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

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

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.</jats:p

Multiplane Calcium Imaging Reveals Disrupted Development of Network Topology in Zebrafish<i>pcdh19</i>Mutants

AbstractFunctional 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 ofPCDH19in humans and model organisms leads to functional deficits, the underlying network defects remain unknown. Here, we employ multiplane, resonant-scanningin vivotwo-photon calcium imaging of developing zebrafish, and use graph theory to characterize the development of resting state functional networks in both wild-type andpcdh19mutant larvae. We find that the brain networks ofpcdh19mutants 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.</jats:p

Fluorescence Imaging of Transgenic Zebrafish Embryos: Figure 1.

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.</jats:p