Abnormal neural crest cell migration after the in vivo knockdown of tenascin‐C expression with morpholino antisense oligonucleotides

Abstract

A key feature of vertebrate development is the formation of the neural crest. In the trunk, neural crest cells delaminate from the neural tube shortly after the fusion of the neural folds and migrate ventrally along specific pathways to form the neurons and glia of the peripheral nervous system. As neural crest cells leave the neural tube during the initial stages of their migration, they express the extracellular matrix glycoprotein tenascin-C, which is also found in the stroma of many tumors. We have studied the possible role for tenascin-C during neural crest morphogenesis in vivo by microinjecting tenascin-C morpholino antisense oligonucleotides into the lumen of the avian neural tube in ovo and electroporating the morpholino antisense oligonucleotides into the precursors of the neural crest. After 24 hr, tenascin-C immunostaining is reduced around the dorsal neural tube in the experimental microinjected embryos (12 of 13) but not in embryos microinjected with control morpholino antisense oligonucleotides (n ‫؍‬ 3) or subjected to electroporation only (n ‫؍‬ 2). In each of the 12 tenascin-C knockdown embryos neural crest cells are seen ectopically in the lumen of the neural tube and in the neuroepithelium; cells that do leave the neural tube after the microinjection fail to disperse laterally from the surface of the neural tube into the somites. The observation that neural crest cells must express tenascin-C to migrate normally is consistent with a role for this glycoprotein in contributing to the invasive behavior of neural crest cells.

Cell biology of embryonic migration

“…(Costell et al, 1999) Tenascin C Extracellular matrix Total NC cells fail to disperse laterally (Tucker, 2001) Laminin γ1 Extracellular matrix Total Death at E5.5, lack of basement membranes (Smyth et al, 1999) Laminin β2 Extracellular matrix Total Postnatal death between P15-30, neuromuscular junctions and glomerular defects (Noakes et al, 1995a;Noakes et al, 1995b;Patton et al, 1997) Laminin α2 Extracellular matrix Total Death by 5 weeks postnatal, severe muscular dystrophy and peripheral neurophathy (Miyagoe et al, 1997) Laminin α2 (dy/dy) Extracellular matrix Spontaneous Adult lethality, severe muscular dystrophy and peripheral nerve dysmyelination (Patton et al, 1999;Patton et al, 1997) Laminin α3 Extracellular matrix Total Death at P2-3, epithelial adhesion defect (Ryan et al, 1999) Laminin α4 Extracellular matrix Total Transient microvascular defect with hemorrhages and misalignment of neuromuscular junctions (Patton et al, 2001;Thyboll et al, 2002) Laminin α5 Extracellular matrix Total Death at E14-E17, with placental vessel, neural (Miner et al, 1998;Miner and Li, 2000) Protein Function Type Phenotype Reference tube, limb, and kidney defects Fibronectin Extracellular matrix Total Death before E14.5, shortened anterior-posterior axes, deformed neural tubes, and defects in mesodermally derived tissues. (George et al, 1993) Collagen XVIII Extracellular matrix Total Eye abnormalities modeling Knoblock syndrome (Fukai et al, 2002) Extracellular matrix Total Basement membrane defects (Utriainen et al, 2004) Collagen XV Extracellular matrix Total Skeletal myopathy and cardiovascular defects (Eklund et al, 2001) betaglycan Extracellular matrix Total Embryonic lethality of heart and liver defects (Stenvers et al, 2003) Connexin 43 Cell-cell adhesion In vitro studies of cells from knockout mice…”

Using Antisense Morpholino Oligos to Knockdown Gene Expression in the Chicken Embryo.

“…This paper will concentrate on the techniques used to introduce morpholinos into chicken embryos and analyze the embryos for the knockdown of the target transcript. To illustrate the method, we will show how this technique was used to knockdown the expression of the extracellular matrix glycoprotein tenascin-C in migrating neural crest cells [14].…”

Tenascins and the Importance of Adhesion Modulation

“…Thus, tenascins are relatively new additions to the extracellular matrix, appearing in the first organisms with a dorsal hollow nerve cord and neural crest cells or neural crest-like properties, as well as a pharyngeal apparatus and notochord. This is intriguing, as tenascin-C is prominently expressed by neural crest cells (Tucker and McKay 1991) and can be required for their normal migration (Tucker 2001), and tenascin-C and its relative tenascin-W are expressed in dense connective tissues like cartilage and bone (e.g., see Mackie et al 1987;Scherberich et al 2004), which may have their origins in the notochord (Zhang and Cohn 2006) and pharyngeal arch mesenchyme (Hecht et al 2008). Thus, Neidhardt et al 2003 the evolution of tenascins is closely tied to the appearance of chordates, and may have played a key role in the development of their novel, defining structures.…”