PDGFB Regulates the Development of the Labyrinthine Layer of the Mouse Fetal Placenta
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Developmental Biology 212, 124–136 (1999) Article ID dbio.1999.9306, available online at http://www.idealibrary.com on PDGFB Regulates the Development of the Labyrinthine Layer of the Mouse Fetal Placenta Rolf Ohlsson,*,1 Pierre Falck,* Mats Hellstro¨m,† Per Lindahl,† Hans Bostro¨m,† Gary Franklin,* Lars A¨ hrlund-Richter,‡ Jeffrey Pollard,§ Philippe Soriano,¶ and Christer Betsholtz† *Department of Animal Development & Genetics, Uppsala University, Norbyva¨gen 18A, S-752 36 Uppsala, Sweden; †Department of Medical Biochemistry, Gothenburg University, Medicinaregatan 9A, S-413 90 Gothenburg, Sweden; ‡Department of Medical Nutrition, Karolinska Institute, Huddinge, Sweden; §Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461; and ¶Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 PDGFB is a growth factor which is vital for the completion of normal prenatal development. In this study, we report the phenotypic analysis of placentas from mouse conceptuses that lack a functional PDGFB or PDGFRb gene. Placentas of both types of mutant exhibit changes in the labyrinthine layer, including dilated embryonic blood vessels and reduced numbers of both pericytes and trophoblasts. These changes are seen from embryonic day (E) 13.5, which coincides with the upregulation of PDGFB mRNA levels in normal placentas. By E17, modifications in shape, size, and number of the fetal blood vessels in the mutant placentas cause an abnormal ratio of the surface areas between the fetal and the maternal blood vessels in the labyrinthine layer. Our data suggest that PDGFB acts locally to contribute to the development of the labyrinthine layer of the fetal placenta and the formation of a proper nutrient–waste exchange system during fetal development. We point out that the roles of PDGFB/Rb signaling in the placenta may be analogous to those in the developing kidney, by controlling pericytes in the labyrinthine layer and mesangial cells in the kidney. Key Words: trophoblasts; labyrinthine; morphology; blood vessels; pericytes. INTRODUCTION types elicit potent mitogenic signals, although only PDGFRb mediates chemotaxis and actin reorganization in certain cell Platelet-derived growth factor (PDGF) is a potent mitogenic types, such as human fibroblasts and porcine aortic endothe- agent for connective tissue cells such as fibroblasts and lial cells (Hammacher et al., 1989). smooth muscle cells and for glial cells such as oligodendrocyte In adults, most of the functions of PDGF relate to progenitors (Heldin and Westermark, 1990; Raines et al., 1990; different responses to injury, such as inflammation, vascu- Raines and Ross, 1993; Calver et al., 1998). PDGF is synthe- lar injury, and wound healing (Heldin and Westermark, sized by a variety of cell types, including vascular endothelial 1990; Ross et al., 1986; Khachigian et al., 1996). Increased cells, monocytes/macrophages, placental cytotrophoblasts, expression of PDGF and PDGF receptors is seen in a and certain neurons. PDGF is a dimeric molecule composed of number of pathological conditions, including atherosclero- disulfide-bonded A and/or B polypeptide chains, PDGFA and sis, connective tissue overgrowth in conjunction with PDGFB, which are the products of two distinct genes (Heldin chronic inflammatory processes, fibrosis, and tumor stroma and Westermark, 1989). PDGF dimers signal via homo- or formation (Raines and Ross, 1993). The successive activa- heterodimers of two receptor types. The PDGF a-receptor tion of the PDGFB and PDGFRb genes during the genesis of (PDGFRa) interacts with both the A and the B chain of PDGF, choriocarcinoma may reflect the establishment of a persis- whereas the PDGF b-receptor (PDGFRb) preferentially binds tent autocrine loop (Holmgren et al., 1994). The most the B chain (Heldin and Westermark, 1990). Both receptor striking illustration of the importance of PDGFB and PDGFRb function can be found, however, during prenatal 1 To whom correspondence should be addressed. Fax: 146-18- development. PDGFB-orPDGFRb-deficient mice die peri- 4712683. E-mail: [email protected]. natally and exhibit specific developmental abnormalities 124 0012-1606/99 PDGF and Placental Development 125 (Leve´en et al., 1994; Soriano, 1994). These include mal- Peroxidase activity was detected by conventional DAB staining formed kidney glomerular tufts and the formation of mi- (Vectastain). Staining of placenta pericytes was done using croaneurysms due to loss of mesangial cells and microvas- antibodies against a-smooth muscle actin (ASMA) or desmin as cular pericytes (Lindahl et al., 1997a, 1998). Most embryos described (Lindahl et al., 1998) develop fatal vascular leakage and rupture of microvessels Morphometric and statistical analysis. Cell numbers within the labyrinthine layer were counted from randomly chosen areas just prior to birth. The hematological status of such mice measuring 0.27 3 0.21 mm. Fetal blood vessels could be distin- includes erythroblastosis, macrocytic anemia, and throm- guished from maternal blood lacunas by the presence of endothelial bocytopenia. The fact that similar phenotypes are observed cells and adjacent isolectin-positive matrix (see Results). The with both mutants suggests failure of PDGFB interaction surface areas (parametric length; 1000 units equivalent to 1 mm2)of with PDGFRb, rather than with PDGFRa. fetal blood vessels and maternal blood lacunas were scored from The communication between the fetus and the mother randomly chosen areas (0.27 3 0.21 mm) in the labyrinthine layer occurs via the placenta in all eutherian mammals. In the and analyzed using the computer program NIH Image 1.58. At least mouse placenta, contact between the fetal and the maternal three different specimens from each of the wild-type, PDGFB- b circulatory systems takes place in the labyrinthine layer, in deficient, and PDGFR -deficient placentas were examined in 9–16 which the two intermingled networks of vessels—fetal areas (from nine different glass slides). Although measurements were analyzed using average and standard deviation of error, they capillaries and maternal lacunas—form a large contact were also tested using the ranking nonparametric Mann–Whitney surface for nutrient exchange and excretion. The labyrin- U test. Comparisons of cell and vessel numbers and parametric thine layer is functionally analogous to the region of the length variations between wild-type versus PDGFB 2/2 and human placenta containing the chorionic villi, which carry PDGFRb 2/2 showed that the probabilities that the measure- the fetal capillaries, and the intervillus spaces, which con- ments are part of the same populations of values were P , 0.0001 tain the maternal blood. We show here that PDGFB- and for all the statistical calculations presented in Fig. 6. The volumes PDGFRb-deficient mouse placentas have a specific defect of the indicated placentas were analyzed by measuring the para- in the labyrinthine layer, characterized by reduced numbers metric surfaces of the labyrinthine and the spongiotrophoblast m of pericytes and labyrinth trophoblasts, dilated fetal blood layers in every third section (each 7 m thin) throughout each vessels, and a reduced contact surface for maternal blood. specimen. The volumes were then calculated using the NIH Image software. Based on the phenotypic similarities between PDGFB- and b b RNA probes. The PDGFR probe was derived from the PDGFR -null placentas and kidney glomeruli, we propose pSVRI clone (Yarden et al., 1986) and was a kind gift from Dr. J. that placental pericytes and kidney mesangial cells may Escobedo. A 5.4-kb insert was subcloned in pBluescript KS have similar functions in the morphogenesis of complex plasmid. RNA polymerases T7 and T3 generated sense and capillary tufts involved in excretion and/or nutrient ex- antisense probes, respectively. The mouse PDGFB probe was change. generated from a cDNA clone isolated from a lgt10 cDNA library of mouse fetal mRNA (Stratagene) using a human PDGFB cDNA probe. The 2.2-kb insert, starting 918 bp downstream of MATERIALS AND METHODS the cap site and ending within exon 7, was subcloned into a pBSKS plasmid. RNA polymerases T7 and T3 were used to generate sense and antisense probes, respectively. The PDGFRa Production of PDGFB-, PDGFA-, PDGFRb-, and PDGFRa- template was a 234-bp segment (Mercola et al., 1990) containing mutant conceptuses. The derivation of mutant mice has already 39 untranslated sequences and was a kind gift from Dr. M. been described (Leve´en et al., 1994; Soriano, 1994; Bostro¨m et al., Mercola. RNA polymerases T7 and T3 were used to generate 1996; Soriano, 1997). 129SV/C57Bl/6 hybrid offspring or embryos antisense and sense probes, respectively. were genotyped by Southern blot analysis or PCR as described Hybridization analysis. Northern blot hybridization analysis (Leve´en et al., 1994; Soriano, 1994; Lindahl et al., 1997a,b). of total cellular RNA, extracted from the dissected placentas and Histological analysis. Placentas were processed for routine juxtaposed decidua, was performed as described (Ohlsson et al., histology by fixation in Bouin’s solution, or in 4% paraformalde- 1989). In situ hybridization analysis was performed on 7-mm hyde in PBS, followed by paraffin embedding and sectioning. Four- paraffin sections of paraformaldehyde- or Bouin’s-solution-fixed to seven-micrometer sections were stained with PAS–Schiffs re- placentas using 35S-labeled antisense riboprobes as described (Ohls- agents and Mayer’s