Cardiovascular and craniofacial defects in Crk-null mice

Abstract

The Crk adaptor protein, which is encoded by two splice variants termed CrkI and CrkII, contains both SH2 and SH3 domains but no catalytic region. It is thought to function in signal transduction processes involved in growth regulation, cell transformation, cell migration, and cell adhesion. Although the function of Crk has been studied in considerable detail in cell culture, its biological role in vivo is still unclear, and no Crk-knockout mouse model has been available. Therefore, we generated a complete null allele of Crk in mice by using the Cre-loxP recombination approach. The majority of Crk-null mice die at late stages of embryonic development, and the remainder succumb shortly after birth. Embryos lacking both CrkI and CrkII exhibited edema, hemorrhage, and cardiac defects. Immunohistochemical examination suggested that defects in vascular smooth muscle caused dilation and rupturing of blood vessels. Problems in nasal development and cleft palate were also observed. These data indicate that Crk is involved in cardiac and craniofacial development and that it plays an essential role in maintaining vascular integrity during embryonic development.

FIG. 1.

Targeted disruption of Crk in mice. (A) Schematic diagram of wild-type, homologous recombinant, floxed, and knockout alleles of Crk. Red triangles represent loxP sites. The relevant restriction sites ScaI (Sc), KpnI (K), SacI (Sa), and ClaI (C) and locations of probes (thick colored bars) for Southern blotting are indicated. (B) Southern blot analysis of the genomic DNA from embryos. Two independent DNA samples from each genotype were digested with SacI and then analyzed. WT, wild type. (C) Western blot analysis of total protein lysates from MEFs. An anti-Crk antibody was used to detect the endogenous CrkI and CrkII proteins in two independent samples from each genotype.

FIG. 2.

General morphology of Crk^−/− embryos. Embryos at E12.5, E13.5, and E15.5 were dissected from mouse uteri and fixed in 4% paraformaldehyde at 4°C overnight. Images of whole mouse embryos were taken before paraffin processing. Red arrows indicate hemorrhagic edema on the snout and the nasion. Yellow arrowheads indicate edema.

FIG. 3.

Histological analysis of Crk^−/− embryos. Wild-type and Crk^−/− embryos at E12.5 (A and B) and E15.5 (D, E, and F) were embedded in paraffin, and sagittal sections were obtained at a thickness of 5 μm. (C) High magnification of the focal edema (blue arrowheads) in panel B. (E and F) Arrows indicate aberrant accumulation of blood cells.

FIG. 4.

Histological analysis of the Crk^−/− embryonic heart. Wild-type (A, C, E, and G) and Crk^−/− (B, D, F, and H) embryos at E15.5 were embedded in paraffin, and sagittal sections were obtained at a thickness of 5 μm. Sections were then stained with hematoxylin and eosin, and anatomically matching images of heart tissue were taken. The ventricles (V) in the Crk^−/− embryos were markedly dilated, and the ventricular walls (double-headed arrows) and interventricular septum (VS) were strikingly thin compared to those structures in the wild-type littermates. Panels C, D, G, and H are high-magnification images of boxes in panels A, B, E, and F, respectively. Bar, 100 μm.

FIG. 5.

CD34 immunostaining of Crk^−/− embryos. Sagittal paraffin sections from E15.5 Crk^−/− and wild-type littermates were stained with anti-CD34 antibody and counterstained with hematoxylin. (A) Two hemorrhagic edemas are present on the nasion (arrowhead) and tip of the snout (arrow). (B) High-magnification view of the hemorrhagic edema on the tip of snout. CD34-labeled endothelial cells are stained brown. The hemorrhagic edema is not lined with endothelial cells (arrow), which suggests the presence of a ruptured blood vessel. The ruptured blood vessel can be seen clearly at higher magnification (C). CD34 immunostaining of the region between the two areas of hemorrhagic edema in a Crk^−/− embryo shown in the box in panel A (E) was compared with that of a corresponding region in a wild-type littermate (D). Hemorrhagic edema (he), mesenchyme (mc), and epidermal layer (ep) are marked. Bar, 50 μm.

FIG. 6.

SMA immunostaining of the head region in Crk^−/− embryos. Sagittal sections from E15.5 Crk^−/− and wild-type littermates were stained with anti-SMA antibody and counterstained with hematoxylin. (A to F) High-magnification views of SMA staining. A, C, and E (wild-type) anatomically correspond to B, D, and F (Crk^−/−), respectively. (G and H) Locations of B, D, and F are indicated as boxes. Defective regions (arrows), hemorrhagic edema (*), nasal cavity (n), upper lip (u), and tongue (t) are marked. Bar, 25 μm.

FIG. 7.

Defective nasal development and cleft palate in Crk^−/− embryos. E15.5 Crk^−/− and wild-type littermates were fixed and embedded in paraffin, and coronal and sagittal sections were obtained. Paraffin sections were stained with hematoxylin and eosin, and matching images of the nasal parts were taken. (A to F) Panels A, B, and C are anatomically matching coronal sections that are anterior to panels D, E, and F, respectively. (G and H) High-magnification views of the edema formed in panels C and E, respectively. (I to N) Panels I, J, and K are anatomically matching sagittal sections, and their high-magnification views of the nasopharynx are shown in panels L, M, and N, respectively. Nasal cavity (nc), nasal septum (ns), cartilage primordium of nasal septum (cn), edema (*), palate (p), cleft palate (cp), nasopharynx (np), and oropharynx (op) are marked. Bar, 250 μm.