Michelle R. Emond

In Vivo Trafficking and Targeting of N-Cadherin to Nascent Presynaptic Terminals

N-cadherin is a prominent component of developing and mature synapses, yet very little is known about its trafficking within neurons. To investigate N-cadherin dynamics in developing axons, we used in vivo two-photon time-lapse microscopy of N-cadherin—green fluorescent protein (Ncad-GFP), which was expressed in Rohon-Beard neurons of the embryonic zebrafish spinal cord. Ncad-GFP was present as either stable accumulations or highly mobile transport packets. The mobile transport packets were of two types: tubulovesicular structures that moved preferentially in the anterograde direction and discrete-punctate structures that exhibited bidirectional movement. Stable puncta of Ncad-GFP accumulated in the wake of the growth cone with a time course. Colocalization of Ncad-GFP puncta with synaptic markers suggests that N-cadherin is a very early component of nascent synapses. Expression of deletion mutants revealed a potential role of the extracellular domain in appropriate N-cadherin trafficking and targeting. These results are the first to characterize the trafficking of a synaptic cell-adhesion molecule in developing axons in vivo. In addition, we have begun to investigate the cell biology of N-cadherin trafficking and targeting in the context of an intact vertebrate embryo.

Protocadherin-19 and N-cadherin interact to control cell movements during anterior neurulation.

Protocadherin-19 and N-cadherin act synergistically during brain morphogenesis in zebrafish.

A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion.

Formation of a Protocadherin-19–N-cadherin complex facilitates adhesion by Protocadherin-19 while suppressing homophilic interactions by N-cadherin.

Protocadherin-19 is essential for early steps in brain morphogenesis

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.

Inhibition of protocadherin-α function results in neuronal death in the developing zebrafish

The pcdhalpha/CNR gene comprises a diverse array of neuronal cell-surface proteins of the cadherin superfamily, although very little is known about their role in neural development. Here we provide the first in-depth characterization of pcdh1alpha in zebrafish. Whole-mount immunocytochemistry demonstrates that a large proportion of endogenous cytoplasmic domain immunoreactivity is present in the nucleus, suggesting that endoproteolytic cleavage and nuclear translocation of the intracellular domain are important aspects of pcdh1alpha activity in vivo. Using whole-mount immunocytochemistry and BAC-based expression of Pcdh1alpha-GFP fusion proteins, we find that Pcdh1alpha does not appear to form stable, synaptic puncta at early stages of synaptogenesis. We also demonstrate that the presence of the Pcdh1alpha cytoplasmic domain is essential for normal function. Truncation of Pcdh1alpha proteins, using splice-blocking antisense morpholinos to prevent the addition of the common intracellular domain to the entire pcdh1alpha cluster, results in neuronal apoptosis throughout the developing brain and spinal cord, demonstrating an essential role for pcdh1alpha in early neural development. This cell death phenotype can be attenuated by the expression of a soluble Pcdh1alpha cytoplasmic domain.

Protocadherin-18b interacts with Nap1 to control motor axon growth and arborization in zebrafish

Interference with Pcdh18b function results in impaired arborization of motor axons in the developing zebrafish. Pcdh18b interacts with Nap1, a regulator of actin assembly. Time-lapse imaging indicates that both Pcdh18b and Nap1 may affect axon arborization by regulating the density of axonal filopodia.

Protocadherins Control the Modular Assembly of Neuronal Columns in the Zebrafish Optic Tectum

δ-Protocadherins partition the zebrafish optic tectum into radial columns of neurons, and the neurons within a column are siblings derived from common neuronal progenitors.

Differential expression, alternative splicing, and adhesive properties of the zebrafish δ1-protocadherins

Protocadherins comprise the largest family within the cadherin superfamily of cell surface receptors. Here, we characterize the δ1-protocadherin subfamily during the development of the zebrafish nervous system. In zebrafish, there are five δ1-protocadherins: pcdh1a, pcdh1b, pcdh7a, pcdh7b, andpcdh9. Each protocadherin gene is highly homologous to its human ortholog. While the expression pattern in the developing CNS is similar for each δ1-protocadherin, with labeling observed in all major subdivisions, the detailed patterns are distinct. In addition, we provide evidence for alternative splicing of the pcdh7b and pcdh9 genes, resulting in variation in their respective cytoplasmic domains. As protocadherins are widely regarded to act as cell adhesion molecules, we used in vitro assays of δ1-pcdh ectodomains to directly test their adhesive properties. We found no evidence for calcium-dependent, homophilic adhesion, contrasting sharply with the behavior of classical cadherins.

Zebrafish calsyntenins mediate homophilic adhesion through their amino-terminal cadherin repeats.

The calsyntenins are atypical members of the cadherin superfamily that have been implicated in learning in Caenorhabditis elegans and memory formation in humans. As members of the cadherin superfamily, they could mediate cell-cell adhesion, although their adhesive properties have not been investigated. As an initial step in characterizing the calsyntenins, we have cloned clstn1, clstn2 and clstn3 from the zebrafish and determined their expression in the developing zebrafish nervous system. The three genes each have broad, yet distinct, expression patterns in the zebrafish brain. Each of the ectodomains mediates homophilic interactions through two, amino-terminal cadherin repeats. In bead sorting assays, the calsyntenin ectodomains do not exhibit homophilic preferences. These data support the idea that calsyntenins could either act as adhesion molecules or as diffusible, homophilic or heterophilic ligands in the vertebrate nervous system.

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.