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Anatomy of the Mouse Placenta Throughout Gestation
Anatomy of the Mouse Placenta Throughout Gestation Sarah M. Isaac Department of Physiology, University of Toronto, Toronto, ON, Canada; LunenfeldTanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada
Michael B. Langford The Queensland Brain Institute, The University of Queensland, St Lucia, QLD, Australia
David G. Simmons The School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia
4
CHAPTER SUMMARY This chapter outlines the morphology of the mouse placenta during development and iden-
tifies characteristic features that can be observed on histological cross-sections.
Abbreviations
C-TGC Canal TGC Ctsq
Cathepsin Q
gd
Gestational day
GlyT
Glycogen trophoblast
Gcm1 Glial cells missing 1 H&E
Hematoxylin and Eosin
S. Lee Adamson
ISH
In situ hybridization
Department of Physiology, University of Toronto, Toronto, ON, Canada; LunenfeldTanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada; Department Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada
Mest
Mesoderm specific transcript
PBS
Phosphate buffered saline
PL-II
Placental lactogen-II (gene name Prl3b1)
PlpN
Prolactin-like protein N (gene name Prl7b1)
P-TGC Parietal TGC SpT Spongiotrophoblast S-TGC Sinusoidal TGC
The Guide to Investigation of Mouse Pregnancy. http://dx.doi.org/10.1016/B978-0-12-394445-0.00004-7 © 2014 Elsevier Inc. All rights reserved.
TGC
Trophoblast giant cells
Tpbpa Mus musculus trophoblast specific protein alpha Specimens for in situ hybridization (ISH) were collected from C57Bl/6 mice purchased from the Animal Resources Centre (Australia). Mice were mated in-house. The morning a vaginal copulation plug was detected was designated as gestation day (gd) 0.5. Placental samples were collected at gd 8.5, 10.5, 13.5, 14.5 (ISH), and 15.5. Placentas were immediately fixed in 4% paraformaldehyde in phosphate buffered saline overnight. Fixed tissues were processed into paraffin blocks and midline cross-sections were cut 5 μm thick. The specimens were stained with H&E to outline general features of the placenta, processed using immunohistochemistry (Blaisdell and Erlebacher, Chapter 49) to stain for CD34, a marker for the basement membrane of endothelial cells, or processed for ISH (Bany and Simmons, Chapter 50). CD34 immunostaining (rat anti-CD34; ab8158, ABCAM, dilution 1:50) and counterstaining with hematoxylin was 69
ANATOMY OF THE MOUSE PLACENTA CHAPTER 4
THE GUIDE TO INVESTIGATION OF MOUSE PREGNANCY
performed by the pathology core laboratory of the Centre for Modeling Human Disease at the Toronto Centre for Phenogenomics. H&E
staining and immunohistochemistry pictures were taken with a BX61 Olympus microscope (Olympus America; Center Valley, PA). ISH
(A)
(B)
pictures were taken using the Aperio Slide Scanner and ImageScope software (Aperio; Vista, CA).
(C)
vys epc epc
ch ch
em al am pys
am
(D)
al pys vys
(E) dec dec jz lab cp
jz lab cp
Plate 1 Hematoxylin and eosin–stained midline sections of the developing mouse placenta. (A) A complete implantation site at gestation day (gd) 8.5 is shown. In this cross-section, the embryo (em), amnion (am), allantois (al), chorion (ch), ectoplacental cone (epc), and the parietal (pys) and visceral yolk sac (vys) membranes are labeled. Other structures are labeled in Edwards et al Chapter 3 (Plate 5 in which the embryo had been removed). (B, C) show enlarged views at gd 8.5. (B) The ectoplacental cone (epc) is outlined by secondary trophoblast giant cells (TGC; arrows). This region differentiates to form the junctional zone and is discussed in detail, including at the ultrastructural level in Bevilacqua et al Chapter 10. The allantois (al) has attached to the bilayered chorion. This region differentiates to form the chorionic plate (cp) and labyrinth. The amnion (am) and yolk sac membranes (pys, vys) are also shown. (C) The remainder of the implantation chamber in A is lined by primary TGC (arrows in C). (D) By gd 10.5, the allantois has become the umbilical cord (arrow). The placenta has differentiated into four distinct layers of the mature placenta; decidua (dec), junctional zone (jz), labyrinth (lab) and chorionic plate. (E) By gd 13.5, the chorionic plate, labyrinth, and junctional zone have enlarged relative to the decidual region. The four layers of the mature placenta are shown in greater detail in Plate 2. Other anatomical structures are labeled in Edwards et al Chapter 3 (Plate 7 gd 10.5; and Plate 10 gd 13.5). Scale bar in A, D, and E = 1000 μm. Scale bar in B and C = 200 μm.
70
H&E (A)
CD34
(D)
(G) dec
dec jz
jz
lab cp
* cp
(B)
lab
(E)
(H)
dec
dec
jz
jz lab
jz (C)
(F)
(I)
jz
lab
* * * *
* cp
*
* lab
* Plate 2 Hematoxylin and eosin and CD34-stained midline sections of the mature placenta at gd 15.5. (A) H&E-stained midline section showing the four layers of the placenta; decidua (dec), junctional zone (jz), labyrinth (lab), and chorionic plate (cp). Enlarged images from this section are shown in B, C, E, and F. (B) The decidua and junctional zones are separated by a layer of parietal trophoblast giant cells (P-TGC; arrows). (C) The cp contains chorionic vessels (asterisks). These vessels connect the fetal arterioles and capillaries in the labyrinth with the vessels of the umbilical cord (not shown). (D) The layers of the placenta are outlined (superimposed on same image shown in A). (E) The junctional zone is made up of three trophoblast cell types: P-TGC (arrows), spongiotrophoblast cells, and glycogen trophoblast cells (in blue and green outlined regions). Glycogen trophoblast cells can be distinguished morphologically by their vacuolated cytoplasm. At gd 15.5, glycogen trophoblast cells have invaded the decidua and are seen both in the junctional zone (outlined in green) and in the decidua (outlined in blue). (F) Finger-like projections from the junctional zone are often seen in the labyrinth (asterisks). (G) CD34-stained midline section showing endothelium stained brown. The fetal vasculature in the labyrinth and the maternal vasculature in the decidua are stained. Note the absence of staining in the avascular junctional zone. An asterisk marks a maternal arterial canal in the labyrinth. It is lined by trophoblast so it is not stained brown. Yolk sac villi (arrows) are particularly prominent in the region of the yolk sac that overlays the placenta. (H) An enlarged image showing the boundary between the avascular junctional zone and the highly vascular labyrinth layer. (I) A higher-power view of the labyrinth to show brown-stained fetal endothelial cells enclosing fetal blood spaces, whereas maternal blood spaces (also referred to as sinusoids) do not stain brown because they are lined with trophoblast cells (red asterisks). Maternal blood spaces can also be distinguished where they are lined by cells with very large nuclei, the sinusoidal trophoblast giant cells (arrows), or when they contain small anuclear red blood cells, which are characteristic of adult cells. Embryonic red blood cells are larger and often nucleated at gd 15.5. Scale bar in A, D, and G = 1000 μm. Scale bar in B, C, E, F, and H = 200 μm. Scale bar in I = 55 μm.
71
ANATOMY OF THE MOUSE PLACENTA CHAPTER 4
THE GUIDE TO INVESTIGATION OF MOUSE PREGNANCY
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
Plate 3 In situ hybridization staining for Cathepsin Q (Ctsq), Glial cells missing 1 (Gcm1), and Mesoderm specific transcript (Mest) show different cell types in the labyrinth in gd 14.5 placentas. (A) Ctsq expression within the labyrinth layer. (B) Canal trophoblast giant cells in the labyrinth do not express Ctsq. (C) Ctsq is expressed by the mononuclear trophoblast giant cells that line the maternal blood spaces (S-TGC; arrows) as do the occasional parietal trophoblast giant cells (seen in A). Other labyrinth cell types, such as the two syncytiotrophoblast layers and the fetal capillary endothelial cells, are negative for Ctsq expression. (D) Gcm1 expression is restricted to the labyrinth. (E,F) Gcm1 is specifically expressed by the syncytiotrophoblast layer II cells of the interhemal membrane. Arrows in F show sinusoidal trophoblast giant cells (S-TGC) in maternal blood spaces. Although not shown here, syncytiotrophoblast layer II cells also express Synb and Cepba, whereas Syna is expressed by syncytiotrophoblast layer I cells. (G) The expression of Mest is restricted to cells of the labyrinth layer. (H–I) However, in this case, Mest expression is localized to the endothelial cells lining the fetal circulation. Arrows in I show S-TGC in maternal blood spaces. Scale bar in A, D, and G = 1 mm; Scale bar in B, C, E, F, H, and I = 100 μm.
72
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
Plate 4 In situ hybridization staining for Prl3b1, Prl7b1, and Mus musculus trophoblast specific protein alpha (Tpbpa) in gd 14.5 placentas. (A) Prl3b1 (PL-II) staining is evident in several trophoblast cell types within the junctional zone and labyrinth layer of the definitive placenta. (B) Within the junctional zone, parietal trophoblast giant cells (PTGC; black arrows) and spongiotrophoblast cells (SpT) are strongly positive for Prl3b1 expression, whereas glycogen trophoblast cells (GlyT) are negative. (C) Within the labyrinth layer (Lab), trophoblast giant cells lining the maternal blood spaces (S-TGC; gray arrow) and maternal arterial canals (C-TGC) express Prl3b1, but syncytiotrophoblast and fetal capillary endothelium do not. (D) Cells within the junctional zone, and also some within the decidual compartment, express Prl7b1 (PlpN) by gd 14.5. (E) Specifically, GlyT and C-TGC are strongly positive for Prl7b1 expression, while SpT remain negative. (F) By gd 14.5, GlyT leave the confines of the junctional zone and migrate into the decidual compartment. Prl7b1-positive GlyT cells can be seen in the junctional zone as well as above the P-TGC layer within the decidual compartment. Prl7b1-negative P-TGCs can be clearly seen (black arrows) demarcating the boundary between the junctional zone and the decidua. (G) Tpbpa (or 4311) expression is routinely used to identify cells of the junctional zone. (H, I) Both SpT and GlyT express Tpbpa, although P-TGC do not. Scale bar in A, D, and G = 1 mm; Scale bar in B, C, E, F, H, and I = 100 μm.
73
Michael B. Langford The Queensland Brain Institute, The University of Queensland, St Lucia, QLD, Australia
David G. Simmons The School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia
4
CHAPTER SUMMARY This chapter outlines the morphology of the mouse placenta during development and iden-
tifies characteristic features that can be observed on histological cross-sections.
Abbreviations
C-TGC Canal TGC Ctsq
Cathepsin Q
gd
Gestational day
GlyT
Glycogen trophoblast
Gcm1 Glial cells missing 1 H&E
Hematoxylin and Eosin
S. Lee Adamson
ISH
In situ hybridization
Department of Physiology, University of Toronto, Toronto, ON, Canada; LunenfeldTanenbaum Research Institute of Mount Sinai Hospital, Toronto, ON, Canada; Department Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada
Mest
Mesoderm specific transcript
PBS
Phosphate buffered saline
PL-II
Placental lactogen-II (gene name Prl3b1)
PlpN
Prolactin-like protein N (gene name Prl7b1)
P-TGC Parietal TGC SpT Spongiotrophoblast S-TGC Sinusoidal TGC
The Guide to Investigation of Mouse Pregnancy. http://dx.doi.org/10.1016/B978-0-12-394445-0.00004-7 © 2014 Elsevier Inc. All rights reserved.
TGC
Trophoblast giant cells
Tpbpa Mus musculus trophoblast specific protein alpha Specimens for in situ hybridization (ISH) were collected from C57Bl/6 mice purchased from the Animal Resources Centre (Australia). Mice were mated in-house. The morning a vaginal copulation plug was detected was designated as gestation day (gd) 0.5. Placental samples were collected at gd 8.5, 10.5, 13.5, 14.5 (ISH), and 15.5. Placentas were immediately fixed in 4% paraformaldehyde in phosphate buffered saline overnight. Fixed tissues were processed into paraffin blocks and midline cross-sections were cut 5 μm thick. The specimens were stained with H&E to outline general features of the placenta, processed using immunohistochemistry (Blaisdell and Erlebacher, Chapter 49) to stain for CD34, a marker for the basement membrane of endothelial cells, or processed for ISH (Bany and Simmons, Chapter 50). CD34 immunostaining (rat anti-CD34; ab8158, ABCAM, dilution 1:50) and counterstaining with hematoxylin was 69
ANATOMY OF THE MOUSE PLACENTA CHAPTER 4
THE GUIDE TO INVESTIGATION OF MOUSE PREGNANCY
performed by the pathology core laboratory of the Centre for Modeling Human Disease at the Toronto Centre for Phenogenomics. H&E
staining and immunohistochemistry pictures were taken with a BX61 Olympus microscope (Olympus America; Center Valley, PA). ISH
(A)
(B)
pictures were taken using the Aperio Slide Scanner and ImageScope software (Aperio; Vista, CA).
(C)
vys epc epc
ch ch
em al am pys
am
(D)
al pys vys
(E) dec dec jz lab cp
jz lab cp
Plate 1 Hematoxylin and eosin–stained midline sections of the developing mouse placenta. (A) A complete implantation site at gestation day (gd) 8.5 is shown. In this cross-section, the embryo (em), amnion (am), allantois (al), chorion (ch), ectoplacental cone (epc), and the parietal (pys) and visceral yolk sac (vys) membranes are labeled. Other structures are labeled in Edwards et al Chapter 3 (Plate 5 in which the embryo had been removed). (B, C) show enlarged views at gd 8.5. (B) The ectoplacental cone (epc) is outlined by secondary trophoblast giant cells (TGC; arrows). This region differentiates to form the junctional zone and is discussed in detail, including at the ultrastructural level in Bevilacqua et al Chapter 10. The allantois (al) has attached to the bilayered chorion. This region differentiates to form the chorionic plate (cp) and labyrinth. The amnion (am) and yolk sac membranes (pys, vys) are also shown. (C) The remainder of the implantation chamber in A is lined by primary TGC (arrows in C). (D) By gd 10.5, the allantois has become the umbilical cord (arrow). The placenta has differentiated into four distinct layers of the mature placenta; decidua (dec), junctional zone (jz), labyrinth (lab) and chorionic plate. (E) By gd 13.5, the chorionic plate, labyrinth, and junctional zone have enlarged relative to the decidual region. The four layers of the mature placenta are shown in greater detail in Plate 2. Other anatomical structures are labeled in Edwards et al Chapter 3 (Plate 7 gd 10.5; and Plate 10 gd 13.5). Scale bar in A, D, and E = 1000 μm. Scale bar in B and C = 200 μm.
70
H&E (A)
CD34
(D)
(G) dec
dec jz
jz
lab cp
* cp
(B)
lab
(E)
(H)
dec
dec
jz
jz lab
jz (C)
(F)
(I)
jz
lab
* * * *
* cp
*
* lab
* Plate 2 Hematoxylin and eosin and CD34-stained midline sections of the mature placenta at gd 15.5. (A) H&E-stained midline section showing the four layers of the placenta; decidua (dec), junctional zone (jz), labyrinth (lab), and chorionic plate (cp). Enlarged images from this section are shown in B, C, E, and F. (B) The decidua and junctional zones are separated by a layer of parietal trophoblast giant cells (P-TGC; arrows). (C) The cp contains chorionic vessels (asterisks). These vessels connect the fetal arterioles and capillaries in the labyrinth with the vessels of the umbilical cord (not shown). (D) The layers of the placenta are outlined (superimposed on same image shown in A). (E) The junctional zone is made up of three trophoblast cell types: P-TGC (arrows), spongiotrophoblast cells, and glycogen trophoblast cells (in blue and green outlined regions). Glycogen trophoblast cells can be distinguished morphologically by their vacuolated cytoplasm. At gd 15.5, glycogen trophoblast cells have invaded the decidua and are seen both in the junctional zone (outlined in green) and in the decidua (outlined in blue). (F) Finger-like projections from the junctional zone are often seen in the labyrinth (asterisks). (G) CD34-stained midline section showing endothelium stained brown. The fetal vasculature in the labyrinth and the maternal vasculature in the decidua are stained. Note the absence of staining in the avascular junctional zone. An asterisk marks a maternal arterial canal in the labyrinth. It is lined by trophoblast so it is not stained brown. Yolk sac villi (arrows) are particularly prominent in the region of the yolk sac that overlays the placenta. (H) An enlarged image showing the boundary between the avascular junctional zone and the highly vascular labyrinth layer. (I) A higher-power view of the labyrinth to show brown-stained fetal endothelial cells enclosing fetal blood spaces, whereas maternal blood spaces (also referred to as sinusoids) do not stain brown because they are lined with trophoblast cells (red asterisks). Maternal blood spaces can also be distinguished where they are lined by cells with very large nuclei, the sinusoidal trophoblast giant cells (arrows), or when they contain small anuclear red blood cells, which are characteristic of adult cells. Embryonic red blood cells are larger and often nucleated at gd 15.5. Scale bar in A, D, and G = 1000 μm. Scale bar in B, C, E, F, and H = 200 μm. Scale bar in I = 55 μm.
71
ANATOMY OF THE MOUSE PLACENTA CHAPTER 4
THE GUIDE TO INVESTIGATION OF MOUSE PREGNANCY
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
Plate 3 In situ hybridization staining for Cathepsin Q (Ctsq), Glial cells missing 1 (Gcm1), and Mesoderm specific transcript (Mest) show different cell types in the labyrinth in gd 14.5 placentas. (A) Ctsq expression within the labyrinth layer. (B) Canal trophoblast giant cells in the labyrinth do not express Ctsq. (C) Ctsq is expressed by the mononuclear trophoblast giant cells that line the maternal blood spaces (S-TGC; arrows) as do the occasional parietal trophoblast giant cells (seen in A). Other labyrinth cell types, such as the two syncytiotrophoblast layers and the fetal capillary endothelial cells, are negative for Ctsq expression. (D) Gcm1 expression is restricted to the labyrinth. (E,F) Gcm1 is specifically expressed by the syncytiotrophoblast layer II cells of the interhemal membrane. Arrows in F show sinusoidal trophoblast giant cells (S-TGC) in maternal blood spaces. Although not shown here, syncytiotrophoblast layer II cells also express Synb and Cepba, whereas Syna is expressed by syncytiotrophoblast layer I cells. (G) The expression of Mest is restricted to cells of the labyrinth layer. (H–I) However, in this case, Mest expression is localized to the endothelial cells lining the fetal circulation. Arrows in I show S-TGC in maternal blood spaces. Scale bar in A, D, and G = 1 mm; Scale bar in B, C, E, F, H, and I = 100 μm.
72
(A)
(D)
(G)
(B)
(E)
(H)
(C)
(F)
(I)
Plate 4 In situ hybridization staining for Prl3b1, Prl7b1, and Mus musculus trophoblast specific protein alpha (Tpbpa) in gd 14.5 placentas. (A) Prl3b1 (PL-II) staining is evident in several trophoblast cell types within the junctional zone and labyrinth layer of the definitive placenta. (B) Within the junctional zone, parietal trophoblast giant cells (PTGC; black arrows) and spongiotrophoblast cells (SpT) are strongly positive for Prl3b1 expression, whereas glycogen trophoblast cells (GlyT) are negative. (C) Within the labyrinth layer (Lab), trophoblast giant cells lining the maternal blood spaces (S-TGC; gray arrow) and maternal arterial canals (C-TGC) express Prl3b1, but syncytiotrophoblast and fetal capillary endothelium do not. (D) Cells within the junctional zone, and also some within the decidual compartment, express Prl7b1 (PlpN) by gd 14.5. (E) Specifically, GlyT and C-TGC are strongly positive for Prl7b1 expression, while SpT remain negative. (F) By gd 14.5, GlyT leave the confines of the junctional zone and migrate into the decidual compartment. Prl7b1-positive GlyT cells can be seen in the junctional zone as well as above the P-TGC layer within the decidual compartment. Prl7b1-negative P-TGCs can be clearly seen (black arrows) demarcating the boundary between the junctional zone and the decidua. (G) Tpbpa (or 4311) expression is routinely used to identify cells of the junctional zone. (H, I) Both SpT and GlyT express Tpbpa, although P-TGC do not. Scale bar in A, D, and G = 1 mm; Scale bar in B, C, E, F, H, and I = 100 μm.
73