Paxillin expression between E7.5 and E10. Paxillin^+/− or paxillin^+/+ embryos were isolated between E6.5 and E10 and stained with X-Gal. In some cases they were counterstained with hematoxylin and eosin or immunostained with a neurofilament antibody. (A and B) Expression at E7.5 is seen only in the extraembryonic region (EEM) and not in the embryonic region (EM), and wild-type embryos show no staining. Sectioning of stained E7.5 embryos (B) indicates that the ectoplacental cone (EPC), chorion (Ch), allantois (All), and amnion (Amn) express paxillin. Expression at E8.5 to E10 (C to I) is detected in the embryo proper. At E8.5, paxillin expression is detected in many structures including mesodermally derived components such as the headfold (Hf), the notochord (NC), and the heart (Ht). Sectioning of E8.5 X-Gal-stained embryos (D and E) indicates that expression is observed in the underlying cephalic mesenchyme (CM) but is not detected in the neural tube (NT). Little staining is detected in the somites (S). At E9 and E10 (F to I), paxillin is expressed ubiquitously, with more intense staining observed in areas derived from neural crest cells including dorsal root ganglion (DRG). The brown staining represents neurofilament-positive structures (H). Sections of E10.5 embryos indicate strong staining of the notochord and the neural crest cells (NCC) emanating from the neural tube (I).

Paxillin mutant cells have abnormal focal adhesions and are defective in lamellipodium formation. (A) Paxillin and Hic-5 immunoblots of control cells, paxillin^−/− cells, or paxillin^−/− cells rescued with myc-tagged paxillin. (B) Cells were plated on fibronectin-coated coverslips overnight and stained with antibodies to vinculin or the paxillin-related protein Hic-5 to look at focal adhesions. Cells were stained with phalloidin or cortactin to look at stress fibers and the membrane cytoskeleton.

Paxillin-deficient cells migrate more slowly. (A) A confluent monolayer of control or paxillin-deficient cells was wounded with a yellow tip and photographed at the indicated time points (in hours). (B) A confluent monolayer of control, paxillin^−/− cells, or paxillin^−/− cells in which paxillin was re-expressed (rescue) was wounded with a yellow tip and photographed at the indicated time points (in hours). The pictures are representative of results obtained in four independent experiments.

Targeting of the paxillin locus and generation of the null mutation. (A) The targeting vector used to generate the null mutation is diagrammed. SA, adenovirus splice acceptor; βgeo, β-galactosidase-neomycin resistance gene fusion; pGK, phosphoglucokinase promoter; DTA, diphtheria toxin A subunit; bPA, bovine(poly A). Primers used for genotyping are diagrammed (a, b, and c). Exons are represented by black boxes. Exons 2 and 3 were deleted and encode amino acids 5 to 74. Restriction sites used to generate the targeting vector are indicated. RI, EcoRI; H_II, HindII; H_III, HindIII (see Materials and Methods for more detail). (B) Loss of paxillin expression in paxillin^−/− cells. Lysates prepared from wild-type, paxillin^+/−, or paxillin^−/− embryos were subjected to SDS-PAGE, immobilized on nitrocellulose, and immunoblotted with an antibody to paxillin.

Paxillin-null embryos display defects in multiple mesodermally derived structures. Timed matings were performed with paxillin^+/− mice and embryos isolated at E7.5 or E8.5. Genotypes of embryos were determined by PCR either with yolk sac DNA or from scraping of sections (data not shown). (A and B) At E7.5 in paxillin^−/− embryos, the amnion (Amn) is collapsed on the embryo and the allantois (All) is misshapen and moving anteriorly rather than dorsally towards the chorion. (C and D) At E8.5, paxillin mutants are significantly smaller overall. No obvious heart structure is present (Ht), and mutants have an abnormal headfold (Hf) and allantois (All). (E to H) Histological sections of wild-type and paxillin-null embryos. The headfold region is smaller than normal, but expected cell populations are present adjacent to the neural groove (NG), including neural epithelium (NE) and cephalic mesenchyme (CM). In contrast, the mutant embryo lacks a cardiogenic plate (CP) and the endodermal lining of the foregut (FG) remains open. The allantois (All) also appears to be abnormal. (G) A more caudal section of a wild-type embryo, in which the notochord (NC) and rostral extension of dorsal aorta (DA) are visible. Adjacent to the primitive streak (PS), mesodermal cells (M) are visible. (H) More caudal section of a paxillin-null embryo, in which the discrepancy in overall headfold size, compared to that of the wild type, is more pronounced. Cephalic mesenchymal (CM) cells appear condensed. Mutant embryos also lack an organized notochord, and the dorsal aorta is absent. Paxillin-null embryos also lack organized somites (in more caudal sections; not shown). In contrast, the primitive streak (PS) and adjacent mesodermal (M) cells appear normal.

Paxillin-deficient cells have a delayed rate of spreading. Control or paxillin-deficient cells were plated on fibronectin-coated bacterial dishes for the indicated periods of time and photographed by phase contrast. At least three independent fields were analyzed for each time point. Dark cells are considered to be spread, while bright cells are unspread. The photograph is representative of results obtained in four independent experiments. In the experiment shown at the 10-min time point, 76% of control cells were spread while only 34% (middle panel) and 20% (lower panel) of cells in the two different knockout cultures were spread at this time point. At 45 min almost 100% of the control cells were spread, while 55% of the knockout cells in one case (middle panel) and 39% of the knockout cells in the other case (lower panel) were spread. By 60 min, 60% of the cells were spread in all cultures.

Fibronectin-induced phosphorylation of FAK, Cas, and MAPK and FAK localization are defective in paxillin-deficient cells. Cells were plated on fibronectin-coated plates for the indicated time points (in minutes). Lysates were prepared and analyzed for total cell phosphotyrosine (A) or tyrosine phosphorylation of specific substrates (B). The total cell lysate blot was probed with an antibody to Src to ensure equal loading. The pTyr blots of FAK and Cas immunoprecipitations were reprobed with an antibody to FAK or Cas to check for levels. (C) Cells were also costained with antibodies to FAK and Hic-5 to determine if loss of paxillin affects FAK localization. While Hic-5 and FAK colocalized to focal adhesions in control cells, a few Hic-5-positive focal adhesions which were negative for FAK in paxillin mutant cells were observed (arrows). (D) Total cell lysates were prepared from knockout or rescued cells plated on fibronectin for the indicated time points and analyzed with phospho-specific antibodies to MAPK. Blots were reprobed for MAPK to check for equal loading. In this experiment, comparison of the knockout cells to the rescued cells indicates that there is a twofold difference in MAPK activation at the 10- and 20-min time points.