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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02422

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PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage

¹ Department of Pathology and Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
² Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany
³ Institute of Molecular Pathology, University of Copenhagen, 2100 Copenhagen, Denmark

View larger version (40K): [in a new window]   Fig. 1. Strategy of PINCH1 gene disruption and embryos from PINCH1 heterozygous intercrosses. (A) Partial map of the wild-type PINCH1 allele, PINCH1neo, PINCH1-floxed allele (PINCH1fl), and the PINCH1-null allele after Cre-mediated recombination. Exons and loxP sequences are indicated as rectangles and triangles, respectively. The DNA fragment length obtained after Southern blotting and the internal (probe 1) and external (probe 2) probes are indicated. Restriction sites are: B1, BamHI; B2, BglII. (B) Southern blot of PINCH1neo tail DNA digestion with BamHI and hybridization with probe 2. The sizes of the wild-type (13 kb) and targeted (4 kb) bands are indicated. (C) PCR genotyping of E7.5 embryos derived from heterozygous intercrosses. (D) Whole-mount morphology of E7.5 wild-type and remnants of PINCH1-null embryos.

View larger version (61K): [in a new window]   Fig. 2. Peri-implantation lethality in the absence of PINCH1 expression. (A) Haematoxylin and Eosin staining of sections from E6.5 implantation chambers from heterozygous intercrosses shows that the presumptive mutant chambers contained either degenerated embryos (middle image) or blood cells (right image). d, decidua; epc, ectoplacental cone; ee, extraembryonic ectoderm; e, ectoderm; m, mesoderm. (B) TUNEL staining on sections from E6.5 decidual chambers shows multiple apoptotic cells in the remnants of the PINCH1-null embryo proper (marked region). Control embryos were TUNEL negative. The embryos were identified on consecutive sections stained with DAPI.

View larger version (77K): [in a new window]   Fig. 3. Endodermal detachment and disorganized F-actin in PINCH1-null EBs. (A,B) Phase-contrast images of wild-type EB (A) showing the epiblast layer consisting of pseudostratified columnar epithelial cells, and PINCH1-null EB (B) showing a multi-layered and polygonal epiblast cell layer sometimes associated with incomplete cavitation. (C,D) Immunofluorescence of PINCH1 (green) and laminin 1 (red) revealed PINCH1 expression at the basement membrane zone of normal endoderm and epiblast and in the nucleus of some endodermal cells (C). PINCH1-null EBs lack PINCH1 signals but show a linear laminin 1 staining (D). Nuclei are counterstained with DAPI (blue). (E,F) ß1 integrin (green) is condensed in the basal plasma membranes of normal endoderm and epiblast where it colocalizes with laminin 1 (red; arrowheads; E). Signals for ß1 integrin are similarly strong in PINCH1-null EBs (F). (G,H) F-actin (red) forms an apical belt and bundles parallel to lateral plasma membranes in the normal, polarized epiblast (G). PINCH1-null epiblast contains actin aggregates at the basal plasma membranes and lacks the parallel actin bundles (H).

View larger version (104K): [in a new window]   Fig. 4. Abnormal epiblast polarity and disruption of endodermal tight junctions in PINCH1-null EBs. (A,B) In normal EBs the cis-Golgi matrix protein GM130 (red) is localized to the apical region of the epiblast and to the basolateral region of the endoderm. GM130 is also observed in the apical side of the remaining ICM cells facing the cavity. In PINCH1-null EBs, GM130 is peri-nuclear in the endoderm, and sometimes apical in the epiblast although in a non-linear fashion (B). Arrowheads showing a large segment of endoderm detached from the basement membrane. (C,D) E-cadherin is localized to the lateral plasma membrane and concentrated at the apex of the epiblast (C). PINCH1-null EBs show reduced apical E-cadherin signals in the epiblast and often miss segments of endodermal cells (asterisk; D). (E,F) ZO-1 is found in the apex of the cell-cell junctions of normal endoderm (E, arrowheads). In PINCH1-null endoderm, ZO-1 is found at the basal or entire apical and lateral plasma membranes (F, arrowhead).

View larger version (109K): [in a new window]   Fig. 5. Apoptosis in PINCH1-null EBs. (A,B) Cell proliferation assessed by Ki67 staining (red) is seen in most epiblast and some endodermal cells from both wild-type and PINCH1-null EBs. (C,D) Immunostaining for cleaved caspase-3 (green) reveals apoptotic cells in the central portion of normal EBs leading to cavitation. Immunosignals were occasionally detected in the epiblast but rarely in the endoderm (C). PINCH1-null EBs have apoptotic endodermal cells with condensed nuclei (D). (E,F) Immunostaining for Ser473 phosphorylated form of PKB/Akt shows singly positive cells in the epiblast and endoderm layer both in sections derived from normal (E) and PINCH1-null EBs (F).

View larger version (35K): [in a new window]   Fig. 6. ILK expression in PINCH1-null EBs. (A) Western blot analysis of PINCH1 and ILK in PINCH1-null and Ilk-null EBs. (B) Northern blot analysis of PINCH1 in control and PINCH1-null cells. (C) Immunostaining of ILK in wild-type and PINCH1-null EBs. (D) Immunostaining of ILK in Ilk-null EBs.

View larger version (101K): [in a new window]   Fig. 7. Ultrastructural changes in PINCH1-null EBs as seen by Methylene Blue staining. (A-C) (A) Section of a typical wild-type EB with vacuole-containing endodermal cells (en), pseudo-stratified epiblast (ep) and a sharply defined central cavity (cv). (B,C) In PINCH1-null EBs the endoderm is frequently detached (arrow in C) and cells of the ICM and epiblast show different staining intensities (B). (D) Ultrastructural analysis of wild-type EBs revealed cuboidal endodermal cells (en), a thin basement membrane (bm) and an elongated epiblast (ep; D). (E,F) PINCH1-null endodermal and epiblast cells are connected through thin plasma membrane extensions. The epiblast cells are non-polarized, irregular (E) and the endoderm is often detached from the basement membrane (F). Normal endoderm has symmetric tight junctions (arrowheads in G; the inset shows 2 x magnification of the tight junction marked by an arrow). (H) In PINCH1-null endoderm, tight junctions often assemble at the middle or basal portion of cell-cell contacts. Sometimes asymmetric tight junctions form in PINCH1-null endoderm with one lateral plasma membrane of a cell not in direct contact with that of another (inset).

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