Quantitation of mitotic cells in the neural tube of zebrafish embryos using automated nuclei counting.

Here, we provide a protocol to automate the quantification of the number of phospho-histone H3-positive cells in the developing nervous system of zebrafish using a custom MATLAB script to identify labeled nuclei. We describe steps for fixation, immunolabeling, and imaging of zebrafish embryos. We then detail the analysis steps using Fiji and MATLAB. This protocol can be used for fixed, immunolabeled tissue, as shown here, or for live samples, such as cells expressing a histone-GFP fusion protein. For complete details on the use and execution of this protocol, please refer to Biswas et al.1.

δ-Protocadherins regulate neural progenitor cell division by antagonizing Ryk and Wnt/ß-catenin signaling.

The division of neural progenitor cells provides the cellular substrate from which the nervous system is sculpted during development. The δ-protocadherin family of homophilic cell adhesion molecules is essential for the development of the vertebrate nervous system and is implicated in an array of neurodevelopmental disorders. We show that lesions in any of six, individual δ-protocadherins increases cell divisions of neural progenitors in the hindbrain. This increase is due to mis-regulation of Wnt/ß-catenin signaling, as this pathway is upregulated in δ-protocadherin mutants and inhibition of this pathway blocks the increase in cell division. Furthermore, the δ-protocadherins can be present in complex with the Wnt receptor Ryk, and Ryk is required for the increased proliferation in protocadherin mutants. Thus, δ-protocadherins are novel regulators of Wnt/ß-catenin signaling that may control the development of neural circuits by defining a molecular code for the identity of neural progenitor cells and differentially regulating their proliferation.

Proximity-dependent Proteomics Reveals Extensive Interactions of Protocadherin-19 with Regulators of Rho GTPases and the Microtubule Cytoskeleton.

Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.

A CreER mouse to study melanin concentrating hormone signaling in the developing brain.

The neuropeptide, melanin concentrating hormone (MCH), and its G protein-coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1-Cre) exists, there is a need for an inducible Mchr1-Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1-Cre expression pattern are recapitulated by the Mchr1-CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1-CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1-CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences.

Protocadherins control the modular assembly of neuronal columns in the zebrafish optic tectum.

Cell-cell recognition guides the assembly of the vertebrate brain during development. δ-Protocadherins comprise a family of neural adhesion molecules that are differentially expressed and have been implicated in a range of neurodevelopmental disorders. Here we show that the expression of δ-protocadherins partitions the zebrafish optic tectum into radial columns of neurons. Using in vivo two-photon imaging of bacterial artificial chromosome transgenic zebrafish, we show that pcdh19 is expressed in discrete columns of neurons, and that these columnar modules are derived from proliferative pcdh19(+) neuroepithelial precursors. Elimination of pcdh19 results in both a disruption of columnar organization and defects in visually guided behaviors. These results reveal a fundamental mechanism for organizing the developing nervous system: subdivision of the early neuroepithelium into precursors with distinct molecular identities guides the autonomous development of parallel neuronal units, organizing neural circuit formation and behavior.

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.

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

The proper assembly of neural circuits during development requires the precise control of axon outgrowth, guidance, and arborization. Although the protocadherin family of cell surface receptors is widely hypothesized to participate in neural circuit assembly, their specific roles in neuronal development remain largely unknown. Here we demonstrate that zebrafish pcdh18b is involved in regulating axon arborization in primary motoneurons. Although axon outgrowth and elongation appear normal, antisense morpholino knockdown of pcdh18b results in dose-dependent axon branching defects in caudal primary motoneurons. Cell transplantation experiments show that this effect is cell autonomous. Pcdh18b interacts with Nap1, a core component of the WAVE complex, through its intracellular domain, suggesting a role in the control of actin assembly. Like that of Pcdh18b, depletion of Nap1 results in reduced branching of motor axons. Time-lapse imaging and quantitative analysis of axon dynamics indicate that both Pcdh18b and Nap1 regulate axon arborization by affecting the density of filopodia along the shaft of the extending axon.

In vivo imaging of synaptogenesis in zebrafish.

The embryonic zebrafish is a nearly ideal model system in which to use time-lapse imaging to study the development of the vertebrate nervous system in vivo. The embryos are small and transparent, they develop externally and rapidly, and the embryonic central nervous system is relatively simple and highly stereotyped. With the refinement of green fluorescent protein (GFP) as a genetically encoded fluorescent tag of neuronal proteins, along with advances in imaging technology, it is possible to follow the cellular and molecular events underlying development as they occur in the living embryo. This article describes strategies for imaging synapse formation in the embryonic zebrafish.

Fluorescence imaging of transgenic zebrafish embryos.

The embryonic zebrafish is a nearly ideal model system in which to use time-lapse imaging to study the development of the vertebrate nervous system in vivo. The embryos are small and transparent, they develop externally and rapidly, and the embryonic central nervous system is relatively simple and highly stereotyped. With the refinement of green fluorescent protein (GFP) as a genetically encoded fluorescent tag of neuronal proteins, along with advances in imaging technology, it is possible to follow the cellular and molecular events underlying development as they occur in the living embryo. This protocol describes methods for imaging synapse formation in embryonic zebrafish. Injection of DNA into early embryos is followed by mounting of the transgenic embryos in agarose and then time-lapse data collection.

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

During embryonic morphogenesis, adhesion molecules are required for selective cell-cell interactions. The classical cadherins mediate homophilic calcium-dependent cell adhesion and are founding members of the large and diverse cadherin superfamily. The protocadherins are the largest subgroup within this superfamily, yet their participation in calcium-dependent cell adhesion is uncertain. In this paper, we demonstrate a novel mechanism of adhesion, mediated by a complex of Protocadherin-19 (Pcdh19) and N-cadherin (Ncad). Although Pcdh19 alone is only weakly adhesive, the Pcdh19-Ncad complex exhibited robust adhesion in bead aggregation assays, and Pcdh19 appeared to play the dominant role. Adhesion by the Pcdh19-Ncad complex was unaffected by mutations that disrupt Ncad homophilic binding but was inhibited by a mutation in Pcdh19. In addition, the complex exhibited homophilic specificity, as beads coated with Pcdh19-Ncad did not intermix with Ncad- or Pcdh17-Ncad-coated beads. We propose a model in which association of a protocadherin with Ncad acts as a switch, converting between distinct binding specificities.

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

The protocadherins comprise the largest subgroup within the cadherin superfamily, yet their cellular and developmental functions are not well understood. In this study, we demonstrate that pcdh 19 (protocadherin 19) acts synergistically with n-cadherin (ncad) during anterior neurulation in zebrafish. In addition, Pcdh 19 and Ncad interact directly, forming a protein-protein complex both in vitro and in vivo. Although both molecules are required for calcium-dependent adhesion in a zebrafish cell line, the extracellular domain of Pcdh 19 does not exhibit adhesive activity, suggesting that the involvement of Pcdh 19 in cell adhesion is indirect. Quantitative analysis of in vivo two-photon time-lapse image sequences reveals that loss of either pcdh 19 or ncad impairs cell movements during neurulation, disrupting both the directedness of cell movements and the coherence of movements among neighboring cells. Our results suggest that Pcdh 19 and Ncad function together to regulate cell adhesion and to mediate morphogenetic movements during brain development.

AMPA receptor subunits define properties of state-dependent synaptic plasticity.

Many synapses undergo immediate and persistent activity-dependent changes in strength via processes that fall under the umbrella of synaptic plasticity. It is known that this type of synaptic plasticity exhibits an underlying state dependence; that is, as synapses change in strength they move into distinct 'states' that are defined by the mechanism and ability to undergo future plasticity. In this study, we have investigated the molecular mechanisms that underlie state-dependent synaptic plasticity. Using intracellular application of peptides that mimic the C-terminal tail sequences of GluR1 and GluR2 AMPA receptor subtypes, combined with paired recordings of minimal synaptic connections, we have shown that AMPA receptor subtypes present in the membrane at a given time confer some properties of plasticity states. These data show that during synaptic plasticity, AMPA receptor subtypes are differentially stabilized by postsynaptic density proteins in or out of the postsynaptic membrane, and this differential synaptic expression of different AMPA receptor subtypes defines distinct synaptic states.

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-alpha 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.

Blocking polysynaptic inhibition via opioid receptor activation isolates excitatory synaptic currents without triggering epileptiform activity in organotypic hippocampal slices.

The abundance of synaptic connectivity in the cultured hippocampal slice preparation allows measurements of the unitary excitatory connection between pairs of pyramidal neurons using simultaneous presynaptic and postsynaptic intracellular recordings. However, the useful yield of these recordings can be greatly reduced by the presence of polysynaptic inhibition that occludes the measurement of the monosynaptic excitatory postsynaptic current (EPSC). We have found that the traditional method of eliminating contaminating synaptic inhibition with GABA receptor antagonists is of limited usefulness because the recurrent excitatory connections in organotypic slices cause epileptiform bursting in the absence of inhibitory function. This bursting obscures EPSCs to an even greater extent than the normally occurring polysynaptic inhibitory transmission. Here, we report a new method for isolating monosynaptic EPSCs using the mu-opioid agonist peptide DAMGO to reduce polysynaptic inhibition during these recordings. Activation of mu-opioid receptors is known to hyperpolarize inhibitory neurons. We found that DAMGO application reduces the amplitude and frequency of polysynaptic inhibition, allowing isolation of the excitatory connection between the two neurons being recorded. Furthermore, because inhibitory function is not completely eliminated by DAMGO application, epileptiform bursting very rarely develops. Therefore, the use of DAMGO to prevent polysynaptic inhibition without causing epileptiform bursting provides a useful tool to substantially increase the yield of experiments measuring the unitary excitatory connection between pyramidal neurons in the cultured hippocampal slice preparation.

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.