Isolation and Characterization of Chondroitin Sulfate Proteoglycans from Embryonic Quail That Influence Neural Crest Cell Behavior

Abstract

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.

β1 integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells

“…We carefully examined the presence of various ECM components in E11.5 embryonic guts, including aggrecan, laminin α1 (LN1; LAMA1), laminin α5 (LNα5; LAMA5), collagen IV (ColIV), vitronectin (VN; VTN), FN, tenascin-W (TNW; tenascin-N -Mouse Genome Informatics) and TNC. Aggrecan, a protein sulphate proteoglycan that inhibits NCC adhesion and migration in other systems (Perris et al, 1996;Kerr and Newgreen, 1997), was not detected in E11.5 gut walls (not shown). LN1 and ColIV were present in the basal laminae around the gut epithelium, around blood vessels and at the base of the serosal layer (Fig.…”

Thrombospondin-1 and neural crest cell migration

“…If TSP-1 is directing neural crest cells into the rostral somite, it would be only one actor in a large cast of characters that influence neural crest morphogenesis. Numerous molecules concentrated in the caudal somite have been proposed to act as a barrier to neural crest cell migration, including collagen type IX (Ring et al, 1996), proteoglycans (Perris et al, 1991;Landolt et al, 1995;Henderson et al, 1997;Kerr and Newgreen, 1997), and PNA-binding glycoproteins (Oakley and Tosney, 1991;Oakley et al, 1994). Ephrins and their receptors are also expressed in patterns that are consistent with a role in inhibiting neural crest cells from the caudal somite (Krull et al, 1997;Robinson et al, 1997;Wang and Anderson, 1997).…”

“…Grafting studies have shown that the selective migration of neural crest cells into the rostral somite is under regulation by factors found in the somite itself (reviewed in Erickson and Reedy, 1998). Specific factors include collagen type IX (Ring et al, 1996), versican (Landolt et al, 1995;Henderson et al, 1997), and other chondroitin sulfate proteoglycans (Perris et al, 1991;Kerr and Newgreen, 1997), as well as PNA-binding glycoproteins (Oakley and Tosney, 1991). These repulsive molecules are believed to inhibit neural crest cells from the caudal sclerotome.…”

Regional differences in neural crest morphogenesis

“…Some of these are extracellular matrix (ECM) molecules: chondroitin-6-sulfate proteoglycan (CSPG), 33,34 peanut agglutinin (PNA)-binding molecules, 34,35 collagen type IX, 36 versican 37 and F-spondin. 38 T-cadherin, a cell-cell adhesion molecule, is expressed on posterior sclerotome cells.…”