Additive dominant effect of a SOX10 mutation underlies a complex phenotype of PCWH.

Distinct classes of SOX10 mutations result in peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease, collectively known as PCWH. Meanwhile, SOX10 haploinsufficiency caused by allelic loss-of-function mutations leads to a milder non-neurological disorder, Waardenburg-Hirschsprung disease. The cellular pathogenesis of more complex PCWH phenotypes in vivo has not been thoroughly understood. To determine the pathogenesis of PCWH, we have established a transgenic mouse model. A known PCWH-causing SOX10 mutation, c.1400del12, was introduced into mouse Sox10-expressing cells by means of bacterial artificial chromosome (BAC) transgenesis. By crossing the multiple transgenic lines, we examined the effects produced by various copy numbers of the mutant transgene. Within the nervous systems, transgenic mice revealed a delay in the incorporation of Schwann cells in the sciatic nerve and the terminal differentiation of oligodendrocytes in the spinal cord. Transgenic mice also showed defects in melanocytes presenting as neurosensory deafness and abnormal skin pigmentation, and a loss of the enteric nervous system. Phenotypes in each lineage were more severe in mice carrying higher copy numbers, suggesting a gene dosage effect for mutant SOX10. By uncoupling the effects of gain-of-function and haploinsufficiency in vivo, we have demonstrated that the effect of a PCWH-causing SOX10 mutation is solely pathogenic in each SOX10-expressing cellular lineage in a dosage-dependent manner. In both the peripheral and central nervous systems, the primary consequence of SOX10 mutations is hypomyelination. The complex neurological phenotypes in PCWH patients likely result from a combination of haploinsufficiency and additive dominant effect.

A sharp cadherin-6 gene expression boundary in the developing mouse cortical plate demarcates the future functional areal border.

The mammalian cerebral cortex can be tangentially subdivided into tens of functional areas with distinct cyto-architectures and neural circuitries; however, it remains elusive how these areal borders are genetically elaborated during development. Here we establish original bacterial artificial chromosome transgenic mouse lines that specifically recapitulate cadherin-6 (Cdh6) mRNA expression profiles in the layer IV of the somatosensory cortex and by detailing their cortical development, we show that a sharp Cdh6 gene expression boundary is formed at a mediolateral coordinate along the cortical layer IV as early as the postnatal day 5 (P5). By further applying mouse genetics that allows rigid cell fate tracing with CreERT2 expression, it is demonstrated that the Cdh6 gene expression boundary set at around P4 eventually demarcates the areal border between the somatosensory barrel and limb field at P20. In the P6 cortical cell pellet culture system, neurons with Cdh6 expression preferentially form aggregates in a manner dependent on Ca(2+) and electroporation-based Cdh6 overexpression limited to the postnatal stages perturbs area-specific cell organization in the barrel field. These results suggest that Cdh6 expression in the nascent cortical plate may serve solidification of the protomap for cortical functional areas.

Targeting Neurons with Functional Oxytocin Receptors: A Novel Set of Simple Knock-In Mouse Lines for Oxytocin Receptor Visualization and Manipulation.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

Bacterial artificial chromosomes as analytical basis for gene transcriptional machineries.

Bacterial Artificial Chromosomes (BACs) had been minimal components of various genome-sequencing projects, constituting perfect analytical basis for functional genomics. Here we describe an enhancer screening strategy in which BAC clones that cover any genomic segments of interest are modified to harbor a reporter cassette by transposon tagging, then processed to carry selected combinations of gene regulatory modules by homologous recombination mediated systematic deletions. Such engineered BAC-reporter constructs in bacterial cells are ready for efficient transgenesis in mice to evaluate activities of gene regulatory modules intact or absent in the constructs. By utilizing the strategy, we could speedily identify a critical genomic fragment for spatio-temporally regulated expression of a mouse cadherin gene whose structure is extraordinarily huge and intricate. This BAC-based methodology would hence provide a novel screening platform for gene transcriptional machineries that dynamically fluctuate during development, pathogenesis and/or evolution.

An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

Retina-specific GTPase accelerator RGS11/G beta 5S/R9AP is a constitutive heterotrimer selectively targeted to mGluR6 in ON-bipolar neurons.

Members of the R7 family of the regulators of G-protein signaling (R7 RGS) proteins form multi-subunit complexes that play crucial roles in processing the light responses of retinal neurons. The disruption of these complexes has been shown to lead to the loss of temporal resolution in retinal photoreceptors and deficient synaptic transmission to downstream neurons. Despite the well established role of one member of this family, RGS9-1, in controlling vertebrate phototransduction, the roles and organizational principles of other members in the retina are poorly understood. Here we investigate the composition, localization, and function of complexes containing RGS11, the closest homolog of RGS9-1. We find that RGS11 forms a novel obligatory trimeric complex with the short splice isoform of the type 5 G-protein beta subunit (G beta 5) and the RGS9 anchor protein (R9AP). The complex is expressed exclusively in the dendritic tips of ON-bipolar cells in which its localization is accomplished through a direct association with mGluR6, the glutamate receptor essential for the ON-bipolar light response. Although association with both R9AP and mGluR6 contributed to the proteolytic stabilization of the complex, postsynaptic targeting of RGS11 was not determined by its membrane anchor R9AP. Electrophysiological recordings of the light response in mouse rod ON-bipolar cells reveal that the genetic elimination of RGS11 has little effect on the deactivation of G alpha(o) in dark-adapted cells or during adaptation to background light. These results suggest that the deactivation of mGluR6 cascade during the light response may require the contribution of multiple GTPase activating proteins.

Redundant type II cadherins define neuroepithelial cell states for cytoarchitectonic robustness.

Individual cell shape and integrity must precisely be orchestrated during morphogenesis. Here, we determine function of type II cadherins, Cdh6, Cdh8, and Cdh11, whose expression combinatorially demarcates the mouse neural plate/tube. While CRISPR/Cas9-based single type II cadherin mutants show no obvious phenotype, Cdh6/8 double knockout (DKO) mice develop intermingled forebrain/midbrain compartments as these two cadherins' expression opposes at the nascent boundary. Cdh6/8/11 triple, Cdh6/8 or Cdh8/11 DKO mice further cause exencephaly just within the cranial region where mutated cadherins' expression merges. In the Cdh8/11 DKO midbrain, we observe less-constricted apical actin meshwork, ventrally-directed spreading, and occasional hyperproliferation among dorsal neuroepithelial cells as origins for exencephaly. These results provide rigid evidence that, by conferring distinct adhesive codes to each cell, redundant type II cadherins serve essential and shared roles in compartmentalization and neurulation, both of which proceed under the robust control of the number, positioning, constriction, and fluidity of neuroepithelial cells.

Analysis of mouse Cdh6 gene regulation by transgenesis of modified bacterial artificial chromosomes.

Classic cadherins are cell adhesion molecules whose expression patterns are dynamically modulated in association with their diverse functions during morphogenesis. The large size and complexity of cadherin loci have made it a challenge to investigate the organization of cis-regulatory modules that control their spatiotemporal patterns of expression. Towards this end, we utilized bacterial artificial chromosomes (BACs) containing the Cdh6 gene, a mouse type II classic cadherin, to systematically identify cis-regulatory modules that govern its expression. By inserting a lacZ reporter gene into the Cdh6 BAC and generating a series of modified variants via homologous recombination or transposon insertions that have been examined in transgenic mice, we identified an array of genomic regions that contribute to specific regulation of the gene. These regions span approximately 350 kb of the locus between 161-kb upstream and 186-kb downstream of the Cdh6 transcription start site. Distinct modules independently regulate compartmental expression (i.e. forebrain, hindbrain rhombomeres, and spinal cord) and/or cell lineage-specific expression patterns (i.e. neural crest subpopulations such as Schwann cells) of Cdh6 at the early developmental stages. With respect to regulation of expression in neural crest cells, we have found that distinct regions contribute to different aspects of expression and have identified a short 79-bp region that is implicated in regulating expression in cells once they have emigrated from the neural tube. These results build a picture of the complex organization of Cdh6 cis-regulatory modules and highlight the diverse inputs that contribute to its dynamic expression during early mouse embryonic development.

Genetic labeling of mouse rhombomeres by Cadherin-6::EGFP-BAC transgenesis underscores the role of cadherins in hindbrain compartmentalization.

Cadherin-6 (Cdh6) is a type II classic cadherin cell adhesion molecule whose expression delineates specific sets of rhombomeres during early mouse development. Here we establish a stable BAC transgenic mouse line in which enhanced green fluorescent protein (EGFP) recapitulates Cdh6 expression within the embryonic hindbrain. When the transgenic posterior hindbrain region at the embryonic (E) day 8.75-E9.75 that contains EGFP-positive rhombomere (r) 6-7 and EGFP-negative r4-5 cells was dissociated to single cells and incubated in suspensions, they were found to make aggregates only in the presence of calcium ion with the EGFP-Cdh6-positive/negative populations segregating each other in an aggregate. We further demonstrated that EGFP-Cdh6 expression boundary in the transgenic hindbrain at E9.5 shifted rostrally by the treatment that allows Hox gene expression anteriorize in the whole embryo culture system. These results suggest the role of Cdh6 in coordinating mouse hindbrain compartments possibly under the control of Hox gene regulatory network.

Cadherin-6 gene regulatory patterns in the postnatal mouse brain.

Cadherin-6 (Cdh6, K-cadherin) is a synaptic adhesion molecule the expression of which demarcates restricted sets of neuronal circuitries in postnatal mouse brains. While roles for the cadherins in the formation and/or modulation of synaptic junctions have been implicated, that which drives cadherin expression along functional brain circuits has remained elusive. Here we investigate the genetic control of Cdh6 expression by applying a method that permits systematic integration of a reporter cassette into bacterial artificial chromosomes with extensive coverage of the huge Cdh6 gene locus, whereby the reporter activities are efficiently evaluated in stable transgenic mouse lines. Such screenings revealed that divisible genomic segments differentially control each brain region or nucleus specific expression of Cdh6 at the right phases for circuit formation. These separable regulatory modules for cadherin expressions tended to be grouped by working connectivities, suggesting their developmental and/or evolutional value in elaborating brain circuitry.

Balanced expression of various TrkB receptor isoforms from the Ntrk2 gene locus in the mouse nervous system.

A brain-derived neurotrophic factor (BDNF) receptor TrkB involves three spliced variants, namely the tyrosine kinase domain (TK) intact (+) and two TK(-) isoforms T1 and T2, yet their precise roles are largely unknown. Here we extensively map the mRNA expression patterns of BDNF and TrkB variants, further to gain insights in TK(-) specific functions during mouse development. Consequently, we found that TK(+), T1 and T2 were expressed in distinct regions of the mouse nervous system at the embryonic and postnatal stages, implicating separable functions of TK(-) forms. Additionally we uncovered five expressed segments in the intron between T2 and T1 specific exons, and one of these segments was revealed to code novel TK(-) receptors with unique responsiveness in vitro. These results suggest dynamic modes of expression from the Ntrk2 gene locus and multiple roles of TK(-) forms in the developing mouse nervous system.

Classic cadherin expressions balance postnatal neuronal positioning and dendrite dynamics to elaborate the specific cytoarchitecture of the mouse cortical area.

A unique feature of the mammalian cerebral cortex is in its tangential parcellation via anatomical and functional differences. However, the cellular and/or molecular machinery involved in cortical arealization remain largely unknown. Here we map expression profiles of classic cadherins in the postnatal mouse barrel field of the primary somatosensory area (S1BF) and generate a novel bacterial artificial chromosome transgenic (BAC-Tg) mouse line selectively illuminating nuclei of cadherin-6 (Cdh6)-expressing layer IV barrel neurons to confirm that tangential cellular assemblage of S1BF is established by postnatal day 5 (P5). When we electroporate the cadherins expressed in both barrel neurons and thalamo-cortical axon (TCA) terminals limited to the postnatal layer IV neurons, S1BF cytoarchitecture is disorganized with excess elongation of dendrites at P7. Upon delivery of dominant negative molecules for all classic cadherins, tangential cellular positioning and biased dendritic arborization of barrel neurons are significantly altered. These results underscore the value of classic cadherin-mediated sorting among neuronal cell bodies, dendrites and TCA terminals in postnatally elaborating the S1BF-specific tangential cytoarchitecture. Additionally, how the "protocortex" machinery affects classic cadherin expression profiles in the process of cortical arealization is examined and discussed.

Dynamic expression patterns of G protein-regulated inducer of neurite outgrowth 1 (GRIN1) and its colocalization with Galphao implicate significant roles of Galphao-GRIN1 signaling in nervous system.

GRIN1 (Gprin 1) is a signaling molecule coexpression of which with constitutively active form of Galphao can stimulate neurite extensions in Neuro2a cells, yet its in vivo roles remain elusive. Here, we examine expression profiles of GRIN1 during mouse development by in situ hybridization (ISH) and immunohistochemistry. ISH analysis revealed that GRIN1 expression was limited to the nervous system at all developmental stages tested: in the central nervous system, GRIN1 expression occurred within the entire embryonic mantle zones, while it became restricted to sets of nuclei at postnatal to adult stages. Immunohistochemistry using a GRIN1-specific antibody demonstrated that GRIN1 colocalized with Galphao at neuronal dendrites and axons, but it was not detected in glial cells. These results suggest that Galphao-GRIN1 pathway could mediate significant roles in neuronal migration and differentiation at embryonic stages and exert functions in wiring and/or maintenance of specific neural circuitries at postnatal to adult stages.