Overview

    The Krauss lab is interested in the mechanisms by which cell-cell contact regulates signal transduction pathways during development, and how such processes may go awry in disease. We have focused much of our effort on a small group of complex and multifunctional receptor-like proteins of the Ig superfamily. Cdo and Boc have Ig and FnIII repeats in their ectodomains and long, divergent cytoplasmic tails that resemble nothing else in the databases. Fruit fly orthologs are Ihog and Boi. Cdo promotes skeletal myogenesis in vivo and in vitro (Figure 1). During myoblast differentiation, the Cdo intracellular region binds to the p38 MAPK pathway scaffold protein JLP and, via JLP, p38 itself. Cdo also interacts with Bnip-2, a protein that binds the small GTPase, Cdc42. Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38 activity and cell differentiation (Figure 2). These results reveal a novel linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling.

    Cdo binds in a cis manner (in the plane of the same cell membrane) to the cell-cell adhesion molecule N-cadherin, and to the netrin and RGM receptor, neogenin. Cdo is likely to function as a non-ligand binding co-receptor for N-cadherin and neogenin during myogenesis, with N-cadherin ligation the likely initiator of Cdo-dependent p38 signaling. Cdo and Boc also function as both components and targets of the Hedgehog signaling and feedback network. Cdo and Boc bind directly to Sonic hedgehog (Shh) and promote Shh signaling. Mice lacking Cdo or Boc display tissue-specific loss-of-Shh function phenotypes. Cdo-null animals display holoprosencephaly (HPE). HPE is the most common defect of human forebrain development and is associated with haploinsufficiency for genes encoding Shh pathway components. Clinical expression of HPE is extremely variable, but it is rarely associated with defects in other SHH-dependent structures, such as the limbs. Mice lacking Cdo display HPE with strain-specific severity and without limb defects (Figure 3), modeling human HPE and implicating modifier genes as a cause of variability. Boc-null mice are viable and fertile, but removal of Boc from Cdo mutant mice worsens the latter’s phenotype.

    Taking these results together, Cdo and Boc appear to function as components of multiple cell surface protein complexes to influence, within specific contexts, signaling by cadherins, netrins and Shh. A long-term goal is to understand these multiple functions at a detailed mechanistic level and to discern whether higher order cross-regulation exists that may tie these mechanisms together.

Figure 1. Cdo promotes skeletal myogenesis in vivo and in vitro. Top panels: Whole mount in situ hybridization of E9.5 wild-type (+/+) and Cdo-/- (-/-) embryos reveals that, while sclerotome development proceeds normally (as visualized with a probe against Pax1), myotomes develop inefficiently (as visualized with a probe against myogenin).  Likewise, at E10.5, myogenin-positive cells have infiltrated the developing limb (arrowheads) in the +/+ embryo, but have barely begun to do so in the -/- embryo. Bottom panels: Primary myoblasts isolated from Cdo+/+ and Cdo-/- mice were cultured in differentiation medium and stained with an antibody to myosin heavy chain (MHC, in brown). Note that the Cdo+/+ cells form large myotubes with many nuclei, while the Cdo-/- cells fuse inefficiently. Furthermore, although Cdo-/- cells are positive for MHC by immunocytochemistry, Western blot analysis reveals they produce lower levels than do the control Cdo+/+ cells (not shown).

Figure 2: Model of Cdo-mediated p38 MAPK activation during myogenic differentiation. Cdo binds directly to JLP and, via JLP, to p38 MAPK. Cdo also binds to Bnip-2 and, via Bnip-2, Cdc42. Formation of a Cdo/Bnip-2/Cdc42 complex promotes or stabilizes activation of Cdc42, which in turn triggers signals culminating in phosphorylation and activation of p38 bound to Cdo via JLP. Cdo interacts with itself and is shown as a dimer; JLP and Bnip-2 are shown as binding to different Cdo proteins of the dimer for convenience and does not preclude the possibility that JLP and Bnip-2 bind the same Cdo protein simultaneously.

Figure 3: Cdo mutant mice display variable HPE phenotypes dependent on genetic background. A. Frontal views of the midfacial region of four-week old adult mice of the indicated Cdo genotypes on the 129/Sv background. The Cdo-/- animal in the right panel shows microforms of HPE, including a single, central maxillary incisor (arrows) and a dysgenic philtrum (arrowheads). Cdo-/- mice of the 129/Sv background show such craniofacial midline phenotypes with ~50% penetrance. B. Whole embryos of the indicated Cdo genotypes on the C57BL/6 background at embryonic day (E) 15.5. The arrows indicate that, while the Cdo+/+ embryo has two nostrils and a normal midface, the Cdo-/- embryo displays a single nostril and hypoplastic midface. C. Sections of E11.5 embryos of the indicated Cdo genotypes on the C57BL/6 background. Note the paired lateral ventricles in the Cdo+/- (control) embryo and the single ventricle in the Cdo-/- embryo. Cdo-/- mice of the C57Bl/6 background show the more severe HPE phenotypes shown in B and C with ~80% penetrance. Reprinted with permission from Cole and Krauss, 2003 and Zhang et al., 2006.

© 2008 Mount Sinai Medical Center