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Ena/vasodilator-stimulated phosphoprotein (VASP) proteins regulate the geometry of the actin cytoskeleton, thereby influencing cell morphology and motility. Analysis of invertebrate mutants implicates Ena/VASP function in several actin-dependent processes such as axon and dendritic guidance, cell migration, and dorsal closure. In vertebrates, genetic analysis of Ena/VASP function is hindered by the broad and overlapping expression of the three highly related family members Mena (Mammalian enabled), VASP, and EVL (Ena-VASP like). Mice deficient in either Mena or VASP exhibit subtle defects in forebrain commissure formation and platelet aggregation, respectively. In this study, we investigated the consequence of deleting both Mena and VASP.Mena-/-VASP-/-double mutants die perinatally and display defects in neurulation, craniofacial structures, and the formation of several fiber tracts in the CNS and peripheral nervous system.

Zyxin is a LIM domain protein that is present at sites of cell-substratum and cell-cell adhesion, where it is proposed to dock proteins involved in cytoskeletal organization and dynamics (7, 8, 11, 16). Biochemical studies have identified α-actinin, members of the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) family, Vav, members of the cysteine-rich protein family, the LATS1 tumor suppressor, and members of the p130cas family as binding partners for zyxin (12, 19, 23, 24, 42, 43, 47, 56). The molecular architecture of zyxin is compatible with the protein's postulated role as a scaffold for the assembly of multimeric protein complexes. Zyxin displays three tandemly arrayed LIM domains, double zinc finger structures that support specific protein interactions (37, 47). In addition, zyxin exhibits at least one nuclear export signal that regulates its excursions between the nucleus and the cytoplasm (35, 36). Zyxin also displays four proline-rich ActA repeats that can interact directly with members of the Ena/VASP family (14, 19, 34). Members of the Ena/VASP family are concentrated at focal adhesions, the leading edge of lamellipodia, and filopodial tips (19, 42). Ena/VASP proteins are involved in modulation of actin assembly and organization (4, 6, 30, 41, 48) and are required for normal fibroblast and neuron migration (5, 20). Recent work has illustrated that the proper subcellular targeting of Ena/VASP family members is essential for them to fulfill their normal roles in vivo (1, 5, 6). The Ena/VASP binding capacity of zyxin has been studied in detail, and several lines of evidence suggest that zyxin plays an important role in localizing members of this family to sites of cell-substratum adhesion. For example, microinjection of peptides that cause displacement of zyxin from focal adhesions also results in loss of Ena/VASP family members from these sites (15). In complementary experiments, targeting of zyxin to membranes or mitochondria leads to recruitment of Ena/VASP proteins (14, 18). Moreover, microinjection of peptide inhibitors that interfere with the binding of Ena/VASP proteins to partners with ActA repeats, such as zyxin, disturbs the subcellular distribution of Ena/VASP proteins and affects cell spreading and cell-cell adhesion (14, 52). Collectively, these studies suggest that failure of Ena/VASP proteins to localize properly results in defects in actin-based cell functions and implicate zyxin as an important contributor to the proper localization of Ena/VASP proteins. The physiological consequences of disturbing Ena/VASP function have been characterized in vertebrates by analysis of mice in which Mena (mammalian Ena) or VASP functions have been compromised by gene disruption or expression of mutant constructs. Although mice that lack either Mena or VASP are viable and fertile, compromising the functions of murine Ena/VASP family members results in disturbed platelet function, nervous system development, and epithelial sheet formation (3, 21, 29, 52). Here we pursued a reverse genetic strategy to probe the role of zyxin in the mouse. We generated mice that lack zyxin function and analyzed the importance of zyxin for embryonic development and adult functions, with particular attention to processes that are known to depend on members of the Ena/VASP family. Mice that lack zyxin are both viable and fertile and do not display defects in processes that are compromised when either VASP or Mena functions are eliminated. The existence of multiple zyxin family members may obscure our ability to detect the contribution of zyxin to these processes.

Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.

Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.