1. Volume 6, Issue 1, Pages 109-116 (July 2000)
Essential Role of p38α MAP Kinase in Placental but Not Embryonic Cardiovascular Development 
Ralf H. Adams, Almudena Porras, Gema Alonso, Margaret Jones, Kristina Vintersten, Simona Panelli, Amparo Valladares, Lidia Perez, Rüdiger Klein, Angel R. Nebreda 
Molecular Cell 
Volume 6, Issue 1, Pages (July 2000)
DOI: /S (05)

2. Figure 1 Gene Targeting of p38α
(A) Restriction map of genomic p38α sequences and construction of the targeting vector. Two exons of the p38α gene were deleted (gray boxes). The amino acid sequences encoded by three p38α exons, the p38α probe used for Southern blotting, and the positions of cassettes encoding β-galactosidase (LacZ), neomycin resistance (neo), and thymidine kinase (TK) are indicated. E, EcoRI; H, HindIII; X, XbaI.
(B) Genomic analysis of embryonic stem cells. Genomic DNA was isolated from p38α+/+ and p38α+/− ES cells, digested with HindIII, and analyzed by Southern blotting using the 5′ p38α flanking probe. Relative molecular masses of wild-type and targeted alleles are 9 kb and 4.5 kb, respectively.
(C) Absence of p38α protein in p38α−/− embryos. Total cell lysates from p38α+/+, p38α+/−, and p38α−/− E10.5 embryos were probed for p38α, p42Erk2, and MKK6 proteins by immunoblotting.
Molecular Cell 2000 6, DOI: ( /S (05) )

3. Figure 2 Inhibition of p38α Signaling in p38α−/− Cardiomyocytes
(A) Activation of p38 and p42/p44Erk MAP kinases in cardiomyocytes. p38α+/− and p38α−/− cardiomyocytes were incubated in 0.5% serum overnight and then either left untreated (control) or activated with UV irradiation (254 nm lamp at 9 cm from plate, 10 min), anisomycin (10 μg/ml, 30 min), A23187 Ca2+ ionophore (10 μM, 6 hr), PMA (1 μM, 30 min), or H2O2 (200 μM, 10 min). Total cell lysates were analyzed by immunoblotting with antibodies that specifically recognize either p38s (middle panel) or Erks (lower panel) phosphorylated both on the Thr and Tyr residues of their activation loop. The upper panel shows an immunoblot of the same lysates probed for p38α protein.
(B) Activation of MAPKAP kinase-2 in cardiomyocytes. MAPKAP kinase-2 was immunoprecipitated from the indicated cardiomyocyte lysates and assayed for in vitro kinase activity using Hsp27 as a substrate.
(C) Activation of Hsp27-phosphorylating kinases in cardiomyocytes. p38α+/− and p38α−/− cardiomyocyte lysates from the same experiment as in (A) were directly assayed for in vitro kinase activity using Hsp27 as a substrate.
Molecular Cell 2000 6, DOI: ( /S (05) )

4. Figure 3
Expression of p38α during Embryonic Development as Detected by β-Galactosidase Activity
(A and B) E10.5 p38α+/− embryo after whole-mount LacZ staining. X-gal labeling is absent in a wild-type littermate (B) processed in parallel to the heterozygote embryo shown in (A). LacZ expression is abundant but most prominent in somites (so), limb buds (lb), and branchial arches (ba).
(C) p38α expression in the endoderm (en), mesoderm (me), and endothelial cells (ec) of E10.5 yolk sacs as seen in transverse sections.
(D and E) β-galactosidase is expressed in the embryonic part of the placenta. Staining in the contact area between a p38α+/− E10.5 embryo and its p38α+/+ mother is shown (D). No color reaction was obtained for identically treated p38+/+ embryos (E).
(F) Section through a whole-mount stained placenta (as shown in D). The chorionic plate (cp), the labyrinthine trophoblast layer (lt), and a few cells in the labyrinthine layer (lb) are LacZ positive. Label is most prominent in the yolk sac (large arrow) and absent in the spongiotrophoblast layer (sp).
(G) Expression of p38α in the E10.5 heart. Endocard and myocard (arrows) of both right (rv) and left ventricles (lv) are stained.
(H) Backside view of an isolated E17.5 heart. Ventricles (rv, lv) and right (ra) and left atrium (la) are prominently labeled by β-galactosidase reaction.
Scale bar in (H) is 500 μm (A and B); 20 μm (C); 500 μm (D and E); 20 μm in (F); 70 μm in (G); or 1000 μm (H).
Molecular Cell 2000 6, DOI: ( /S (05) )

5. Figure 4
Phenotypic Analysis of p38α Mutant Embryos and Placenta at E10.5
(A) Phenotype of freshly dissected embryos. Note severe growth retardation of homozygous mutant embryo (right) compared to a heterozygous littermate (left).
(B and C) Anti-desmin staining highlighting the myocardial cell population in transverse sections of a control heart (B). In contrast, myocardial cells were largely absent in homozygous mutants (C).
(D and E) Head vasculature as visualized by whole-mount anti-PECAM-1 immunohistochemistry. Numerous blood vessels of different diameters were visible in heads of control embryos (D) whereas vasculature was reduced and lacking large vessels in homozygous mutants (E).
(F–I) Hematoxylin/eosin-stained sections of E10.5 placentas. (F) Layer organization of control placentas. Chorionic plate (cp), labyrinthine trophoblast (lt), labyrinthine (lb), and spongiotrophoblast layers (sp) are indicated. (G) Abnormal layering in p38α−/− placentas. Note dramatically reduced size of the labyrinthine layer whereas the area containing labyrinthine trophoblasts appears thickened. (H) Extensive intermingling of maternal (asterisks, containing enucleated erythrocytes) and embryonic blood vessels (arrows, identified by nucleated erythrocytes) in labyrinthine layers of control placentas. (I) Blood vessels of p38α−/− embryos (arrows) were found not to penetrate into the labyrinthine layer, resulting in severe disorganization of this area.
Scale bar in (F) is 700 μm (A), 90 μm (B and C), 250 μm (D and E), 100 μm (F and G), or 40 μm (H and I).
Molecular Cell 2000 6, DOI: ( /S (05) )

6. Figure 5 Tetraploid Rescue of Defects in Extraembryonic Tissues
(A) Yolk sac of an E12.5 heterozygous embryo. Most or all endodermal and mesodermal cells are LacZ positive.
(B) Chimeric yolk sac at E12.5 resulting from a tetraploid rescue experiment. Contribution of tetraploid wild-type cells that are not expressing β-galactosidase is easily detectable (arrow).
(C and D) Rescue of defects in extraembryonic tissue as visualized in hematoxylin/eosin-stained sections. Large embryonic blood vessels (open arrows) are penetrating deep into well-developed labyrinthine layers in placentas of both p38α-deficient (D) and control (C) embryos. Several maternal blood vessels containing only enucleated blood cells are indicated by black arrows.
(E and F) Appearance of freshly dissected p38α+/− and p38α−/− E15.5 embryos after successful rescue of defects in extraembryonic tissues. Note that null mutants can now survive and no longer exhibit growth retardation (F) compared to control (E).
(G) Absence of p38α protein in rescued E18.5 embryos. Hind limb lysates from p38α+/+, p38α+/−, and p38α−/− embryos were probed for p38α and MKK6 proteins by immunoblotting.
Scale bar in (E) is 50 μm (A and B), 200 μm (C and D), or 2400 μm (E and F).
Molecular Cell 2000 6, DOI: ( /S (05) )

7. Figure 6 Absence of Cardiovascular Defects in Rescued p38α−/− Embryos
(A and B) Anti-desmin-stained transverse sections through the hearts of E12.5 embryos. No reduction of myocard is visible in p38α−/− hearts (B) compared to the wild-type control (A).
(C and D) Hematoxylin/eosin-stained sections of tranverse sections through the primary head vein (phv, arrow) near the trigerminal ganglion (tg) in E12.5 heads. No overt differences can be detected between control animals (C) and p38α-deficient homozygotes (D).
Scale bar in (C) is 240 μm (A and B) or 150 μm (C and D).
Molecular Cell 2000 6, DOI: ( /S (05) )