Fritz G. Rathjen

Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations

The neural cell recognition molecule L1 is a member of the immunoglobulin superfamily implicated in embryonic brain development. L1 is engaged in complex extracellular interactions, with multiple binding partners on cell surfaces and in the extracellular matrix. It is the founder of a neural group of related cell surface receptors that share with L1 a highly conserved cytoplasmic domain that associates with the cytoskeleton. Phenotypic analyses of human patients with mutations in the L1 gene and characterizations of L1-deficient mice suggest that L1 is important for embryonic brain histogenesis, in particular the development of axon tracts.

The axonal recognition molecule F11 is a multifunctional protein: specific domains mediate interactions with Ng-CAM and restrictin.

F11 is a glycosyl phosphatidylinositol-anchored axonal surface glycoprotein belonging to a neural subgroup of the immunoglobulin superfamily. In this report, we demonstrate that the F11 protein displays three distinguishable activities: binding to the cell recognition molecule Ng-CAM, interaction with the extracellular matrix glycoprotein restrictin (RN), and a neurite outgrowth-promoting activity. By analyzing deletion mutants expressed in transfected COS cells, epitope mapping of monoclonal antibodies, and neurite outgrowth assays, we reveal that these activities can be localized to distinct regions within the F11 protein. The Ng-CAM-binding site resides in the first two immunoglobulin-like domains of F11, whereas the RN-binding site resides in the second or third domain. A neurite outgrowth-promoting activity of F11 characterized by in vitro culture of tectal cells is independent of F11-Ng-CAM and F11-RN binding.

Structure/function relationships of axon-associated adhesion receptors of the immunoglobulin superfamily

Evidence is accumulating that axonal members of the Ig superfamily (IgSF) interact in a complex manner with other axonal Ig-like proteins and with proteins of the extracellular matrix. Studies investigating the structure/function relationships of these proteins have highlighted the importance of Ig-like domains near the amino terminus (N-proximal) as both necessary and sufficient for homophilic and heterophilic binding. Although efforts have been made in the past year to correlate the structure and neurite-outgrowth-promoting ability of axonal IgSF members, this work is still at an early stage.

The chicken neural extracellular matrix molecule restrictin: Similarity with EGF-, fibronectin type III-, and fibrinogen-like motifs

Restrictin is a chick neural extracellular matrix protein implicated in neural cell attachment and found to be associated with the cell surface recognition protein F11. Here we show by cDNA cloning that restrictin is a large multidomain protein composed of 4 structural motifs. At the N-terminus restrictin contains a cysteine-rich segment of about 140 aa that might link restrictin monomers into oligomers. This region is followed by 4.5 epidermal growth factor-like repeats and then by 9 consecutive motifs that are similar to fibronectin type III motifs. At the C-terminus restriction is related to the beta and gamma chains of fibrinogen, including similarity to a calcium-binding segment. Restrictin shows substantial sequence similarity with tenascin (cytotactin) throughout the polypeptide, and like tenascin, it forms oligomeric structures, as revealed by electron microscopy of immunoaffinity-purified restriction. The cell attachment site of restrictin is mapped to the C-terminal region by antibody perturbation experiments.

Axonal glycoproteins with immunoglobulin- and fibronectin type III-related domains in vertebrates : structural features, binding activities, and signal transduction

Abstract: The L1‐ and F11‐like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III‐like domains. Their Ig‐like and fibronectin type III‐related domains are likely to be composed of seven β‐strands arranged in two opposing β‐sheets of highly similar topology. Whereas the F11‐like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 ‐like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre‐mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 ‐like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 ‐like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III‐like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.

Pathological missense mutations of neural cell adhesion molecule L1 affect homophilic and heterophilic binding activities

Mutations in the gene for neural cell adhesion molecule L1 (L1CAM) result in a debilitating X‐linked congenital disorder of brain development. At the neuronal cell surface L1 may interact with a variety of different molecules including itself and two other CAMs of the immunoglobulin superfamily, axonin‐1 and F11. However, whether all of these interactions are relevant to normal or abnormal development has not been determined. Over one‐third of patient mutations are single amino acid changes distributed across 10 extracellular L1 domains. We have studied the effects of 12 missense mutations on binding to L1, axonin‐1 and F11 and shown for the first time that whereas many mutations affect all three interactions, others affect homophilic or heterophilic binding alone. Patient pathology is therefore due to different types of L1 malfunction. The nature and functional consequence of mutation is also reflected in the severity of the resultant phenotype with structural mutations likely to affect more than one binding activity and result in early mortality. Moreover, the data indicate that several extracellular domains of L1 are required for homophilic and heterophilic interactions.

Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins Fll and Nr-CAM/Bravo

F11 and Nr-CAM/Bravo are two axon-associated glycoproteins belonging to different subgroups of the immunoglobulin superfamily. In this report we have investigated the interaction of both proteins using neurite outgrowth and binding assays. Antibody blocking experiments demonstrate that neurite extension of tectal cells on immobilized F11 is mediated by Nr-CAM/Bravo. Binding studies further reveal a direct heterophilic interaction between F11 and Nr-CAM/Bravo. This activity can be mapped to the amino-terminal second or third immunoglobulin-like domain within F11 with domain-specific monoclonal antibodies and deletion mutant proteins expressed on COS cells. Furthermore, perturbation experiments with domain-specific monoclonal antibodies demonstrate that this region is required for adhesion and neurite extension.

Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development.

The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.

N-Cadherin is essential for retinal lamination in the zebrafish

N‐Cadherin is one of the major Ca2+‐dependent cell adhesion proteins in the developing nervous system. Here, we analyze eye development in the zebrafish N‐cadherin loss‐of‐function mutant parachutepaR2.10 (pacpaR2.10). The zebrafish visual system is fully developed by the time pacpaR2.10 mutants show lethality at day 5. Already at 24 hr postfertilization (hpf), mutant retinal cells are more disorganized and more rounded than in wild‐type. At later stages, mutant retinae display a severe lamination defect with rosette formation (mostly islands of plexiform layer tissue surrounded by inner nuclear layer or photoreceptor cells), even though all major classes of cell types appear to be present as determined by histology. Of interest, electron microscopy reveals that the islands of plexiform layer tissue contain a normal amount of synapses with normal morphology. Although mutant photoreceptor cells are sometimes deformed, all typical structural components are present, including the membranous discs for rhodopsin storage. The lens fibers of the pacpaR2.10 mutants develop completely normally, but in some cases, lens epithelial cells round up and become multilayered. We conclude that cell adhesion mediated by N‐cadherin is of major importance for retinal lamination and involved in maintenance of the lens epithelial sheet, but is not essential for the formation of photoreceptor ultrastructure or for synaptogenesis. Developmental Dynamics 226:000–000, 2003.

The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord

Sensory axonal projections into the spinal cord display a highly stereotyped pattern of T- or Y-shaped axon bifurcation at the dorsal root entry zone (DREZ). Here, we provide evidence that embryonic mice with an inactive receptor guanylyl cyclase Npr2 or deficient for cyclic guanosine monophosphate–dependent protein kinase I (cGKI) lack the bifurcation of sensory axons at the DREZ, i.e., the ingrowing axon either turns rostrally or caudally. This bifurcation error is maintained to mature stages. In contrast, interstitial branching of collaterals from primary stem axons remains unaffected, indicating that bifurcation and interstitial branching are processes regulated by a distinct molecular mechanism. At a functional level, the distorted axonal branching at the DREZ is accompanied by reduced synaptic input, as revealed by patch clamp recordings of neurons in the superficial layers of the spinal cord. Hence, our data demonstrate that Npr2 and cGKI are essential constituents of the signaling pathway underlying axonal bifurcation at the DREZ and neuronal connectivity in the dorsal spinal cord.