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.

Multiplane Calcium Imaging Reveals Disrupted Development of Network Topology in Zebrafish pcdh19 Mutants.

Functional brain networks self-assemble during development, although the molecular basis of network assembly is poorly understood. Protocadherin-19 (pcdh19) is a homophilic cell adhesion molecule that is linked to neurodevelopmental disorders, and influences multiple cellular and developmental events in zebrafish. Although loss of PCDH19 in humans and model organisms leads to functional deficits, the underlying network defects remain unknown. Here, we employ multiplane, resonant-scanning in vivo two-photon calcium imaging of developing zebrafish, and use graph theory to characterize the development of resting state functional networks in both wild-type and pcdh19 mutant larvae. We find that the brain networks of pcdh19 mutants display enhanced clustering and an altered developmental trajectory of network assembly. Our results show that functional imaging and network analysis in zebrafish larvae is an effective approach for characterizing the developmental impact of lesions in genes of clinical interest.

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.

The Cadherin Superfamily in Neural Circuit Assembly.

The cadherin superfamily comprises a large, diverse collection of cell surface receptors that are expressed in the nervous system throughout development and have been shown to be essential for the proper assembly of the vertebrate nervous system. As our knowledge of each family member has grown, it has become increasingly clear that the functions of various cadherin subfamilies are intertwined: they can be present in the same protein complexes, impinge on the same developmental processes, and influence the same signaling pathways. This interconnectedness may illustrate a central way in which core developmental events are controlled to bring about the robust and precise assembly of neural circuitry.

δ-Protocadherins: Organizers of neural circuit assembly.

The δ-protocadherins comprise a small family of homophilic cell adhesion molecules within the larger cadherin superfamily. They are essential for neural development as mutations in these molecules give rise to human neurodevelopmental disorders, such as schizophrenia and epilepsy, and result in behavioral defects in animal models. Despite their importance to neural development, a detailed understanding of their mechanisms and the ways in which their loss leads to changes in neural function is lacking. However, recent results have begun to reveal roles for the δ-protocadherins in both regulation of neurogenesis and lineage-dependent circuit assembly, as well as in contact-dependent motility and selective axon fasciculation. These evolutionarily conserved mechanisms could have a profound impact on the robust assembly of the vertebrate nervous system. Future work should be focused on unraveling the molecular mechanisms of the δ-protocadherins and understanding how this family functions broadly to regulate neural development.

Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy.

Non-clustered δ-protocadherins are homophilic cell adhesion molecules essential for the development of the vertebrate nervous system, as several are closely linked to neurodevelopmental disorders. Mutations in protocadherin-19 (PCDH19) result in a female-limited, infant-onset form of epilepsy (PCDH19-FE). Over 100 mutations in PCDH19 have been identified in patients with PCDH19-FE, about half of which are missense mutations in the adhesive extracellular domain. Neither the mechanism of homophilic adhesion by PCDH19, nor the biochemical effects of missense mutations are understood. Here we present a crystallographic structure of the minimal adhesive fragment of the zebrafish Pcdh19 extracellular domain. This structure reveals the adhesive interface for Pcdh19, which is broadly relevant to both non-clustered δ and clustered protocadherin subfamilies. In addition, we show that several PCDH19-FE missense mutations localize to the adhesive interface and abolish Pcdh19 adhesion in in vitro assays, thus revealing the biochemical basis of their pathogenic effects during brain development.

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.

Motoneuron development influences dorsal root ganglia survival and Schwann cell development in a vertebrate model of spinal muscular atrophy.

Low levels of the survival motor neuron protein (SMN) cause the disease spinal muscular atrophy. A primary characteristic of this disease is motoneuron dysfunction and paralysis. Understanding why motoneurons are affected by low levels of SMN will lend insight into this disease and to motoneuron biology in general. Motoneurons in zebrafish smn mutants develop abnormally; however, it is unclear where Smn is needed for motoneuron development since it is a ubiquitously expressed protein. We have addressed this issue by expressing human SMN in motoneurons in zebrafish maternal-zygotic (mz) smn mutants. First, we demonstrate that SMN is present in axons, but only during the period of robust motor axon outgrowth. We also conclusively demonstrate that SMN acts cell autonomously in motoneurons for proper motoneuron development. This includes the formation of both axonal and dendritic branches. Analysis of the peripheral nervous system revealed that Schwann cells and dorsal root ganglia (DRG) neurons developed abnormally in mz-smn mutants. Schwann cells did not wrap axons tightly and had expanded nodes of Ranvier. The majority of DRG neurons had abnormally short peripheral axons and later many of them failed to divide and died. Expressing SMN just in motoneurons rescued both of these cell types showing that their failure to develop was secondary to the developmental defects in motoneurons. Driving SMN just in motoneurons did not increase survival of the animal, suggesting that SMN is needed for motoneuron development and motor circuitry, but that SMN in other cells types factors into survival.

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.

Protocadherins, not prototypical: a complex tale of their interactions, expression, and functions.

The organization of functional neural circuits requires the precise and coordinated control of cell-cell interactions at nearly all stages of development, including neuronal differentiation, neuronal migration, axon outgrowth, dendrite arborization, and synapse formation and stabilization. This coordination is brought about by the concerted action of a large number of cell surface receptors, whose dynamic regulation enables neurons (and astrocytes) to adopt their proper roles within developing neural circuits. The protocadherins (Pcdhs) comprise a major family of cell surface receptors expressed in the developing vertebrate nervous system whose cellular and developmental roles are only beginning to be elucidated. In this review, we highlight selected recent results in several key areas of Pcdh biology and discuss their implications for our understanding of neural circuit formation and function.

Temporal requirement for SMN in motoneuron development.

Proper function of the motor unit is dependent upon the correct development of dendrites and axons. The infant/childhood onset motoneuron disease spinal muscular atrophy (SMA), caused by low levels of the survival motor neuron (SMN) protein, is characterized by muscle denervation and paralysis. Although different SMA models have shown neuromuscular junction defects and/or motor axon defects, a comprehensive analysis of motoneuron development in vivo under conditions of low SMN will give insight into why the motor unit becomes dysfunctional. We have generated genetic mutants in zebrafish expressing low levels of SMN from the earliest stages of development. Analysis of motoneurons in these mutants revealed motor axons were often shorter and had fewer branches. We also found that motoneurons had significantly fewer dendritic branches and those present were shorter. Analysis of motor axon filopodial dynamics in live embryos revealed that mutants had fewer filopodia and their average half-life was shorter. To determine when SMN was needed to rescue motoneuron development, SMN was conditionally induced in smn mutants during embryonic stages. Only when SMN was added back soon after motoneurons were born, could later motor axon development be rescued. Importantly, analysis of motor behavior revealed that animals with motor axon defects had significant deficits in motor output. We also show that SMN is required earlier for motoneuron development than for survival. These data support that SMN is needed early in development of motoneuron dendrites and axons to develop normally and that this is essential for proper connectivity and movement.

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.

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.