We used a genetic approach to address the expression and function of the mammalian Nck SH2/SH3 adaptors. To this end, we engineered mouse strains with mutations that place the β-galactosidase gene under the control of the Nck1 or Nck2 endogenous promoter and at the same time introduce deletions into the Nck1 or Nck2 coding sequence. Analysis of the resulting embryos indicates that the two Nck genes have a largely overlapping pattern of expression, are jointly required for embryonic development and, in particular, are important for placentation and axial rotation. Nck1−/− Nck2−/− embryos die in utero around gestational day 9.5 and exhibit, among other defects, severe alterations in mesoderm-derived structures, particularly in the organization of the notochord. Embryonic fibroblast lines derived from Nck1−/− Nck2−/− embryos have altered migratory properties and show defects in lamellipodium formation, revealing a significant physiological role for Nck in regulating the actin filament network, which may pertain to its in vivo functions.
With regard to expression, we have compared Nck1 and Nck2 in adult and embryonic tissues. For adult tissues, we analyzed protein extracts derived from either Nck1−/−
mice. Unlike the more widely expressed Nck1, Nck2 was not detectable in the liver and in skeletal muscle and was present at low levels in brain extracts. This finding was confirmed by β-galactosidase staining of adult brain of heterozygous Nck1IRES-lacZ
, which showed a more restricted pattern of expression for Nck2 (data not shown). Both Nck1 and Nck2 were found at high levels in adult thymus and spleen, a finding consistent with observations implicating Nck in antigen-specific T-cell (63
) and B-cell (39
) receptor signaling. The potential role of Nck adaptors in the nervous and immune systems cannot currently be addressed because of the early lethality of Nck1−/−
embryos but is under investigation by conditional inactivation of the Nck genes.
The expression of Nck1 and Nck2 during early embryonic development was investigated by β-galactosidase staining of mice heterozygous for the NckIRES-lacZ or Nck2tau-lacZ alleles. At E7.5, both Nck genes were expressed in the allantois and headfold but, at later stages, Nck1 remained broadly expressed, whereas Nck2 appeared mainly restricted to developing nervous system, endoderm, and mesodermal derivatives, particularly the notochord and somites. Although inactivation of Nck1 or Nck2 was compatible with normal pre- and postnatal development, mutation of both Nck1 and Nck2 resulted in early embryonic lethality, indicating that the functions of Nck1 and Nck2 are overlapping, mutually compensatory, and essential for embryonic development.
embryos exhibited alterations in mesoderm-derived structures, such as notochord and somites, and neural ectoderm. These defects may be directly due to the loss of Nck function in these structures. Alternatively, the abnormal development of the notochord may be the primary defect, which causes secondary abnormalities in patterning of the neural tube and somatic derivatives (5
). Our finding that late but not early notochord development (two to four somites) was affected by loss of Nck function suggests that the Nck genes products may not be required for initial notochord formation but rather prevent notochord degeneration and/or promote anterior progression of mesodermal precursor cells from the primitive streak or node. This hypothesis is also supported by preliminary findings that Nck is expressed at the late-streak stage in the node (data not shown), the source of notochord progenitors (3
). These observations warrant further investigation by chimeric analysis.
The mechanisms responsible for axial rotation in the embryo are not completely understood. Several lines of evidence suggest that the faster growth rate of the neural tube compared to the underlying endodermal structures (26
) and notochord development (27
) may be decisive factors. It is tempting to speculate that in Nck1−/−
embryos the fragmentation, midline displacement, and bifurcation of the notochord may disrupt the hard core around which the embryo normally revolves, thereby inhibiting the physiologic turning of the embryo. The phenotype of Nck1−/−
embryos is strikingly similar to that of Csk−/−
) and partially overlaps with that of fibronectin−/−
), α5 integrin−/−
), and focal adhesion kinase−/−
) mice but is distinct from that of Nik−/−
). Although embryos deficient for components of the fibronectin pathway lack somites and exhibit a more severe phenotype, Csk−/−
, α5 integrin−/−
, and Nck1−/−
mutants share abnormalities in the notochord, axial rotation, and chorioallantoic fusion. In contrast, the mesodermal defect of Nik−/−
embryos is targeted to the somites and spares the integrity of the notochord and processes such as chorioallantoic fusion and axial rotation. Furthermore, the defect of Nik−/−
embryos is cell nonautonomous and results from defective migration of mesodermal precursor from the primitive streak. It is tempting to speculate that the binding of Nck to components of the integrin signaling pathway (for a review, see reference 7
) may underlie the defects common to Nck1−/−
, α5 integrin−/−
, and FAK−/−
embryos by regulating the migratory properties of mesodermal precursors through cell adhesion.
To pursue the potential role of Nck in cell migration suggested by the mutant phenotype, we analyzed the actin filament network of fibroblast cell lines from Nck1−/−
E8.5 embryos by platinum replica electron microscopy. Cell migration has been proposed to require four steps: extension of actin-rich protrusions, such as lamellipodia, formation of new adhesions, cell body contraction, and tail detachment (32
MEFs exhibit defective migration and differ morphologically from Nck1-rescued controls under basal conditions and during recovery after ATP depletion. The reduced density, length, and disorganization of actin filaments at the leading edge and its adjacent region indicate that Nck plays a role in lamellipodial formation. The defective migration of Nck1−/−
fibroblasts was confirmed upon Cre-mediated deletion of Nck2 in Nck1−/−
fibroblasts, further supporting the hypothesis that Nck genes are essential in cytoskeletal plasticity (unpublished results). Interactions of Nck with proteins such as Pak, N-WASP, and WAVE (4
) may underlie this phenomenon. It will be of interest to analyze the contribution of each of these Nck-mediated pathways in physiologic cell movement. N-WASP mutant mice have been generated and display prominent neural tube and cardiac abnormalities and alterations in the actin-based motility of certain intracellular pathogens (53
). However, the general formation of actin-containing structures appeared to be unaffected. The relatively mild phenotype of N-WASP-deficient cells in comparison to Nck-deficient fibroblasts is consistent with the view that Nck proteins also regulate other mediators of actin polymerization, such as WAVE.
In conclusion, our results shed light on the in vivo functions of mammalian Nck1 and Nck2 and reveal an essential role in embryogenesis. The involvement of Nck in actin reorganization and cell motility in culture provides a cellular basis for understanding the in vivo functions of these SH2/SH3 adaptors.