Search

Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both the nervous system and cardiac development are dependent on agrin modulation of sonic hedgehog (shh) and fibroblast growth factor (Fgf) signaling pathways. As both ethanol and cannabinoids impact these signaling molecules, we examined their role on zebrafish heart development. Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 and 2 agonist concentrations in the absence or presence of morpholino oligonucleotides that disrupt agrin or shh expression. In situ hybridization was employed to analyze cardiac marker gene expression. Exposure to cannabinoid receptor agonists disrupted midbrain-hindbrain boundary development, but had no effect on heart development, as assessed by the presence of cardiac edema or the altered expression of cardiac marker genes. In contrast, exposure to 1.5% ethanol induced cardiac edema and the altered expression of cardiac marker genes. Combined exposure to agrin or shh morpholino and 0.5% ethanol disrupted the cmlc2 gene expression pattern, with the restoration of the normal expression following shh mRNA overexpression. These studies provide evidence that signaling pathways critical to heart development are sensitive to ethanol exposure , but not cannabinoid s, during early zebrafish embryogenesis.

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ 9 -THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.

The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Olfactomedins comprise a diverse family of secreted glycoproteins, which includes noelin, tiarin, pancortin and gliomedin, implicated in development of the nervous system, and the glaucoma-associated protein myocilin. Here we show in zebrafish that olfactomedin-2 (OM2) is a developmentally regulated gene, and that knockdown of protein expression by morpholino antisense oligonucleotides leads to perturbations of nervous system development. Interference with OM2 expression results in impaired development of branchiomotor neurons, specific disruption of the late phase branchiomotor axon guidance, and affects development of the caudal pharyngeal arches, olfactory pits, eyes and optic tectum. Effects of OM2 knockdown on eye development are likely associated with Pax6 signaling in developing eyes, as Pax6.1 and Pax6.2 mRNA expression patterns are altered in the eyes of OM2 morphants. The specific absence of most cartilaginous structures in the pharyngeal arches indicates that the observed craniofacial phenotypes may be due to perturbed differentiation of cranial neural crest cells. Our studies show that this member of the olfactomedin protein family is an important regulator of development of the anterior nervous system.

Neuroepithelium is an apicobasally polarized tissue that contains neural stem cells and gives rise to neurons and glial cells of the central nervous system. The cleavage orientation of neural stem cells is thought to be important for asymmetric segregation of fate-determinants, such as Numb. Here, we show that an intermediate filament protein, transitin, colocalizes with Numb in the cell cortex of mitotic neuroepithelial cells, and that transitin anchors Numb via a physical interaction. Detailed immunohistological and time-lapse analyses reveal that basal Numb-transitin complexes shift laterally during mitosis, allowing asymmetric segregation of Numb-transitin to one of the daughter cells, even when the cell cleavage plane is perpendicular to the ventricular surface. In addition, RNA interference (RNAi) knockdown of the transitin gene reveals its involvement in neurogenesis. These results indicate that transitin has important roles in determining the intracellular localization of Numb, which regulates neurogenesis in the developing nervous system of avian embryos.

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.

Recent studies have begun to investigate the role of agrin in brain and suggest that agrin's function likely extends beyond that of a synaptogenic protein. Particularly, it has been shown that agrin is associated with the pathological lesions of Alzheimer's disease (AD) and may contribute to the formation of beta-amyloid (Abeta) plaques in AD. We have extended the analysis of agrin's function in neurodegenerative diseases to investigate its role in Parkinson's disease (PD). Alpha-synuclein is a critical molecular determinant in familial and sporadic PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated macromolecules. In the studies reported here, we show that agrin binds to alpha-synuclein in a heparan sulfate-dependent (HS-dependent) manner, induces conformational changes in this protein characterized by beta-sheet structure, and enhances insolubility of alpha-synuclein. We also show that agrin accelerates the formation of protofibrils by alpha-synuclein and decreases the half-time of fibril formation. The association of agrin with PD lesions was also explored in PD human brain, and these studies shown that agrin colocalizes with alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain. These studies indicate that agrin is capable of accelerating the formation of insoluble protein fibrils in a second common neurodegenerative disease. These findings may indicate shared molecular mechanisms leading to the pathophysiology in these two neurodegenerative disorders.

Purpose: The inner limiting membrane (ILM) and the vitreous body (VB) are major parts of the extracellular matrix of the eye. The present study was undertaken to investigate the synthesis and turnover of the ILM and VB in chick and human embryonic and postembryonic eye development., Methods: The abundance of ILM and VB proteins was determined by Western blot analysis using samples from chick and human VB of different ages. The mRNA expression of the ILM proteins in lens was determined by in situ hybridization and RT-PCR., Results: Based on the abundance of mRNA expression, the prominent sources of ILM and VB proteins in chick eyes are the lens and ciliary body. In chick, ILM and VB matrix proteins were most abundant in embryonic VB, and their concentration declined precipitously after hatching. Most ILM and VB proteins were no longer detectable in the adult VB. In humans, a similar developmentally regulated expression of ILM and VB proteins in VB was detected: The highest concentrations of ILM and VB proteins were detected in fetal VB, the lowest in the adult VB. The decline in ILM and VB protein synthesis occurred within the first 2 years of life., Conclusions: The abundance of ILM and VB proteins in the embryonic VB, their sharp decline at postembryonic stages, and their very low abundance in the adult VB show that ILM and VB are assembled during embryogenesis and are maintained throughout life with minimum turnover.

Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.

Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three Ser-Gly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly., (Copyright 2000 Academic Press.)

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

The present study shows that collagen XVIII is, next to perlecan and agrin, the third basal lamina heparan sulfate proteoglycan (HSPG) and the first collagen/proteoglycan with heparan sulfate side chains. By using monoclonal antibodies to an unidentified HSPG in chick, 14 cDNA clones were isolated from a chick yolk sac library. All clones had a common nucleotide sequence that was homologous to the mRNA sequences of mouse and human collagen XVIII. The deduced amino acid sequence of the chick fragment shows an 83% overall homology with the human and mouse collagen XVIII. Similar to the human and mouse homologue, the chick collagen XVIII mRNA has a size of 4.5 kilobase pairs. In Western blots, collagen XVIII appeared as a smear with a molecular mass of 300 kDa. After treatment with heparitinase, the protein was reduced in molecular mass by 120 kDa to a protein core of 180 kDa. Collagen XVIII has typical features of a collagen, such as its existence, under non-denaturing conditions, as a non-covalently linked oligomer, and a sensitivity of the core protein to collagenase digestion. It also has characteristics of an HSPG, such as long heparitinase-sensitive carbohydrate chains and a highly negative net charge. Collagen XVIII is abundant in basal laminae of the retina, epidermis, pia, cardiac and striated muscle, kidney, blood vessels, and lung. In situ hybridization showed that the main expression of collagen XVIII HSPG in the chick embryo is in the kidney and the peripheral nervous system. As a substrate, collagen XVIII moderately promoted the adhesion of Schwann cells but had no such activity on peripheral nervous system neurons and axons.

A novel agrin isoform was identified based on the isolation of an agrin cDNA from E9 chick brain that lacked 21 base pairs (bp) in the NH2-terminal encoding region of the agrin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) of E9 chick brain mRNA confirmed the existence of this agrin isoform in brain, although the novel splice variant represents a minor fraction of agrin mRNA in brain. However, upon analysis of chick brain astrocyte mRNA, smooth muscle mRNA, and cardiac muscle mRNA by RT-PCR, we show that this novel agrin isoform is the predominant agrin isoform in these non-neuronal cell populations. We extended our analyses to examine the expression of this agrin mRNA isoform during chick development and show that the agrin mRNA lacking this 21-bp exon is up-regulated with brain development, consistent with the increase in glial number during brain development, while the agrin isoform that does not undergo splicing and thus contains the 21-bp exon is down-regulated in brain development. Because the 21-bp exon is inserted in the region of chick agrin which encodes the putative signal sequence of agrin, with the signal peptidase site immediately preceding the putative first amino acid of the mature protein being deleted as a result of splicing, these data raise the interesting possibility that the presence or absence of this alternatively spliced exon may differentially regulate processing of the agrin protein in neuronal and non-neuronal cells, respectively.

In the present study we have identified the extracellular matrix protein agrin as a major heparan sulfate proteoglycan (HSPG) in embryonic chick brain. Using monoclonal antibodies and a polyclonal antiserum to the core protein of a previously identified HSPG from embryonic chick brain, our expression screened a random-primed E9 chick brain cDNA library. Twelve cDNAs were isolated that were shown to be identical to the chick extracellular matrix protein agrin. Western blot analysis and immunocytochemistry confirmed that agrin is a HSPG that is identical with the HSPG from embryonic chick brain. A polyclonal antiserum to recombinant agrin protein recognized agrin as a diffuse band of over 400 kDa in extracts from brain and vitreous humor. The agrin immunoreactivity on the blot was shifted to a defined band of approximately 250 kDa after treatment of the samples with heparitinase or nitrous acid, and this banding pattern was indistinguishable from immunoreactivity obtained with antibodies to the brain HSPG. We also show that agrin binds tightly to anion exchange beads, indicating that the molecule is highly negatively charged, which is a hallmark of all proteoglycans. Furthermore, the agrin antiserum recognizes the affinity purified HSPG from chick brain and vitreous humor. Immunocytochemistry demonstrated that agrin is expressed in developing brain, and is especially abundant in developing axonal tracts, in a distribution identical to the staining of the brain HSPG with monoclonal antibodies. We also show that the anti-HSPG antibodies stain the synaptic site of the neuromuscular junction, in agreement with agrin expression. Thus, our studies demonstrate that chick agrin is a HSPG that is prominent in the embryonic chick brain. Since previous studies from our laboratories have shown that this proteoglycan interacts with neural cell adhesion molecule, our studies raise the interesting possibility that neural cell adhesion molecule and agrin are interactive partners that may regulate a variety of cell adhesion processes during neural development, including synaptogenesis.

Two biological domains have been identified in the amino terminal region of the neural cell adhesion molecule (NCAM): a homophilic-binding domain, responsible for NCAM-NCAM interactions, and a heparin-binding domain (HBD). It is not known whether these two domains exist as distinct structural entities in the NCAM molecule. To approach this question, we have further defined the relationship between NCAM-heparin binding and cell adhesion. A putative HBD consisting of two clusters of basic amino acid residues located close to each other in the linear amino acid sequence of NCAM has previously been identified. Synthetic peptides corresponding to this domain were shown to bind both heparin and retinal cells. Here we report the construction of NCAM cDNAs with targeted mutations in the HBD. Mouse fibroblast cells transfected with the mutant cDNAs express NCAM polypeptides with altered HBD (NCAM-102 and NCAM-104) or deleted HBD (HBD-) at levels similar to those of wild-type NCAM. Mutant NCAM polypeptides purified from transfected cell lines have substantially reduced binding to heparin and fail to promote chick retinal cell attachment. Furthermore, whereas a synthetic peptide that contains both basic amino acid clusters inhibits retinal-cell adhesion to NCAM-coated dishes, synthetic peptides in which either one of the two basic regions is altered to contain only neutral amino acids do not inhibit this adhesion. These results confirm that this region of the NCAM polypeptide does indeed mediate not only the large majority of NCAM's affinity for heparin but also a significant portion of the cell-adhesion-mediating capability of NCAM.

The C1H3 monoclonal antibody recognizes two distinct developmentally regulated cell surface antigens, with molecular masses of 170,000 and 140,000 daltons, in embryonic chick neural retina (Cole, G. J., and Glaser, L. (1984) Proc. Natl. Acad. Sci. U. S. A., in press). In vitro, the 170,000-dalton polypeptide is released by retinal cells into the surrounding culture medium and is present in material sedimentable at 100,000 X g. This pelletable material contains particles designated as adherons (Schubert, D., LaCorbiere, M., Klier, F. G., and Birdwell, G. (1983) J. Cell Biol. 96, 990-998) which promote cell-substratum adhesion of chick neural retina cells. In the present study, evidence is provided that the C1H3 monoclonal antibody inhibits cell adhesion to adheron-coated dishes when bound either to cells or to the adherons. The failure of other monoclonal antibodies, that bind to retinal cells with equal abundance, to disrupt adhesion demonstrates that the effect is specific. These data suggest that the neural-specific 170,000-dalton C1H3 polypeptide is the neural cell-adhesion molecule which is responsible for the ability of adherons to bind to cells.

Previous studies have reported that the cell-binding region of the neural cell adhesion molecule (N-CAM) resides in a 65,000-D amino-terminal fragment designated Frl (Cunningham, B. A., S. Hoffman, U. Rutishauser, J. J. Hemperly, and G. M. Edelman, 1983, Proc. Natl. Acad. Sci. USA, 80:3116-3120). We have reported the presence of two functional domains in N-CAM, each identified by a specific mAb, that are required for cell-cell or cell-substratum adhesion (Cole, G. J., and L. Glaser, 1986, J. Cell Biol., 102:403-412). One of these domains is a heparin (heparan sulfate)-binding domain. In the present study we have determined the topographic localization of the heparin-binding fragment from N-CAM, which has been identified by our laboratory. The B1A3 mAb recognizes a 25,000-D heparin-binding fragment derived from chicken N-CAM, and also binds to a 65,000-D fragment, presumably Frl, produced by digestion of N-CAM with Staphylococcus aureus V8 protease. Amino-terminal sequence analysis of the isolated 25,000-D heparin-binding domain of N-CAM yielded the sequence: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly. This sequence is identical to the previously reported amino-terminal sequence for murine and bovine N-CAM. Thus, the 25,000-D polypeptide fragment is the amino-terminal region of the N-CAM molecule. We have also shown that the B1A3 mAb recognizes not only chicken N-CAM but also rat and mouse N-CAM, indicating that the heparin-binding domain of N-CAM is evolutionarily conserved among different N-CAM forms. Additional peptide-mapping studies indicate that the second cell-binding site of N-CAM is located in a polypeptide region at least 65,000 D from the amino-terminal region. We conclude that the adhesion domains on N-CAM identified by these antibodies are physically distinct, and that the previously identified cell-binding domain on Frl is the heparin-binding domain.

Embryonal chick neural retina cells release into the culture medium a complex of proteins and glycosaminoglycans, termed adherons, that promote cell to substratum adhesion. A monoclonal antibody (C1H3) blocks adheron-mediated cell to substratum adhesion and specifically binds to a 170,000-mol-wt protein present in retinal adherons (Cole, G.J., and L. Glaser, 1984, J. Biol. Chem., 259:4031-4034). The 170,000-mol-wt protein also can be identified in embryonic chick brain and peripheral nervous tissue. In the neural retina, C1H3 also binds to a second antigen with a molecular weight of 140,000 that is absent in the brain. Embryonic brain, therefore, provides a source for the immunopurification of the 170,000-mol-wt protein. Brain adherons also contain the 170,000-mol-wt protein, and cell to substratum adhesion mediated by these adherons is blocked by the C1H3 monoclonal antibody. The 170,000-mol-wt protein in the brain is therefore functionally identical to that in the retina. To demonstrate that adheron-mediated cell to substratum adhesion is caused by cell binding to the 170,000-mol-wt protein, we showed that (a) protease digestion, but not glycosaminoglycan hydrolase digestion of adherons, blocked their ability to bind cells to substratum; (b) the immunopurified 170,000-mol-wt protein blocks adheron-mediated cell to substratum adhesion; and (c) cells can bind to immunopurified 170,000-mol-wt protein bound to glass surfaces.

Cell-substratum adhesion in the embryonic chicken nervous system has been shown to be mediated in part by a 170,000-mol-wt polypeptide that is a component of adherons. Attachment of retinal cells to the 170,000-mol-wt protein is inhibited by the C1H3 monoclonal antibody and by heparan sulfate (Cole, G. J., D. Schubert, and L. Glaser, 1985, J. Cell Biol., 100:1192-1199). In the present study we have demonstrated that the 170,000-mol-wt C1H3 polypeptide is immunologically identical to the neural cell adhesion molecule N-CAM, and that the 170,000-mol-wt component of N-CAM is preferentially secreted by cells as a component of adherons. We have identified a monoclonal antibody, designated B1A3, that inhibits heparin binding to N-CAM and cell-to-substratum adhesion. A 25,000-mol-wt heparin (heparan sulfate)-binding domain of N-CAM has been identified by limited proteolysis, and this fragment promotes cell attachment when bound to glass surfaces. The fragment also partially inhibits cell binding to adherons when bound to retinal cells, and the B1A3 monoclonal antibody inhibits retinal cell attachment to substrata composed of intact N-CAM or the heparin-binding domain. These data are the first evidence that N-CAM is a multifunctional protein that contains both cell-and heparin (heparan sulfate)-binding domains.

Cell-cell interactions are of critical importance during neural development, particularly since the migration of neural cells and the establishment of functional interactions between growing axons and their target cells has been suggested to depend upon cell recognition processes. Neurone-neurone adhesion has been well studied in vitro, and is mediated in part by the neural cell adhesion molecule N-CAM. N-CAM-mediated cell-cell adhesion has been postulated to occur by a homophilic binding mechanism, in which N-CAM on the surface of one cell binds to N-CAM on a neighbouring cell. Studies in our laboratory have identified a cell surface glycoprotein, now known to be N-CAM, which participates in cell-substratum interactions in the developing chicken nervous system. Although this adhesion involves a homophilic binding mechanism, the binding of the cell surface proteoglycan heparan sulphate to the glycoprotein is also required. This raises the question of whether the binding of heparan sulphate to N-CAM is also required for cell-cell adhesion. Here we show that the binding of retinal probe cells to retinal cell monolayers is inhibited by heparin, a functional analogue of heparan sulphate, but not by chondroitin sulphate. Monoclonal antibodies that recognize two different domains on N-CAM, the homophilic-binding and heparin-binding domains, inhibit cell-cell adhesion. The heparin-binding domain isolated from N-CAM by selective proteolysis also inhibits cell-cell adhesion when bound to the probe cells.

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.

A fluorescence-activated cell sorter screening method has been used to identify hybridomas that secrete monoclonal antibodies that can bind to viable subpopulations of embryonic chicken neural retina cells. One monoclonal antibody, C1H3, recognizes two nervous tissue-specific polypeptides that exhibit distinct developmental patterns. The monoclonal antibody reacts with a 140-kilodalton (kDa) polypeptide that is present at early stages of development (day 7) but is detected by immunoblotting in only negligible amounts at later times (day 17). In contrast, a 170-kDa polypeptide is first detectable by immunoblotting at day 10 and is the predominant C1H3 antigen at day 17. Analysis of proteolytic fragments of the two proteins indicates that the polypeptides are distinct molecules that share a common antigenetic determinant. Both polypeptides are neural-specific; the 140-kDa polypeptide appears to be retina-specific, while the 170-kDa polypeptide is also present in other areas of the nervous system. Metabolic labeling of retina cells in situ at early embryonic stages reveals only the synthesis of the 140-kDa polypeptide. When such cells are dissociated and labeled in vitro, they synthesize primarily the 170-kDa polypeptide. Thus, the differential rate of synthesis of these two polypeptides is controlled by environmental factors that possibly include cell-cell contacts or an unknown systemic factor. The 140-kDa polypeptide is a unique marker for early neural retina cells.