surface, supporting nutritive transfer between the mother and fetus prior to the establishment of the placenta (28). Alpha fetoprotein (Afp), apolipoprotein E (Apoe), and amnionless (Amn), the latter encoding a protein that is part of the cubulin B12 receptor complex (30,31), were expressed in pve, in contrast to Ihh that was also expressed in dve and in the placental rests (see Figs 4 and 5). The distal extent of expression of Afp marked the boundary between pve and dve (yellow arrows, Fig. 5A). Expression of Apoe was also found in selected placental mesenchymal and trophoblast cells (Fig. 4), consistent with the known ability of the placenta to transport maternal cholesterol in the form of lipoprotein complexes to the fetal circulation (32–34). However, no overlap of expression of Apoe and Ihh was detected within the placenta (not shown).


Figure 4054
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  		Figure 4. Expression of selected genes in extraembryonic membranes. Hematoxylin and eosin (top) and dark-field (middle, bottom) images of 35S in situ hybridization of antisense probes for Ihh, Afp, Amn or Apoe to sections from two different fetal placentas and yolk sacs at E11.5. Ihh is expressed in yolk sac pve, dve and placental rests, with the boundary between pve and dve marked by yellow arrows. Expression of Afp and Amn is detected only in yolk sac pve. Apoe is also expressed in pve, as well as in selected placental trophoblast and mesenchymal cells. A comparison of silver grain staining of the Apoe bright-field section with an adjacent section stained with hematoxylin and eosin demonstrated no overlap of Apoe mRNA expression with placental cells in the rests (not shown).

 


Figure 4055
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  		Figure 5. Expression of selected genes in extraembryonic endodermal lineages and in the IPY. (A) Dark-field images of 35S in situ hybridization of antisense probes for Afp, Ihh and S100g to sections of fetal placenta and yolk sac membranes at E8.5. The top panel shows an H&E image taken from an adjacent section. Higher magnification of the region in the box is shown in the remainder of the images. The separation between the visceral (pve, dve) and parietal endoderm (pe) is an artifact of fixation; however, it enables better visualization of the boundary between pve and dve. Note the expression of Ihh and S100g in pve and dve but not in parietal endoderm (pe) at E8.5. There is no detectable expression of either gene in the placenta at E8.5. The junction of the pve and dve is defined as the distal extent of expression of Afp (yellow arrow), which is expressed only in the pve. AL, allantoic mesoderm; CP, chorionic plate; EC, exocoelomic cavity. (B) At E10.5, expression of Afp continues to be found solely in pve, whereas Ihh and S100g share identical expression patterns in pve, dve and in the rests in placental allantoic mesoderm (yellow arrowheads). Pdgfr{alpha} is also expressed in dve and placental rests, as well as in pe (yellow arrow), with very weak, but detectable expression in pve. VYS, visceral yolk sac including mesoderm and endoderm. Sense probes for all of the genes produced no signals (not shown).

 
We then examined two marker genes known to be expressed in the IPY. Expression of the calcium-binding protein S100g (formerly known as Cabp-9k) has been reported in fetal-facing cells of the IPY (26,27), whereas platelet-derived growth factor receptor {alpha} (Pdgfr{alpha}) is expressed in cells from both of the epithelial surfaces of the IPY (35,36). As shown in Figure 5, at both E8.5 and E10.5, expression of S100g in pve and dve paralleled that of Ihh. Expression of Pdgfr{alpha} was not detected in the extraembryonic endoderm at E8.5 (not shown), although it has been reported to be expressed in pve, dve and parietal endoderm (pe) in earlier post-implantation embryos (37). At E10.5, Pdgfr{alpha} demonstrated strong expression in dve and pe, as well as very weak, but reproducible, expression in pve (Fig. 5B). All three of these genes had identical patterns of strong expression in rests within the allantoic mesoderm (Fig. 5B, yellow arrowheads). In contrast, expression of the pve markers Afp, Amn and Apoe was never found within the placental rests at any time-point examined. At E15.5, expression of Pdgfr{alpha} was noted on both epithelial surfaces of the IPY, whereas S100g, like Ihh, was expressed only on the fetal-facing IPY surface (see Fig. 6A–C).


Figure 4056
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  		Figure 6. Morphology of and marker gene expression in the IPY. (A) Structure of the IPY at E15.5. The boxed region at the left is shown at higher magnification to the right where the columnar, fetal-facing epithelial cells can easily be distinguished from the flatter appearing cells on the maternal surface. EC, exocoelomic cavity; IPY, intraplacental yolk sac; VYS, visceral yolk sac including mesoderm and endoderm; pe, parietal endoderm; ‘*’, SD. (B) Bright-field (left) and dark-field (right) images of 35S in situ hybridization of an antisense probe for S100g to sections of fetal placenta adjacent to those shown in (A). Like Ihh, S100g demonstrates strong expression only in the fetal-surface of the IPY. It also shows continued weak expression in visceral endoderm (ve), with the boundary between pve versus dve not distinguished in this image. (C) Bright-field (left) and dark-field (right) images of 35S in situ hybridization of an antisense probe for Pdgfr{alpha} to sections of fetal placenta as in (B). Note the expression of the gene in epithelial cells of both surfaces of the IPY, as well as in pe (yellow arrow). (D) Hematoxylin/eosin stained sections of the edge of the placenta at E11.5 (left) or E12.5 (right). In each image, cells that appear identical to the placental rests are ‘budding’ from the dve (arrowheads). At E12.5, note the continuity of cells that form the dve with the fetal-facing surface of the IPY. At this stage and position, the morphology of the two epithelial surfaces of the IPY appears indistinguishable.

 
Finally, further support that the placental ‘rests’ were derived from cells of dve is provided in Figure 6D, where the arrowheads mark cells that were ‘budding’ from the dve and appeared identical to the placental ‘rests’ shown in Figures 2 and 3. A summary of the patterns of expression of all of the genes examined in extraembryonic endodermal lineages and in the placenta as shown in Table 1.


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  		Table 1. Expression patterns of endoderm marker genes in extraembryonic tissues between E8.5 and E15.5

 
Hedgehog signaling in the fetal placenta of Nsdhl mutant embryos. We next examined Ptch1 expression in placentas from affected Bpa8H males. Eleven affected male embryos and placentas from six litters in a cross between a Bpa8H/+ female and Ptch1-lacZ male were examined at E10.5 after X-gal staining in comparison with normal male littermates. Placentas from six affected males demonstrated no detectable X-gal staining, whereas in another four, placental staining was markedly decreased. A representative pair of affected and normal E10.5 embryos and placentas is shown in Figure 7A. A single affected placenta, among those examined at E10.5, demonstrated normal X-gal staining (not shown), and this affected embryo was of normal size and had the mildest phenotype at E10.5 of any detected in this cross. No X-gal staining was noted in placentas from two Bpa8H affected males examined at E11.5 (Fig. 7B). Finally, while normal Ihh expression was detected in visceral endoderm of affected Bpa8H males, there were no rests found (not shown) nor Ihh expression in the placenta itself in 11/11 affected males from five litters studied at E10.5 (Fig. 7C).


Figure 4057
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  		Figure 7. Ptch1 and Ihh expression in affected male Bpa8H embryos and placentas. (A) Ptch1 expression as detected by X-gal staining in a representative E10.5 mutant embryo (top) and fetal placenta (bottom). For comparison, an embryo and placenta from a normal male littermate are shown to the right. Note the open thoracic neural tube defect in the affected male (yellow arrowhead) and Ptch1 expression in the ZPA of the forelimb bud in both embryos (yellow arrows). There is no detectable X-gal staining on the fetal surface of the affected placenta. (B) X-gal staining in a representative E11.5 mutant male embryo (top) and placenta (bottom) in comparison with a normal male littermate. The affected embryo is dead and the bottom was cut during dissection. Note the expanded domain of expression of Ptch1 in the forelimb paddle of the normal embryo compared with E10.5 (yellow arrow). Magnification in (A) and (B) is 15X. (C) 35S in situ hybridization of an antisense Ihh probe to E10.5 sectioned yolk sacs and fetal placentas. Two pairs of normal and affected male littermates obtained from different litters are shown. Note the presence of intraplacental clusters of Ihh positive cells in the normal (X/Y) but not affected (XBpa8H/Y) males. On adjacent hematoxylin/eosin stained sections, there were also no placental rests noted in the affected embryos (not shown).

 
We also observed that the Ptch+/–, Bpa8H/Y embryos appeared much smaller, and most had severe defects in forebrain development and occasional neural tube defects not noted in Bpa8H male embryos from conventional matings to B6CBA males (Fig. 7A and B). Severe forebrain defects with holoprosencephaly and cyclopia are found in Shh–/– embryos, consistent with the critical role of SHH signaling in the development of the CNS (38). The severe CNS and forebrain defects in our cross may have resulted from haploinsufficiency for Ptch1, in addition to Nsdhl deficiency.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
A critical time in mammalian development is the fusion of the fetal allantois with the placental chorion. Subsequently, reciprocal inductive signals between allantoic mesoderm and trophoblast ectodermal lineages result in chorioallantoic branching and differentiation of chorionic trophoblasts [reviewed in (39–41)]. The transcription factor GCM1 is expressed by selected trophoblast cells by E8.0 and is required for proper branching and formation of syncytiotrophoblasts (42,43). Cells of the allantoic mesoderm penetrate the chorionic plate at these branch points and differentiate into endothelial cells that line the fetal vessels of the developing labyrinth. They also contribute to the mesenchymal cell population. Defects in both the trophoblast lineage and in allantoic mesoderm can produce a similar phenotype of a flat chorion lacking a well-developed labyrinth with fetal vessels. However, the series of events that follow fusion, including the initiation of branching of the labyrinth, the regulation of expression of Gcm1 and the proliferation and migration of mesodermally derived fetal endothelial cells, are not well understood.

We have demonstrated hedgehog signaling in the early murine fetal placenta following fusion of the allantois with the chorion. We defined signaling as upregulation of Ptch1 gene expression using a Ptch-lacZ transgene (24). We believe that the low level of Ptch1 gene expression in the fetal placenta, requiring 48 h of X-gal staining, is the reason that a role for hedgehog signaling in this tissue was not previously recognized. We have shown that the endogenous signaling ligand is IHH, produced by rests of cells found in allantoic mesoderm of E9.5 and later stage embryos. The pattern of expression of marker genes analyzed (Table 1) strongly supports the hypothesis that the placental rests originate from cells derived from dve and that these cells also form or contribute to the formation of the IPY. We believe that the rests bud from dve as shown in Figure 6D and migrate into the allantoic mesoderm following chorioallantoic fusion. Further support for this model is our consistent observation that the rests are initially noted only at the edges of the placenta, close to the dve. As gestation proceeds and the number of rests increases, they are found progressively closer to the center of the allantoic mesoderm (see Fig. 3B). Although we cannot exclude a possible alternative model in which the growing placenta ‘engulfs’ cells of the dve, our observations favor the ‘migration’ of rests toward the center of the placental surface. Our demonstration of Ihh expression in the fetal-facing epithelial cells of the IPY also strongly suggests that this structure serves other functions in addition to a role in calcium transport.

The sites of Ptch1 and Ihh expression in the normal fetal placenta, our observation of markedly reduced or absent hedgehog signaling in NSDHL-deficient placentas, and the morphological defects noted in Nsdhl mutants, all strongly implicate the hedgehog pathway in the process of invasion of mesodermally derived cells of the post-fusion allantois. This invasion, we believe, is necessary for proliferation of labyrinth trophoblast cells, allowing further migration of mesodermal cells destined to line fetal vessels. This hypothesis is consistent with our finding that the placental defects in Nsdhl/+ females are non-cell autonomous since the secreted hedgehog signal can be received by multiple allantoic mesodermal cells. Most heterozygous Bpa8H/+ placentas subsequently recover by an as yet unknown mechanism, although there is some prenatal loss of affected female embryos, particularly for the most severe Bpa1H allele (5). The severity of the placental abnormalities in heterozygous Bpa females results from normal variation in the pattern of X-inactivation. These data do represent a clear difference in gene expression between Nsdhl mutant and wild-type placentas and the first in vivo evidence of a hedgehog signaling defect in an inherited disorder of cholesterol synthesis.

We believe that the primary defect in the Nsdhl mutant alleles studied is the lack of the ‘signal’ that normally results in the migration of Ihh-expressing dve cells. Again, based on the male lethality of mutant Nsdhl alleles and paternal X-inactivation in most extraembryonic lineages in female embryos (including the dve), that signal likely originates in the allantoic mesodermal cells themselves, although we cannot exclude the possibility that it originates in another mesodermally derived extraembryonic cell type. Fusion of the allantois with the placental chorion would lead to the expression of the primary signaling molecule. It is also possible that the primary defect in affected placentas is a response in allantoic mesoderm to a signal from trophoblast or endodermal cells.

Further studies are needed to elucidate how a defect in a cholesterol biosynthetic enzyme can perturb cell migration and/or cell signaling. Cooper et al. noted normal processing of SHH in MEFs derived from mouse models of SLOS and lathosterolosis, despite depletion of 50–65% of the cellular cholesterol. Total sterol levels in the lipid-depleted cells were reduced from 25 to 35% (16). These results are consistent with the ability of 7-dehydrocholesterol (7DHC), the sterol intermediate that accumulates in SLOS, to substitute for cholesterol in hedgehog autoprocessing (44). Harsher treatment of CHO cells to deplete cellular cholesterol did result in nearly complete inhibition of intracellular processing of a transfected and tagged mouse Shh construct (45). In vivo, normal processing of SHH was found late in gestation in a targeted mouse model of SLOS (46). This finding and the survival of SLOS mouse models until birth (47,48) likely reflect the ability of 7DHC to substitute for cholesterol in cell membranes (49), as well as in hedgehog processing. In addition, there is active transfer of substantial amounts of maternal cholesterol to all tissues of the rodent fetus, except the brain (34,50).

Subtle defects in plasma membrane function have been detected, however, in model membranes or in cells cultured from SLOS patients. A unique role for cholesterol in plasma membranes is its incorporation with glycosphingolipids into lipid rafts. These lipid microdomains are more densely packed and have a more ordered structure than other regions of the plasma membrane, and they are enriched in proteins involved in cell signaling (51,52). 7DHC and lathosterol promote the formation of and are incorporated into artificial lipid rafts generated in vitro as well as cholesterol itself (53,54). In contrast, lanosterol does not promote raft formation (54), consistent with the pronounced effects of the C-4 methyl groups on membrane fluidity (55). In vivo, rafts prepared from the brains of rats treated with the 7DHC reductase inhibitor AY9944 were also indistinguishable from those containing cholesterol (49). However, the overall protein composition of such rafts differed from that found in control rat brains, and Tulenko et al. noted increased fluidity in plasma membranes prepared from fibroblasts obtained from patients with SLOS (56). These researchers also noted functional differences in the plasma membranes of SLOS-cultured cells, including increased calcium permeability, suppression of inositol phosphate signaling, and markedly reduced plasma membrane Na+/K+ ATPase activity. All of these physiological processes are associated with caveolae, a subset of lipid rafts (52).

It has been reported that PTCH associates directly with caveolin-1, the major protein component of caveolae (57), whereas SMO associates with and is internalized through non-raft-associated clathrin-coated pits (58). Both of these multipass transmembrane proteins are likely to interact with sterols: PTCH contains a ‘sterol-sensing domain’ found in several proteins involved in the regulation of lipid metabolism (59), whereas SMO has been shown to bind the sterol analog cyclopamine (60). Further, Cooper et al. (16) noted a defective response to hedgehog signaling in MEFs generated from mouse models of SLOS and lathosterolosis, probably at the level of SMO.

Thus, there is accumulating evidence that perturbations of cellular sterols significantly impact plasma membrane structure and function. Given the dramatic structural differences between cholesterol and the C-4 mono- and dimethyl-sterols that accumulate in NSDHL deficiency (2), we believe that the hedgehog signaling abnormalities that occur in mutant placentas result from direct effects on one or more proteins that associate with sterols and/or altered properties of the plasma membrane that secondarily affect one or more signaling pathways. Current studies in the laboratory are exploring additional possible genes that may be involved in this process, including Wnts, Bmps or Fgfs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse strains
The current proper designation for a mutant Nsdhl allele, such as Bpa8H, is NsdhlBpa-8H; for simplicity, they will be denoted in the text without the gene symbol. Heterozygous mutant females would be denoted as Bpa8H/+, for example. The origins and breeding of the Bpa8H/+ and X-linked lacZ alleles have been described (3,5). Heterozygous mice containing a lacZ reporter under the control of the Ptch1 promoter (Ptch1tm1Mps) (24) were purchased from The Jackson Laboratory (strain #003081). Genotyping of Ptch1-lacZ mice was performed by PCR as recommended. Heterozygous Ptch1-lacZ males, on a mixed B6/129 background, were crossed with F1 hybrid B6CBA females (The Jackson Laboratory, strain #001201) and were at the N3–N5 backcross generation in the experiments performed here. Bpa8H/+ females are maintained by mating to B6CBA males, and no differences in phenotype were observed in any of the crosses involving Ptch1 mice at different backcross generations.

Analyses of embryos and placentas
Embryos were collected after timed matings using a 12 h light/dark cycle (6 am–6 pm), with noon on the day of the vaginal plug defined as E0.5. Embryos, yolk sacs and placentas were dissected away from maternal decidua under a Nikon SMZ-10A dissecting microscope (Melville, NY, USA) and photographed using a SPOT digital camera. Genotyping of individual embryos was typically performed after PCR amplification of yolk sac DNA with primers for the Scmx locus for sex determination (61) and for Nsdhl using a coupled PCR assay with restriction enzyme digest that distinguishes wild-type and mutant Bpa8H chromosomes (3).

For routine histology, tissue was fixed in Bouin's fixative and 5 µm serial sections cut manually on a Leica RM 2155 microtome (Bannockburn, IL, USA) and examined following hematoxylin/eosin staining. Whole placentas and embryos were stained for beta-galactosidase (LacZ) activity using X-gal and standard protocols (6). To visualize Ptch1-lacZ expression in the placenta, the staining time in the X-gal solution was extended to 48 h, whereas embryos were typically stained for 18–24 h. The X-gal-stained placentas were fixed in 10% neutral-buffered formalin overnight and paraffin embedded sections were taken at 5 µm and counterstained with nuclear fast red.

RT-PCR was performed on E10.5 whole embryos or fetal placentas dissected away from maternal deciduas, yolk sac, and umbilical cord. Total RNA was prepared using TRIZOL Reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription was performed using the SuperScript First-Strand Synthesis kit (Invitrogen) followed by 35 cycles of PCR (30 s at 95°C, 1 min at 55°C and 1 min at 72°C) using the following gene-specific primers:

Ptch1: 5'-TGCTCGCTCTGGAACACATG-3'.

5'-TCGGTTTAGGCCATTGGCTG-3'.

Ihh: 5'-AGAGAGGCTGCCCGTAGAT-3'.

5'-TTCCCCAGTCCCAGGTAGT-3'.

Dhh: 5'-ACTTTTCCCTGCAGGTGAG-3'.

5'-GGTCTATTGCTGACAGGTTG-3'.

Shh: 5'-GTACTCACAGTGAGGGAAAG-3'.

5'-AGTTAGGTATTGATCTCTCA-3'.

Gapdh: 5'-CGGAGTCAACGGATTTGGTCGTAT-3'.

5'-GAAGATGGTGATGGGCTTCC-3'.

In situ hybridizations were performed on 4% paraformaldehyde fixed and sectioned placentas and yolk sacs as described (62). Sense and antisense RNA probes for Ihh (nucleotides 893–2176 GenBank accession no. NM_010544), Afp (19–596 GenBank accession no. NM_007423), ApoE (3–1049 GenBank accession no. NM_009696), Amn (1275–1664 GenBank accession no. NM_033603), Pdgfr{alpha} (71–940 GenBank accession no. M57683) and S100g (35) were transcribed from partial mouse cDNAs cloned into Bluescript KS+ (Stratagene) or pCRII-TOPO (Invitrogen). RNA probes were synthesized from linearized sense and antisense cDNA templates using the Riboprobe Combination System (Promega) and labeled with [35S]-UTP (Amersham).

Immunohistochemistry was performed using a goat polyclonal antibody to CD31/PECAM-1 (Santa Cruz Biotechnologies; catalog #sc-1506), at a dilution of 1:200. Horseradish peroxidase-staining was performed as recommended using reagents from ScyTek Laboratories.


   ACKNOWLEDGEMENTS
 
The authors thank Heithem El-Hodiri for careful review of the manuscript, Erica Mazzone for help with translation of reference (25) and David Cunningham for both review of the manuscript and help with interpretation of the translation of reference (25). This work was supported by NIH R01 HD38572 to G.E.H and by funds from the Columbus Children's Research Institute.

Conflict of Interest statement. None declared.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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D. Cunningham, T. Talabere, N. Bir, M. Kennedy, K. L. McBride, and G. E. Herman
Significant contributions of the extraembryonic membranes and maternal genotype to the placental pathology in heterozygous Nsdhl deficient female embryos
Hum. Mol. Genet., November 11, 2009; (2009) ddp502v2.
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